Marcin Sarewicz

Sebastian Pintscher

Rafal Pietras

Arkadiusz Borek

Łukasz Bujnowicz

Guy Hanke

William A Cramer

Giovanni Finazzi

Artur Osyczka

Rafał Pietras

Borek

Catalytic Reactions and Energy Conservation in the Cytochrome bc 1 and b 6 f Complexes of Energy-Transducing Membranes

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

UQH2 Qos

Ubiquinol bound at the strong-binding niche UQH2 Qow Ubiquinol bound at the weak binding niche UQ Qos Ubiquinone bound at the strong-binding niche UQ Qow Ubiquinone bound at the weak binding niche USQ Ubisemiquinone

Introduction

Cytochromes bc (Cytbc) constitute one of the broadest groups of energy-transducing enzymes, present in almost every living cell. They are key components of both respiratory and photosynthetic electron transport chains and their function is often indispensable for the operation of these chains.

In this review we focus on two important enzymes from that group: cytochrome bc1 (Cytbc1)

-involved in respiration and bacterial photosynthesis and cytochrome b6f (Cytb6f) -studied in plant, algal and cyanobacterial photosynthesis. We summarize a broad range of structural, mechanistic and physiological aspects required for function of these enzymes, presenting both the textbook fundamentals based on the well-established ideas and the new intriguing concepts and attractive hypotheses emerging from more recent studies.

It is remarkable that the extensive knowledge gathered so far on Cytbc has left many key issues not satisfactorily understood and opened new intriguing issues for consideration. Among subjects still intensely debated are: the molecular mechanism of the catalytic electron bifurcation and the basis of high energetic efficiency of the enzyme, the mechanism of proton transfer to and from the catalytic sites, the role of dimer and the inter-monomer electron transfer, and the role of Cytbc in regulation of electron flow and in redox (ROS) signaling.

It should be noted that in some cases there are questions about mechanisms of Cytbc structurefunction have been regarded as beyond doubt. Despite the fact that the catalytic Q cycle, the conceptual framework in which Peter Mitchell described principles of his chemiosmotic theory, is widely accepted for Cytsbc1, its applicability to details of the function of Cytsb6f remain subject to some questions. Although that the scheme showing cyclic electron transfer (CET) appears in every textbook of photosynthesis, the actual pathway through which electrons recycle back to the main electron transport chain remains controversial. It is clear that all these and many other questions related to this important group of enzymes await resolution in future studies.

Even though our intention was to comprehensively cover the knowledge on Cytbc, the breadth of the subject makes it impossible to include all issues which relate to it. The reader is referred to several excellent reviews on this subject that have appeared in the literature. 2 Energy Conserving and Regulatory role of Intracellular Cytochromes-bc

Conversion of solar or chemical energy into a biologically useful form is one of the most fundamental processes of living organisms. As described by chemiosmotic theory [START_REF] Mitchell | Possible Molecular Mechanisms of the Protonmotive Function of Cytochrome Systems[END_REF][START_REF] Mitchell | Coupling of Phosphorylation to Electron and Hydrogen Transfer by a Chemi-Osmotic Type of Mechanism[END_REF] , this process involves generation of a transmembrane electrochemical potential gradient,𝛥𝜇 ~𝐻 + (or proton motive force, pmf) by enzymes which use external sources of energy to couple electron transfer to proton translocation between cellular compartments separated by a membrane (termed energy-transducing membrane) [START_REF] Al-Attar | Energy Transduction by Respiratory Metallo-Enzymes: From Molecular Mechanism to Cell Physiology[END_REF] . PMF forces the ATP-synthase to convert ADP and Pi into ATP, and the energy is stored by maintaining the concentrations of ATP, ADP, and Pi far from their equilibrium values, thus providing the universal source of energy that powers cellular metabolism. [START_REF] Al-Attar | Energy Transduction by Respiratory Metallo-Enzymes: From Molecular Mechanism to Cell Physiology[END_REF] The enzymes that generate pmf are typically organized in chains, such as the mitochondrial and photosynthetic electron transfer chains, where the individual membrane-embedded electron transport protein complexes are linked functionally by components having diffusional freedom to move between complexes: hydrophobic low-molecular weight electron carriers and water-soluble protein electron carriers, respectively, inside and outside the membrane 30,[START_REF] Cramer | Cytochrome Complexes: Evolution, Structures, Energy Transduction, and Signaling[END_REF] . These carriers shuttle electrons between complexes which, overall, catalyze electron flow from a primary electron donor to a final electron acceptor, while the energy released during this process is used to transfer protons across the membrane.

There are two general mechanisms in electron transport chains by which protons are transferred across the energy-transducing membrane. In one mechanism, reduction of a quinone (coupled to proton uptake) occurs at one side of the membrane while quinol oxidation (coupled to proton release) takes place at the opposite side of the membrane (Mitchell's redox loop [START_REF] Mitchell | Possible Molecular Mechanisms of the Protonmotive Function of Cytochrome Systems[END_REF][START_REF] Mitchell | The Protonmotive Q Cycle: A General Formulation[END_REF][START_REF] Mitchell | Protonmotive Redox Mechanism of the Cytochrome B-C1 Complex in the Respiratory Chain: Protonmotive Ubiquinone Cycle[END_REF] ). In the other mechanism, a series of protonation/deprotonation reactions taking place within the specific proton channels of an electron transfer complex (e.g. complex I and IV of mitochondrial electron transport chain) involving successive pK changes of amino acids in the trans-membrane pathway, allow the pumping of protons between the two sides of the membrane without involvement of quinone molecules.

Cytochromes bc complexes (Cyt-bc) comprise a set of the ubiquitous complexes contributing to generation of pmf, through a mechanism involving quinone/quinol oxidation-reduction reactions.

This superfamily of complexes includes cytochrome bc1 (Cytbc1) (ubiquinol: cytochrome c oxidoreductase, EC 7.1.1.8) and cytochrome b6f (Cytb6f) (plastohydroquinone:plastocyanin oxidoreductase, EC 7. 1.1.6). Cytbc1 provides an electronic connection between quinone molecules and water-soluble electron carriers (such as cytochrome c, c2 or c6) catalyzing a net reaction of oxidation of quinol (ubiquinol in mitochondria and several prokaryotic cells or menaquinol some bacteria, see section 4.1) and reduction of water-soluble electron carrier (section 4.2.1). Cytb6f catalyzes an analogous reaction between membrane embedded plastoquinol molecules and the watersoluble plastocyanin (PC) of chloroplasts or Cytc6 (in cyanobacteria and some microalgae) (section 4.2.2). In all cases, the energy released in these electron transfer reactions powers the oxidation of quinol and reduction of quinone, taking place at the distinct catalytic sites located on the opposite sides of the membrane, according to the first mechanism of H + translocation described above.

Cytbc1 participates in respiration in oxygen-utilizing cells and also electron transfer in numerous bacteria which utilize alternative terminal electron acceptors in addition to oxygen (Fig. 1).

In mitochondria, Cytbc1 (also referred to as mitochondrial complex III or respiratory complex III) is a confluence point for reducing equivalents from the various dehydrogenases (mitochondrial complexes I, II) (Fig 1 A). In animal mitochondria, with the sole exception of some parasites, it is essential for mitochondrial respiration, as there is no alternative route to oxidation of ubiquinol by molecular oxygen. This enzyme is not essential in bacteria, where alternative mechanisms to oxidize ubiquinol usually exist in parallel to Cytbc1. On the other hand, Cytbc1 is an essential enzyme, in some photosynthetic bacteria such as Rhodobacter sphaeroides or R. capsulatus for cyclic, non oxygenic, photosynthetic electron transfer in the absence of a terminal electron acceptor but it is not essential when these organisms are grown heterotrophically (Figure 1 B) 34 .

Cytb6f participates in oxygenic photosynthesis in cyanobacteria, and plant and algal chloroplasts. It is a crucial component of the linear electron transfer ('Z scheme' or LET) linking photosystem I (PSI) with photosystem II (PSII) (Figure 1 C). In addition it also participates in cyclic electron transfer (CET) (Fig. 1 C) (see sections 3.6.5 and 10.1).

Apart from the involvement in generating pmf, Cytbc are believed to be potential points of regulation of electron flow. This stems from the fact that their two quinone binding sites are directly connected with the two main redox pools (intra-membrane ubiquinone/plastoquinone and soluble cytochrome c/plastocyanin) of respiration/photosynthesis. Hence, by virtue of catalyzing opposite chemical reactions, these sites may influence one another (the product of one catalytic site becomes a substrate for the second site and vice versa). Another regulatory function of Cytb6f is to trigger changes in the chloroplasts' light harvesting capacity in response to changes in the ambient light intensity by sensing variations in the redox state of the plastoquinone pool. These changes modulate the interaction of Cytb6f with a kinase able to reversibly phosphorylate the light harvesting complexes, leading to their preferential association with one of the two photo centers (PSII and PSI) in a process called 'state transitions' (section 10.3) [START_REF] Rochaix | Protein Kinases and Phosphatases Involved in the Acclimation of the Photosynthetic Apparatus to a Changing Light Environment[END_REF] . Moreover, although the activities of Cytbc1 and Cytb6f complexes are different, because of the above reactions, they are also inferred to be involved in mitochondrial and chloroplast redox signaling. [START_REF] Sena | Physiological Roles of Mitochondrial Reactive Oxygen Species[END_REF] Finally under specific conditions, Cytbc can generate a limited amount of superoxide, as a side reaction of the catalytic cycle (section 7.5). [START_REF] Adam-Vizi | Bioenergetics and the Formation of Mitochondrial Reactive Oxygen Species[END_REF] O2 + e -→ O2 -; Em = -140 mV

It is noted that the specific rate of superoxide (O2 -) production in purified active crystallizable Cytb6f, normalized to the rate of electron transport, is more than an order of magnitude greater than that of the yeast respiratory Cytbc1 38

Overview of Structure and Function of Cytochromes bc

Cytbc1 and Cytb6f share several structural and mechanistic features. Both are homodimers with each monomer containing a set of similarly arranged spatially separated quinone binding sites and redox active cofactors required for completion of the catalytic cycle (fig. 2). The general features common to both complexes, are summarized in sections 3. 1-3.4. Despite these similarities, several important differences exist between them. Therefore the two following sections of this chapter 3.5 and 3.6 discuss separately Cytbc1-and Cytb6f-specific properties.

Catalytic Sites, Redox cofactors, and Cofactor Chains

In each monomer of the Cyt-bc dimeric complex, the hemes and the iron-sulfur 2Fe-2S

Rieske-type cluster (2Fe2S) assemble into two chains of cofactors designed to link the two catalytic quinone binding sites with diffusible pools of substrates (quinone and Cytc/PC). The two quinone binding sites catalyze opposing reactions (quinol oxidation / quinone reduction) as integral parts of the catalytic cycle (see section 3.2).

In Cytbc1, the quinol oxidation site (the Qo site) is linked with Cytc by the c-chain composed of 2Fe2S and heme c1. The b-chain, composed of heme bL/bp and heme bH/bn, connects the Qo/Qp site with the quinone reduction site (the Qi/Qn site) located at the opposite side of the membrane.

Considering the relative values of the redox midpoint potentials of cofactors, the c-chain and the bchain are commonly named the high-and low-potential chain, respectively. A characteristic feature of this arrangement is that the Qo site is located between the cand b-chains, which has crucial consequences for the catalytic Q cycle mechanism (Figure 2).

In Cytb6f, the high-and low-potential chains and the quinone catalytic sites (the Qp and Qn for quinol oxidation and quinone reduction, respectively, see section 11 for explanation on the usage of the names of the catalytic sites in this review and on other nomenclatural issues) are arranged in the similar manner, but some variation in the redox cofactor exist: heme f (Em = +370 mV) is present in the high-potential chain in place of heme c1 and the low-potential chain contains an additional and atypical heme cn (not present in Cytbc1).

Brief Overview of Catalytic Cycle

The mechanism by which Cytbc1 links electron transfer and proton translocation reactions is the proton motive Q cycle. [START_REF] Mitchell | Protonmotive Redox Mechanism of the Cytochrome B-C1 Complex in the Respiratory Chain: Protonmotive Ubiquinone Cycle[END_REF][START_REF] Crofts | The Role of the Quinone Pool in the Cyclic Electron-Transfer Chain of Rhodopseudomonas Sphaeroides: A Modified Q-Cycle Mechanism[END_REF] In engages the high-and low-potential -chains (c-and b-chains, respectively) and the Qo, and the Qi sites. Protons are taken up at the Qi site, carried across the membrane by the quinol (QH2), and released at the Qo site. The Qi site and the Qo site are located toward the electronegative and electropositive sides of the membrane (n and p sides, respectively).

For that reason, the Qi site and Qo of Cytbc1 site are alternatively named Qn and Qp, respectively (see section 11).

The Qo site, being positioned between the cand b-chains, oxidizes QH2 and passes its two electrons in a highly specific manner: one electron is delivered to the c-chain while the other electron is delivered to the b-chain. This process occurs in a so-called bifurcation reaction in which QH2 is oxidized by 2Fe2S of the c-chain and by the heme bL of the b chain. The reduced 2Fe2S passes 1 electron to heme c1, which subsequently reduces diffusible Cytc. The reduced heme bL passes 1 electron to heme bH. The electron is then used for quinone (Q) reduction at the Qi site. This way the Qo-site-mediated oxidation of QH2 delivers one electron to the Qi site at the time, meaning that two cycles of the Qo site are needed to fully reduce a Q to QH2 at the Qi site. Overall, the net oxidation of one QH2 molecule and transfer of two electrons to two molecules of Cytc leads to the uptake of two protons from the n side (mitochondrial matrix or bacterial cytoplasm) and the release of four protons on the p side (intermembrane space of mitochondria or bacterial periplasm). A step-wise scheme of the reaction taking place during the Q cycle in Cytbc1 is shown in figure 3.

In the case of Cytb6f, although details of the Q cycle have been studied less completely and flash-induced reduction of heme bn separate from that of heme bp not defined experimentally [START_REF] Furbacher | On the Question of Interheme Electron Transfer in the Chloroplast Cytochrome B6f in Situ[END_REF] , the general principles of the Q cycle and proton translocation mechanism are believed by most researchers in the field to be essentially the same as for Cytbc1 (see perspective on this issue in section 3.6.4). This includes the bifurcation reaction taking place at the PQ oxidation site (Qp site) with electrons delivered into the high-and low-potential chains. One of the main differences is the presence of an additional cofactor within the low-potential chain. This is an atypical high-spin and relatively high-potential, heme cn covalently attached to the protein through a single thioether bond [START_REF] Stroebel | An Atypical Haem in the Cytochrome B6f Complex[END_REF][START_REF] Kurisu | Structure of the Cytochrome b6f Complex of Oxygenic Photosynthesis: Tuning the Cavity[END_REF] . The planes of hemes bn and cn are oriented perpendicularly and share electrons [START_REF] Zatsman | Heme-Heme Interactions in the Cytochrome B6f Complex: EPR Spectroscopy and Correlation with Structure[END_REF] with each other with the latter being close to the PQ binding niche. Although a role of this heme is still unknown it was proposed that together with heme bn it forms a pair of cofactors that can deliver two electrons to PQ in order to reduce it to PQH2 in a virtually one-step event. This idea, however, has not been experimentally validated yet. Because the heme cn is exposed to the stromal side of the thylakoids, this cofactor could provide an additional entray point for electrons at the level of the Qn site, possibly triggering a different Q cycle mechanism, or providing an oxidant for stromal electrons in cyclic electron flow (see below and discussion in section 3.6.5). Another difference between Cytbc1 and Cytb6f, due to the variation in the composition of the high-potential chain, is the fact that electrons from the 2Fe2S are transferred to heme f which further reduces diffusible PC or Cytc6 (instead of Cytc interacting with Cytc1). (E) Oxidation of QH2 takes place as in case B, but the electron from the low potential chain reduces SQi to QH2, which is associated with uptake of two protons from the n side. (F) Q, QH2 and reduced Cytc diffuse out of the Qo, Qi and Cytc1, respectively, and the cycle starts again. Hemes are shown as rhombuses, the 2Fe2S is shown as yellow-white circles.

Lipid bilayer is marked as green lines. For simplicity, only one monomer is shown and the motion of the head domain of iron-sulfur protein (ISP-HD) was not included. The scheme in general is believed to hold true also for Cytb6f with some modifications at the Qn site associated with the presence of additional heme cn and possible modifications to the electron transfer sequence associated with the involvement of this enzyme in CET (see Fig. 4).

The Q cycle originally proposed by Mitchell 32 considered a mechanistic link between electron bifurcation at the Qo site and electron confurcation at the Qi site. As mentioned above, the bifurcation at the Qo site results in one electron delivered to the Qi site, implying that a completion of confurcation at the Qi site requires the functional link with another redox-active enzyme/cofactor cooperating with Cytbc1 and serving as an additional source of electrons for that site. The second electron to complete reduction of SQ at the Qi site was initially proposed to originate from the activity of another dehydrogenase. Further studies revealed that Cytbc1 does not need to be associated with any source of electrons but QH2 to complete the cycle. Instead, Cytbc1 must oxidize two QH2 molecules at the Qo site to sequentially deliver two electrons to the Qi site [START_REF] Crofts | The Role of the Quinone Pool in the Cyclic Electron-Transfer Chain of Rhodopseudomonas Sphaeroides: A Modified Q-Cycle Mechanism[END_REF] . On the other hand the idea that the complex interacts with another dehydrogenase could be relevant to account for the possible role of Cytb6f in CET. Indeed, it has been proposed that electrons donated by PSI to the NADPH + / ferredoxin pool could be reinjected into the PQ(H2) pool by electron transfer to PQ or putative plastosemiquinone (PSQ) at the Qn site. In such a case, the catalytic cycle of Cytb6f under conditions favoring CET, could operate according to the original Mitchell Q cycle (Fig. 4). The bifurcation reaction taking place at the Qo/Qp site is essential for the energetic efficiency of the catalytic cycle, and has long been considered a unique feature of Cytbc. However, more recent findings indicate that bifurcation reaction may exist in other systems, such as those utilizing flavins [START_REF] Buckel | Based Electron Bifurcation, A New Mechanism of Biological Energy Coupling[END_REF] .

The H-shaped electron transfer system in the dimer

Cytbc1 complexes from mitochondria and bacterial cells have been crystallized and their structures solved to atomic resolution. The mitochondrial Cytbc1 is a symmetrical, oligomeric homodimer, in which each monomer encompasses the three catalytic subunits (Cytb, Cytc1 and ISP) surrounded by a periphery of non-redox, supernumerary subunits. The bacterial enzyme is also dimeric and the overall structure of the catalytic subunits as well as the position of the redox cofactors are highly similar to the mitochondrial enzyme (Fig 5). The major difference is a general lack of the supernumerary subunits in the bacterial enzyme.

One unusual aspect of the dimeric structure is that ISP spans the dimer. This ISP subunit is anchored in one monomer by a single transmembrane helix, while the peripheral, hydrophilic domain that contains 2Fe2S (ISP-HD) is located in the other monomer, where it forms part of the quinol oxidation Qo site [START_REF] Berry | Unanswered Questions about the Structure of Cytochrome Bc1 Complexes[END_REF][START_REF] Xia | Structural Analysis of Cytochrome Bc1 Complexes: Implications to the Mechanism of Function[END_REF][START_REF] Baniulis | Mechanisms of Superoxide Generation and Signaling in Cytochrome Bc Complexes[END_REF] . A comparison of the structures of enzymes crystallized in the absence or presence of the Qo site inhibitors indicated that the ISP-HD occupies different positions with respect to the other parts of the complex. This suggested that movement of ISP-HD between positions proximal to Cytb and Cytc1 is necessary to transfer an electron between the Qo site and heme c1 in the c-chain. This hypothesis was confirmed by site-directed mutagenesis studies which demonstrated that such movement was essential for enzyme activity. However, the mechanistic rationale behind the movement is not clear (see section 3.4).

In view of the topographic arrangement of ISP, the functional module at the level of the Cytbc1 monomer consists of the Cytb and Cytc1 and ISP linked to them through its ISP-HD, while also interacting with Cytb and Cytc1 of the other monomer through its hydrophobic anchor. The same structure feature applies to Cytb6f (see below). [START_REF] Agarwal | Role of Domain Swapping in the Hetero-Oligomeric Cytochrome B6 f Lipoprotein Complex[END_REF] The crystal structures of Cytbc1 revealed that large distances separate the cofactors from different monomers, except for the two hemes bL that are positioned close enough to allow electron transfer between the monomers (Fig 5). The capacity of this electron transfer bridge to connect the monomers on a catalytically-relevant timescale was further proven in studies that exploited cross-inactivated forms of the enzyme [START_REF] Świerczek | An Electronic Bus Bar Lies in the Core of Cytochrome Bc1[END_REF] . The existence of the heme bL-bL electron transfer bridge implicates that cofactor chains in the dimer assemble into an H-shaped electron transfer system that connects the two Qo sites with the two Qi sites. Given that the functional module of each of the monomers provides all necessary elements to support the catalytic Q cycle (the cand b-chains, the Qo and Qi sites), the mechanistic and physiological meaning of the H-shaped electron transfer system remains unclear but intriguing (see section 9 for details).

Cytb6f is also a homodimer. The topographic arrangement of ISP that are intertwined with the dimer and the movement of ISP-HD are conserved, as is the distance between the two hemes bp. In view of the general similarity of cofactor architecture between Cytbc1 and Cytb6f it is reasonable to assume that the characteristic H-shaped electron transfer system is a shared feature of all Cytbc.

Large-scale movement of ISP-HD

The large-scale movement of the extrinsic domain of the iron-sulfur protein (ISP-HD) is an inherent part of the catalytic cycle of Cytbc1 and Cytb6f. The movement alternately brings the 2Fe2S either to the position close to the catalytic Qo/QP site (referred to as the Qo position or the b-position)

or close to heme c1/f, (the c-position) separating in time and space the two electron transfers within the high potential chain: 2Fe2S at the b-position exchanges electrons exclusively with quinol bound at the site, while at the c-position it exchanges electrons exclusively with heme c1/f. This movement was proven obligatory in many studies showing that impairment of the mobility of ISP-HD entails a severe limitation or complete loss of enzymatic activity [START_REF] Tian | Evidence for the Head Domain Movement of the Rieske Iron-Sulfur Protein in Electron Transfer Reaction of the Cytochrome Bc1 Complex[END_REF][START_REF] Darrouzet | Probing the Role of the Fe-S Subunit Hinge Region during Qo Site Catalysis in Rhodobacter Capsulatus Bc1 Complex[END_REF][START_REF] Darrouzet | Uncovering the [2Fe2S] Domain Movement in Cytochrome Bc1 and Its Implications for Energy Conversion[END_REF][START_REF] Darrouzet | Large Scale Domain Movement in Cytochrome Bc1: A New Device for Electron Transfer in Proteins[END_REF][START_REF] Darrouzet | Movement of the Iron-Sulfur Subunit beyond the Ef Loop of Cytochrome b Is Required for Multiple Turnovers of the Bc1 Complex but Not for Single Turnover Qo Site Catalysis[END_REF][START_REF] Sadoski | Use of a Photoactivated Ruthenium Dimer Complex to Measure Electron Transfer between the Rieske Iron-Sulfur Protein and Cytochrome C1 in the Cytochrome Bc1 Complex[END_REF][START_REF] Rajagukguk | Effect of Mutations in the Cytochrome b Ef Loop on the Electron-Transfer Reactions of the Rieske Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF] . This is commonly explained considering the effect of changing in distance between cofactors on electron transfer rate: [START_REF] Page | Natural Engineering Principles of Electron Tunnelling in Biological Oxidation-Reduction[END_REF] the ISP-HD arrested at a specific position permanently (due to the presence of some inhibitors) or for prolonged period of time (due to specific mutations) cannot efficiently shuttle electrons between the Qo/QP site and heme c1/f as specific distances between the cofactors in a frozen configuration are not optimal for rapid electron transfer along the entire cofactor chain. For example, in a configuration with the ISP-HD arrested at the Qo site, a large distance between the 2Fe2S and heme c1 prevents rapid electron transfer between these two cofactors and consequently enzymatic activity is lost.

First structural indications for ISP-HD movement. The possibility that conformational changes might be associated with the catalytic reactions involving 2Fe2S was considered before the first crystallographic structures of Cytbc became . available [START_REF] Brandt | Analysis of Inhibitor Binding to the Mitochondrial Cytochrome c Reductase by Fluorescence Quench Titration. Evidence for a "catalytic Switch" at the Qo Center[END_REF][START_REF] Brandt | Significance of the "Rieske" Iron-Sulfur Protein for Formation and Function of the Ubiquinol-Oxidation Pocket of Mitochondrial Cytochrome c Reductase (Bc1 Complex)[END_REF] . Although the movement itself was not mentioned, a mechanism evoking a "catalytic switch" of 2Fe2S was formulated on the basis of different affinities of MOA-stilbene and UHNQ inhibitors to the Qo site that depended on the redox state of 2Fe2S. The concept of ISP-HD movement during the catalytic cycle was born soon after crystallographic structures with resolved ISP became available [START_REF] Xia | Crystal Structure of the Cytochrome Bc1 Complex from Bovine Heart Mitochondria[END_REF][START_REF] Iwata | Complete Structure of the 11-Subunit Bovine Mitochondrial Cytochrome Bc1 Complex[END_REF][START_REF] Zhang | Electron Transfer by Domain Movement in Cytochrome Bc1[END_REF] . Early structures of chicken Cytbc1 revealed two conformations of ISP-HD [START_REF] Zhang | Electron Transfer by Domain Movement in Cytochrome Bc1[END_REF] , suggesting that it might switch between different positions during the catalytic cycle. This was further substantiated by the mammalian Cytbc1 structures containing all 11 monomer subunits [START_REF] Iwata | Complete Structure of the 11-Subunit Bovine Mitochondrial Cytochrome Bc1 Complex[END_REF] , which clearly showed that ISP-HD can adopt different conformations for which 2Fe2S was found at the b-position, at the c-position or somewhere in between. Further crystallographic and cryo-EM structures of Cytbc originating from many different organisms: bacteria [START_REF] Kurisu | Structure of the Cytochrome b6f Complex of Oxygenic Photosynthesis: Tuning the Cavity[END_REF][START_REF] Berry | X-Ray Structure of Rhodobacter Capsulatus Cytochrome Bc1: Comparison with Its Mitochondrial and Chloroplast Counterparts[END_REF][START_REF] Kleinschroth | X-Ray Structure of the Dimeric Cytochrome Bc1 Complex from the Soil Bacterium Paracoccus Denitrificans at 2.7-Å Resolution[END_REF][START_REF] Esser | Inhibitor-Complexed Structures of the Cytochrome Bc1 from the Photosynthetic Bacterium Rhodobacter Sphaeroides[END_REF][START_REF] Esser | Crystal Structure of Bacterial Cytochrome Bc1 in Complex with Azoxystrobin Reveals a Conformational Switch of the Rieske Iron-Sulfur Protein Subunit[END_REF][65][START_REF] Hasan | Quinone-Dependent Proton Transfer Pathways in the Photosynthetic Cytochrome B6f Complex[END_REF][START_REF] Yan | Intraprotein Transfer of the Quinone Analogue Inhibitor 2,5-Dibromo-3-Methyl-6-Isopropyl-p-Benzoquinone in the Cytochrome B6f Complex[END_REF] , chlorophyta [START_REF] Stroebel | An Atypical Haem in the Cytochrome B6f Complex[END_REF] , fungi [START_REF] Hunte | Structure at 2.3 Å Resolution of the Cytochrome Bc1 Complex from the Yeast Saccharomyces Cerevisiae Co-Crystallized with an Antibody Fv Fragment[END_REF][START_REF] Lange | Crystal Structure of the Yeast Cytochrome Bc1 Complex with Its Bound Substrate Cytochrome C[END_REF][START_REF] Palsdottir | Structure of the Yeast Cytochrome Bc1 Complex with a Hydroxyquinone Anion Qo Site Inhibitor Bound[END_REF][START_REF] Birth | Structural Analysis of Atovaquone-Inhibited Cytochrome Bc1 Complex Reveals the Molecular Basis of Antimalarial Drug Action[END_REF][START_REF] Solmaz | Structure of Complex III with Bound Cytochrome C in Reduced State and Definition of a Minimal Core Interface for Electron Transfer[END_REF] , plants [START_REF] Malone | Cryo-EM Structure of the Spinach Cytochrome b6f Complex at 3.6 Å Resolution[END_REF] to avian [START_REF] Berry | Structure of the Avian Mitochondrial Cytochrome Bc1 Complex[END_REF][START_REF] Crowley | The Role of Molecular Modeling in the Design of Analogues of the Fungicidal Natural Products Crocacins A and D[END_REF][START_REF] Berry | Crystallographic Location of Two Zn 2+ -Binding Sites in the Avian Cytochrome Bc1 Complex[END_REF] and mammalian proteins [START_REF] Esser | Crystallographic Studies of Quinol Oxidation Site Inhibitors: A Modified Classification of Inhibitors for the Cytochrome Bc1 Complex[END_REF][START_REF] Esser | Surface-Modulated Motion Switch: Capture and Release of Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF][START_REF] Gao | Structural Basis for the Quinone Reduction in the Bc1 Complex: A Comparative Analysis of Crystal Structures of Mitochondrial Cytochrome Bc1 with Bound Substrate and Inhibitors at the Qi Site[END_REF] , including humans [START_REF] Guo | Architecture of Human Mitochondrial Respiratory Megacomplex I2III2IV2[END_REF] , were all consistent with the concept that ISP-HD must undergo a constrained diffusion in order to facilitate electron transfer between the Qo/Qp site and Cytc1/Cytf.

One recognized exception comes from recent analysis of the structure of Cyt-bc in supercomplex with cytochrome c oxidase from Mycobacterium smegmatis, which suggests that a globular domain of a diheme Cytc (equivalent of Cytc1) might move instead of ISP-HD to support the catalytic cycle [START_REF] Gong | An Electron Transfer Path Connects Subunits of a Mycobacterial Respiratory Supercomplex[END_REF][START_REF] Wiseman | Structure of a Functional Obligate Complex III2IV2 Respiratory Supercomplex from Mycobacterium Smegmatis[END_REF] Effects of the Qo/Qp site inhibitors. It is recognized that two types of inhibitors exert opposite effects on the average position of ISP-HD seen in crystals [START_REF] Kim | Inhibitor Binding Changes Domain Mobility in the Iron-Sulfur Protein of the Mitochondrial Bc1 Complex from Bovine Heart[END_REF] . The first group of inhibitors includes compounds, such as stigmatellin, famoxadone, nHDBT, atovaquone and DBMIB in Cytb6f, which fix ISP-HD at the b-position and thus are classified as Pf inhibitors (f-for fixed). The second group includes compounds that do not arrest the ISP-HD at a single b-position, but, quite oppositely, expel ISP-HD from the Qo/Qp site forcing the domain to adopt positions remote from the Qo/Qp site, including the c-position [START_REF] Xia | Structural Analysis of Cytochrome Bc1 Complexes: Implications to the Mechanism of Function[END_REF][START_REF] Berry | Structure of the Avian Mitochondrial Cytochrome Bc1 Complex[END_REF][START_REF] Esser | Crystallographic Studies of Quinol Oxidation Site Inhibitors: A Modified Classification of Inhibitors for the Cytochrome Bc1 Complex[END_REF][START_REF] Schoepp | The Qo-Site Inhibitor DBMIB Favours the Proximal Position of the Chloroplast Rieske Protein and Induces a pK-Shift of the Redox-Linked Proton[END_REF] . As they do not prevent the motion of ISP-HD, they are classified as Pm inhibitors (m-for mobile). Typical inhibitors of this group are myxothiazol, MOA-stilbene and strobilurin derivatives (see section 5).

Constrained diffusion or allosteric control.

There are several mechanistic aspects of the movement of ISP-HD which remain the subject of discussion [START_REF] Berry | Unanswered Questions about the Structure of Cytochrome Bc1 Complexes[END_REF] . One of the most important and still unsolved issues concerns a fundamental question of whether the motion is simply a stochastic, thermally-activated process or whether it is rather controlled by a particular state or states of the enzyme during the catalytic cycle [START_REF] Xia | Structural Analysis of Cytochrome Bc1 Complexes: Implications to the Mechanism of Function[END_REF][START_REF] Berry | Observations Concerning the Quinol Oxidation Site of the Cytochrome Bc1 Complex[END_REF][START_REF] Cooley | Protein Conformational Changes Involved in the Cytochrome Bc1 Complex Catalytic Cycle[END_REF][START_REF] Covian | Regulatory Interactions in the Dimeric Cytochrome Bc1 Complex: The Advantages of Being a Twin[END_REF][START_REF] Cooley | Across Membrane Communication between the Qo and Qi Active Sites of Cytochrome Bc1[END_REF][START_REF] Cen | Domain Movement of Iron Sulfur Protein in Cytochrome Bc 1 Complex Is Facilitated by the Electron Transfer from Cytochrome b L to b H[END_REF][START_REF] Sarewicz | Magnetic Interactions Sense Changes in Distance between Heme bL and the Iron-Sulfur Cluster in Cytochrome Bc1[END_REF] . The simplest answer one may consider is that the motion represents a stochastic process of constrained diffusion [START_REF] Darrouzet | Uncovering the [2Fe2S] Domain Movement in Cytochrome Bc1 and Its Implications for Energy Conversion[END_REF][START_REF] Sarewicz | Magnetic Interactions Sense Changes in Distance between Heme bL and the Iron-Sulfur Cluster in Cytochrome Bc1[END_REF][START_REF] Crofts | Mechanism of Ubiquinol Oxidation by the Bc1 Complex: Different Domains of the Quinol Binding Pocket and Their Role in the Mechanism and Binding of Inhibitors[END_REF][START_REF] Crofts | Physicochemical Aspects of the Movement of the Rieske Iron Sulfur Protein during Quinol Oxidation by the Bc1 Complex from Mitochondria and Photosynthetic Bacteria[END_REF] , which is fast enough (~80 000 s -1 ) [START_REF] Millett | Kinetics of Electron Transfer within Cytochrome Bc1 and between Cytochrome Bc1 and Cytochrome C[END_REF] not to limit the overall rate of catalysis [START_REF] Crofts | The Modified Q-Cycle Explains the Apparent Mismatch between the Kinetics of Reduction of Cytochromes c1 and bH in the Bc1 Complex[END_REF] . The stochastic diffusion, in contrast to any mechanism that would rely on a specific element or elements of control imposed on the ISP-HD movement, has the clear advantage of not requiring energy expenditure for "information gain" needed to control the cycle. This seems important given that the primary function of the enzyme is to conserve energy.

However, the general difficulty with understanding the high energetic efficiency of bifurcation taking place at the Qo/Qp site and associated with its efficient suppression of side reactions (see section includes a structure originally resolved by Iwata et al. [START_REF] Iwata | Complete Structure of the 11-Subunit Bovine Mitochondrial Cytochrome Bc1 Complex[END_REF] which was not classified to the group of cpositions and for which distance is ~2.3 nm and angle ~65 o . For this structure, the distance between Bt H161 ( Rh H156) of ISP and heme c1 is short enough to allow the formation of a H-bond between this histidine Nτ atom and the propionate group of heme c1 indicating the most probable conformation predisposed to fast electron exchange between the cluster and heme c1.

Structure-function relationships for the neck region of ISP-HD. While detailed comparisons of structures revealed several elements that might undergo specific conformational changes upon the motion of ISP-HD, the cause-effect relationships of these elements in the context of the mechanism are not fully understood. The most evident structural change concerns the neck region which, depending on the position of ISP-HD, adopts conformations differing in length [START_REF] Xia | Structural Analysis of Cytochrome Bc1 Complexes: Implications to the Mechanism of Function[END_REF][START_REF] Darrouzet | Uncovering the [2Fe2S] Domain Movement in Cytochrome Bc1 and Its Implications for Energy Conversion[END_REF] . When the ISP-HD is at the c-position, the neck forms a small helical structure, whereas docking the domain to the Qo site coincides with the "melting" of this small helix, thus lengthening the connection between the anchor and the ISP-HD (Fig. 6). However, helix formation was seen upon transition of ISP-HD from the "low-affinity fixed position" (group 3) to b-position (group 1) [START_REF] Berry | Unanswered Questions about the Structure of Cytochrome Bc1 Complexes[END_REF] .

The first evidence for involvement of the neck region in ISP-HD mobility can be found in studies showing that mutations in the conserved fragment of the Box 1 sequence of the ISP (ISP: Rh L136) that destroy sensitivity of 2Fe2S to UQ at the Qo site, were mitigated by mutations in the hinge region (ISP: Rh V44 or ISP: Rh A46) 101,102 . Further mutational studies systematically investigated the effects of changing the length and amino acid composition of the neck region on movement of ISP-HD. Elongating the neck by insertion of 1, 2 or 3 alanine residues (+1Ala, +2Ala, or +3Ala mutants, respectively) was found to decrease overall electron transfer through the highpotential chain and the magnitude of this decrease depended on the length of the insertion. In +1Ala, the electron transfer to heme c was found to occur within the millisecond timescale, while in +2Ala and +3Ala -in the second timescale, which is orders of magnitude slower than in the native enzyme (micro to milliseconds). Apparently ISP-HD movement, which in the native enzyme is fast and not rate-limiting, becomes rate-limiting in the mutants [START_REF] Darrouzet | Uncovering the [2Fe2S] Domain Movement in Cytochrome Bc1 and Its Implications for Energy Conversion[END_REF][START_REF] Millett | Kinetics of Electron Transfer within Cytochrome Bc1 and between Cytochrome Bc1 and Cytochrome C[END_REF]103,104 .

Interestingly, +1Ala is still functional in vivo, while +2Ala and +3Ala are not, indicating that some level of functional tolerance to restrictions of the ISP-HD movement exists Detailed analysis of the kinetic data lead to the proposal that these mutants progressively restrict the movement in such a way that the ISP-HD gets arrested for a prolonged period of time at the b-position (for milliseconds in +1Ala or seconds in +2Ala or +3Ala) [START_REF] Darrouzet | Probing the Role of the Fe-S Subunit Hinge Region during Qo Site Catalysis in Rhodobacter Capsulatus Bc1 Complex[END_REF][START_REF] Darrouzet | Large Scale Domain Movement in Cytochrome Bc1: A New Device for Electron Transfer in Proteins[END_REF][START_REF] Sarewicz | Magnetic Interactions Sense Changes in Distance between Heme bL and the Iron-Sulfur Cluster in Cytochrome Bc1[END_REF][105][106][107] . Also, replacement of the neck-region residues with 6 prolines [START_REF] Darrouzet | Probing the Role of the Fe-S Subunit Hinge Region during Qo Site Catalysis in Rhodobacter Capsulatus Bc1 Complex[END_REF]106 all but abolished helix formation and precluded the release of the domain from the b-position. Surprisingly, shortening of the neck region by 1 to 5 amino acids did not lead to loss of catalysis and the EPR spectra obtained for deletion mutants still reflected the ability of 2Fe2S to interact with the UQ and UQH2 bound at the Qo site, albeit with stoichiometry of ISP to Cytb progressively decreasing to 0.2 in 5-residue-deletion mutant [START_REF] Darrouzet | Probing the Role of the Fe-S Subunit Hinge Region during Qo Site Catalysis in Rhodobacter Capsulatus Bc1 Complex[END_REF] .

The conclusion reached from analysis of kinetic data that the alanine insertions arrest the ISP-HD at the b-position was confirmed by EPR studies with oriented membranes [START_REF] Cooley | Across Membrane Communication between the Qo and Qi Active Sites of Cytochrome Bc1[END_REF]100,[108][109][110][111] and pulse EPR with isolated Cytbc1 [START_REF] Sarewicz | Magnetic Interactions Sense Changes in Distance between Heme bL and the Iron-Sulfur Cluster in Cytochrome Bc1[END_REF]107,112,113 . The latter studies allowed direct monitoring of changes in the average position of the ISP-HD with respect to heme bL by measuring the distance-dependent enhancement of reduced 2Fe2S relaxation by oxidized heme bL. The average distance between these two cofactors was found to increase in the following order +2Ala mutant, +1Ala mutant, WT 107 .

These measurements also revealed that the position of ISP-HD in +2Ala is no longer influenced by Pm inhibitors but remains sensitive to the addition of stigmatellin [START_REF] Sarewicz | Magnetic Interactions Sense Changes in Distance between Heme bL and the Iron-Sulfur Cluster in Cytochrome Bc1[END_REF] . This suggests that conformational changes at the neck region can be overwhelmed by the energy of stigmatellin binding, with a large contribution coming from H-bond formation (typical value of energy required to break an H-bond is ~21 kJ/mol) 20. Structure-function relationships for the interaction of ISP-HD with Cytb. Given that the Pf and Pm inhibitors exert different effects on the position of ISP-HD the question arises as to how the Pf inhibitors fix the domain at a position close to the Qo site. One of the most obvious structural effects of the presence of stigmatellin, nHDBT, atovaquone, crocacin D and ascochlorin (group-1 of Pf inhibitors) at the Qo site is formation of the H-bond between Rh H156 ( Bt H161 or Sc H181) of the ISP subunit and the inhibitor. Formation of this bond is not only associated with docking of ISP-HD to the Qo site, but also stabilizes the reduced state of 2Fe2S by significantly raising its redox potential 114- 116 . This suggests stabilization of the reduced cluster under conditions when it forms the H-bond with a molecule occupying the Qo site. Indeed, the observation that the reduced cluster is detected by EPR at the b-position in membranes indicates that interactions of the reduced 2Fe2S and the natural quinone at the Qo site are also stabilized. On the other hand ISP-HD appears to favor the c-position when the cluster is oxidized 100 . This difference in affinity to quinones at the Qo site between the reduced and oxidized cluster may be one of the elements contributing to the mechanism that diminishes the risk of energy-wasting short-circuits (see section 7.4.1). It should be noted however, that some inhibitors (famoxadone, fenamidione or JG-144 inhibitors, all belonging to the group 2 of Pf inhibitors) hold the domain at the b-position without creating an H-bond to Rh H156/ Bt H161 117 . This indicates that factors other than the H-bond between Rh H156/ Bt H161 of ISP and the occupant of the Qo site can also stabilize binding of ISP-HD to Cytb. While formation of this bond would be expected to significantly strengthen the interaction, it is clearly not an absolute requirement for the domain fixation to Cytb or Cytb6.

It seems reasonable to assume that some changes in conformation on the surface of Cytb or Cytb6 contribute to the process of fixing to or release of the domain from the b-position [START_REF] Berry | Unanswered Questions about the Structure of Cytochrome Bc1 Complexes[END_REF][START_REF] Xia | Structural Analysis of Cytochrome Bc1 Complexes: Implications to the Mechanism of Function[END_REF][START_REF] Esser | Surface-Modulated Motion Switch: Capture and Release of Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF][START_REF] Berry | Observations Concerning the Quinol Oxidation Site of the Cytochrome Bc1 Complex[END_REF] . The potential "holding and release lever", if it exists, would be expected to localize to the part of the Cytb/b6 which remains in close contact with the surface of ISP-HD. These regions encompass small helices that are connected to the transmembrane ones by loops: cd1, cd2 and ef loop [START_REF] Cramer | Transmembrane Traffic in the Cytochrome b6f Complex[END_REF][START_REF] Berry | Unanswered Questions about the Structure of Cytochrome Bc1 Complexes[END_REF][START_REF] Rajagukguk | Effect of Mutations in the Cytochrome b Ef Loop on the Electron-Transfer Reactions of the Rieske Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF][START_REF] Esser | Crystal Structure of Bacterial Cytochrome Bc1 in Complex with Azoxystrobin Reveals a Conformational Switch of the Rieske Iron-Sulfur Protein Subunit[END_REF]118 .

However, when structures containing Pf and Pm inhibitors are compared, there is no significant change in the position of helices in Cytb, except for the cd1 helix, which seems to be slightly pushed inside

Cytb by ISP-HD upon interaction of stigmatellin with 2Fe2S [START_REF] Berry | Unanswered Questions about the Structure of Cytochrome Bc1 Complexes[END_REF] . Furthermore, a comparison of the structures of intermediate positions (group 3) and the c-position (group 4) reveals no changes in the cd1 and cd2 helices, suggesting that the motion of ISP-HD between "low-affinity fixed positions" (group 3) and the c-position (group 4) is a free diffusional process constrained by the neck region of the ISP acting as a peptide tether.

It is not clear if the shift of the cd1 helix upon docking ISP-HD at the Qo site is an effect of the inhibitor itself, allowing the 2Fe2S cluster to "come closer" or if the H-bond between the inhibitor and Rh H156 induces "pressure" of ISP-HD on the structure forcing cd1 to move. Such a pressure could be an element of the "spring-loaded" mechanism which proposes that H-bond formation between ISP-HD and a substrate present in the Qo site stores some energy which is released after the reaction and facilitates the dissociation of ISP-HD from the b-position 119 .

The involvement of the cd1 helix in binding of ISP-HD to the Qo site is also implicated by mutational studies in which Rh G167, located at the end of the cd1 and before the cd2 helix, was replaced by proline. This significantly perturbed the docking of ISP-HD to Cytb, which was reflected in an observed shift in the equilibrium distribution of the ISP-HD toward the c-position 120,121 . As a result, the interaction of 2Fe2S with UQ or UQH2 at the Qo site was broken and the enzymatic activity of the Rh G167P mutant dramatically decreased, abolishing the functionality of the enzyme in vivo.

Also superoxide generation by the Qo site increased in this mutant (see section 7.5 for details). Despite these effects, the mutant remained sensitive to stigmatellin. Interestingly, addition of the +1Ala or +2Ala mutation to the Rh G167P mutant was found to mitigate the inhibitory effects of Rh G167P mutation to various extent. A remarkable example was the double Rh G167P/+2Ala mutant in which the equilibrium distribution of ISP-HD was similar to that of single mutant +1Ala mutant, partially restoring sensitivity of the reduced 2Fe2S to UQ at the Qo site. This brought back some level of functionality to the enzyme in vivo even though both single mutations were non-functional. Clearly, the opposite effects of Rh G167P mutation ("pushing" ISP-HD away from the b-position) and the alanine insertions (arresting ISP-HD at the b-position) can partly compensate one another, establishing that dynamics of the docking/release of ISP-HD to/from cytochrome Cytb can be effectively coinfluenced by the specific interactions at the ISP-HD-Cytb interface and the conformational changes taking place within the neck region. A functional link between these two distinct structural domains was also documented in other mutational studies 122 .

Another eye-catching structural element which may somehow influence the ISP-HD movement is the region of the ef loop -(Cytb: Rh 286-292, Bt 262-268) a small peptide fragment connecting the transmembrane E helix, the conserved PEWY sequence and the transmembrane F helix [START_REF] Berry | Unanswered Questions about the Structure of Cytochrome Bc1 Complexes[END_REF][START_REF] Rajagukguk | Effect of Mutations in the Cytochrome b Ef Loop on the Electron-Transfer Reactions of the Rieske Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF][START_REF] Iwata | Complete Structure of the 11-Subunit Bovine Mitochondrial Cytochrome Bc1 Complex[END_REF] . It is considered to be the main "obstacle" that forms a barrier to ISP-HD on its trajectory from the b-to c-position. The role of the ef loop was proposed following the finding that mutations in this region can compensate the effect of +1Ala [START_REF] Darrouzet | Uncovering the [2Fe2S] Domain Movement in Cytochrome Bc1 and Its Implications for Energy Conversion[END_REF][START_REF] Darrouzet | Movement of the Iron-Sulfur Subunit beyond the Ef Loop of Cytochrome b Is Required for Multiple Turnovers of the Bc1 Complex but Not for Single Turnover Qo Site Catalysis[END_REF] . Further mutational studies revealed that bulky side chains located in the middle part of the loop limit the ability of ISP-HD to move outside the Qo site 122 . This leads to the proposal that the ef loop acts as a switch increasing or decreasing the rate of electron transfer between the Qo site and heme c1 [START_REF] Rajagukguk | Effect of Mutations in the Cytochrome b Ef Loop on the Electron-Transfer Reactions of the Rieske Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF]103 , which together with the cd1 helix constitutes the mechanism of binding and release of ISP-HD depending on the redox state of hemes b [START_REF] Esser | Surface-Modulated Motion Switch: Capture and Release of Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF][START_REF] Cooley | Protein Conformational Changes Involved in the Cytochrome Bc1 Complex Catalytic Cycle[END_REF][START_REF] Cooley | Across Membrane Communication between the Qo and Qi Active Sites of Cytochrome Bc1[END_REF][START_REF] Cen | Domain Movement of Iron Sulfur Protein in Cytochrome Bc 1 Complex Is Facilitated by the Electron Transfer from Cytochrome b L to b H[END_REF] .

However, as demonstrated by mutational studies, amino acid side chains such as Rh L286, Rh I292 on the ef loop in fact may serve to resist in transitions between band c-position [START_REF] Rajagukguk | Effect of Mutations in the Cytochrome b Ef Loop on the Electron-Transfer Reactions of the Rieske Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF] . The control mechanisms steering this loop during the catalytic cycle is a matter of discussion. The observed effect of modifications on the motion caused by mutations in the ef loop does not preclude a simple stochastic model for the movement of ISP-HD in which the mobile loop regions undergo random thermal fluctuations rather than specifically-controlled motion.

Not only the redox state of hemes b, but also specific events taking place at the Qi site have been considered as potential factors that influence the mobility of ISP-HD. In view of topographical arrangement of ISP-HD in the dimer, this would imply a highly ordered communication between monomers which would allow transfer of information over ~ 4 nm across Cytb, possibly through the membranous anchor of ISP that ends in proximity to the Qi site. Most of the proposals on this type of allosteric influence are derived from analysis of the effects of antimycin bound at the Qi site.

However, as we discuss below, the interpretation of these effects needs to be treated with caution.

Effect of the Qi site inhibitor, antimycin. Evaluating the influence of antimycin on the basis of crystallographic structures containing antimycin bound at the Qi site is difficult as the majority of the antimycin-containing structures were obtained with stigmatellin bound at the Qo site, which overrides any possible structural effects of antimycin on the Qo site. To our knowledge, until now, there are two PDB crystallographic structures in the database, containing antimycin bound at the Qi and no other inhibitor at the Qo site 117,123 . As discussed by Berry and Huang (ibidem), antimycin does not seem to change the ground state of the structure but might change dynamics of the structure 117 . The dynamic change was considered to accommodate several biochemical studies suggesting that antimycin induces effects that might be associated with changes to the different mobility of the domain.

The first series of experiments was based on the susceptibility to proteolytic cleavage of the neck region (between residues Rh 46 and 47) as a sensor of conformational changes taking place in this region. It was found that the cleavage was substantially limited by fixing of ISP-HD at b-position by Pf inhibitors, when compared to the enzyme with Pm or without any bound inhibitors 124 . On the other hand addition of antimycin increased proteolysis, suggesting that this inhibitor induced changes that increased accessibility of this fragment for thermolysin.

The second series of experiments focused on various EPR methods to monitor the FeS.

Continuous wave (CW) EPR spectra of microscopically disordered samples containing antimycin-or HQNO-supplemented membranes did not show any significant changes in the spectra when compared to the non-inhibited samples 111 . However, analysis of the samples containing ordered layers of membranes revealed that after addition of antimycin, the angular-dependent amplitudes of gx and gy transitions of the 2Fe2S cluster are different from those detected in samples not containing antimycin [START_REF] Cooley | Across Membrane Communication between the Qo and Qi Active Sites of Cytochrome Bc1[END_REF]111 . Pulse EPR measurements with isolated Cytbc1 showed that the presence of antimycin at the Qi site caused an increase in distance between the reduced 2Fe2S and oxidized heme bL [START_REF] Sarewicz | Magnetic Interactions Sense Changes in Distance between Heme bL and the Iron-Sulfur Cluster in Cytochrome Bc1[END_REF] and an increase in the efficiency of spin-lattice relaxation of the 2Fe2S cluster via two-phonon Raman process. All recognized changes associated with the presence of antimycin at the Qi site consistently indicate that this inhibitor induces some structural changes that modify the average position of ISP-HD. This effect was interpreted by some authors as evidence for allosteric inter-and/or intramonomer interactions between the Qi/Qn and Qo/Qp site that lead to specific sequences of a "duty cycle" of Cytbc1/b6f during the catalysis [START_REF] Cooley | Protein Conformational Changes Involved in the Cytochrome Bc1 Complex Catalytic Cycle[END_REF][START_REF] Covian | Regulatory Interactions in the Dimeric Cytochrome Bc1 Complex: The Advantages of Being a Twin[END_REF][START_REF] Cooley | Across Membrane Communication between the Qo and Qi Active Sites of Cytochrome Bc1[END_REF]125,126 .

In our view, the observed effects of antimycin on the ISP-HD are associated with a decrease in the rigidity of the Cytbc1 structure which decreases the energetic barrier for moving from b-to cpositions. This simple concept assumes that the population of ISP-HD domains is distributed over different conformations and various inhibitors, including antimycin, just modify a barrier for this movement [START_REF] Sarewicz | Magnetic Interactions Sense Changes in Distance between Heme bL and the Iron-Sulfur Cluster in Cytochrome Bc1[END_REF] . In fact, pulse EPR measurements revealed that the average position of ISP-HD can be effectively modified by mutations and/or inhibitors and the observed shifts in this position can be placed in the following order (starting from b-position and going further toward the c-position): +2Ala mutant > stigmatellin > Cytb: Rh G167P/ISP:+2Ala double mutant > +1Ala mutant > Cytb: Rh G167P/ISP:+1Ala double mutant > no-inhibitor WT > WT + myxothiazol ≈ Cytb: Rh G167P ≈ WT + antimycin > WT + antimycin + myxotiazol [START_REF] Świerczek | An Electronic Bus Bar Lies in the Core of Cytochrome Bc1[END_REF][START_REF] Sarewicz | Magnetic Interactions Sense Changes in Distance between Heme bL and the Iron-Sulfur Cluster in Cytochrome Bc1[END_REF]120 . In all cases, addition of stigmatellin overrides any other effect, which suggest that the energy of H-bonding between the inhibitor and ISP Rh H156 exceeds the energy associated with changing the structure of the neck region. Although stigmatellin is able to overcome the effects of alanine insertion into the neck region, neither Pm inhibitors (myxotiazol) nor antimycin influence the ISP-HD position in +2Ala mutations [START_REF] Sarewicz | Magnetic Interactions Sense Changes in Distance between Heme bL and the Iron-Sulfur Cluster in Cytochrome Bc1[END_REF] . Gathering all these observations we can propose that the strength of factors that influence the ISP-HD mobility can be put in the following order: Pf inhibitors which form H-bonds > conformation of cd1 helix ( Rh G160P mutation) > increase in the length of the neck region > Pm and Qi inhibitors (myxothiazol, antimycin). The ef loop was not included in this list since no direct spectroscopic measurements of distance distribution of ISP-HD were done for mutants in this particular region. However, comparing the kinetic data and the effect of the redox potential of 2Fe2S, it may be suggested that the energetic barrier of crossing the ef loop by ISP-HD is lower than the barrier imposed by +2Ala, comparable with the effect of +1Ala insertion and higher than the barrier imposed by Pm-type or Qi inhibitors [START_REF] Darrouzet | Movement of the Iron-Sulfur Subunit beyond the Ef Loop of Cytochrome b Is Required for Multiple Turnovers of the Bc1 Complex but Not for Single Turnover Qo Site Catalysis[END_REF][START_REF] Rajagukguk | Effect of Mutations in the Cytochrome b Ef Loop on the Electron-Transfer Reactions of the Rieske Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF] .

We note that most of the structural studies involving EPR spectroscopy have been performed

under conditions for which 2Fe2S was reduced. Although no changes on the surface of the ISP-HD associated with the changes in the redox state of the cluster, were observed [START_REF] Berry | Unanswered Questions about the Structure of Cytochrome Bc1 Complexes[END_REF] , the domain with a reduced 2Fe2S appear to show a greater tendency to occupy the b-position compared to the domains with the oxidized 2Fe2S. This is in line with the observation that Em of 2Fe2S depends on the position of ISP-HD 106 .

Effect of movement on redox potential of the 2Fe2S cluster. There are numerous studies

showing that the Em of 2Fe2S depends on interaction between ISP-HD with the Qo site [START_REF] Darrouzet | Probing the Role of the Fe-S Subunit Hinge Region during Qo Site Catalysis in Rhodobacter Capsulatus Bc1 Complex[END_REF][START_REF] Darrouzet | Large Scale Domain Movement in Cytochrome Bc1: A New Device for Electron Transfer in Proteins[END_REF][START_REF] Darrouzet | Movement of the Iron-Sulfur Subunit beyond the Ef Loop of Cytochrome b Is Required for Multiple Turnovers of the Bc1 Complex but Not for Single Turnover Qo Site Catalysis[END_REF]106,109,113,114,116 . In general, the greater the tendency for ISP-HD to be at the b-position, the more positive the redox potential of 2Fe2S. For the mutants +1Ala, +2Ala and 6Pro, that were discussed above, the Em values of 2Fe2S were found to increase by the same order. This could suggest decreased in water accessibility, which changes pK of Rh H156 ( Bt H161). However, the position of the ISP-HD is not alone in influencing the redox potential of 2Fe2S. Another important factor is the interaction of the domain with the occupant of the Qo site. The most prominent effect is formation of a H-bond with stigmatellin, which increases the midpoint potential by more than 200 mV. Other inhibitors that form a H bond with Rh H156 of the ISP-HD, such as tridecylstigmatellin or UHDBT, exert a weaker effect on the rise in the redox potential 106 . Also, the presence of Q bound at the Qo site appears to slightly contribute to a slight increase in the redox midpoint potential of 2Fe2S 109 . However, it should be noted that the effect of Q bound at the Qo site has been recognized in +nAla mutant but not in WT 109

(see table I in ref 127 ). On the other hand, Pm inhibitors such as myxotiazol or MOA-stilebene slightly decrease the Em value of the 2Fe2S 128 . Thus, it was proposed that H-bonding is the factor which controls the redox potential and stabilizes binding of substrate and enzyme 128 .

3.5

Cytochrome bc1 -specific structural and functional elements

Subunit composition of Cytochrome bc1

All Cyts bc1 contain three protein subunits per monomer with redox prosthetic groups: the diheme Cytb, the monoheme Cytc1 and the ISP (Rieske iron-sulfur protein). Cytb is built of 8 transmembrane helices and contains a relatively high-potential heme bH and a lower potential heme bL. Cytc1 and the ISP are membrane-anchored and their water-soluble, globular domains contain prosthetic groups (high-potential heme c1 and 2Fe2S, respectively). All these three subunits are necessary for the catalytic function of Cytbc1. In some bacteria, such as Paracoccus denitrificans, Rhodospirillum rubrum, or Rhodobacter (R.) capsulatus, Cytbc1 contains only these three subunits.

Other bacteria, including R. sphaeroides, contain a fourth subunit of unknown function that lacks prosthetic groups. 129

In addition to the catalytic subunits, Cytsbc1 of mitochondria contain as many as seven (in some yeast) or eight (in bovine and human) supernumerary subunits. The largest supernumerary subunits associate to the catalytic subunits from the mitochondrial matrix side. The functions of the supernumerary subunits are not known, expect for the established mitochondrial processing peptidase activity of large subunits in plants. The supernumerary subunits are believed to contribute to the increased stability of mitochondrial complexes, but are not required for the electron transfer and proton translocation activities of the enzyme; the three-subunit bacterial enzymes have the same electron transfer and proton translocation functionality as the mitochondrial enzymes.

3.5.2

Structure and spectral properties of hemes bL, bH, c1 and 2Fe2S cluster.

The redox-active components of Cytbc1 in all organisms are evolutionary conserved within the catalytic core. [START_REF] Berry | Structure and Function of Cytochrome Bc Complexes[END_REF] Remarkably, as shown by the crystal structures of Cytbc1 from various organisms, the positions of the stationary cofactors, hemes b and c1, are unchanged among them. [START_REF] Xia | Structural Analysis of Cytochrome Bc1 Complexes: Implications to the Mechanism of Function[END_REF][START_REF] Berry | X-Ray Structure of Rhodobacter Capsulatus Cytochrome Bc1: Comparison with Its Mitochondrial and Chloroplast Counterparts[END_REF][START_REF] Iwata | Complete Structure of the 11-Subunit Bovine Mitochondrial Cytochrome Bc1 Complex[END_REF] The two b-type hemes, (heme bL and bH) of Cytb subunit are embedded within the four-helixbundle, assembling in what appears to be a common (evolutionary conserved) structural motif of many bioenergetic complexes. [130][131][132] In the Cytb subunit, helices named A-D are involved in bundle formation (Fig. 8 A), with helices B and D providing axial ligands for the heme iron of both hemes (Fig. 8 A,B). 133 Both hemes b are axially coordinated by the two conserved histidines (referred to as a bis-His ligation pattern), via bonds between the heme iron and Nε atom of the respective imidazole ring (Fig. 8 B). [START_REF] Berry | X-Ray Structure of Rhodobacter Capsulatus Cytochrome Bc1: Comparison with Its Mitochondrial and Chloroplast Counterparts[END_REF]132 The heme c1, is a c-type heme attached to the globular head of Cytc1 (Fig. 8 C). It is covalently bound to the apoprotein via thioether linkers formed by its two vinyl side chains and two cysteine residues [START_REF] Berry | X-Ray Structure of Rhodobacter Capsulatus Cytochrome Bc1: Comparison with Its Mitochondrial and Chloroplast Counterparts[END_REF][START_REF] Iwata | Complete Structure of the 11-Subunit Bovine Mitochondrial Cytochrome Bc1 Complex[END_REF] , belonging to a characteristic CXXCH motif. 134 The heme iron of heme c1 is axially coordinated by Nε of the conserved histidine from this motif and Sδ of the conserved methionine (referred to as a His-Met ligation pattern). [START_REF] Berry | X-Ray Structure of Rhodobacter Capsulatus Cytochrome Bc1: Comparison with Its Mitochondrial and Chloroplast Counterparts[END_REF]135 Interestingly, in some bacterial species, the ligation of heme c1 is secured by an additional pair of cysteines, that form a disulfide bond to stabilize the distant loop of the globular part of Cytc1. 135 However, the freedom retained for conformational rearrangement results in a partial weakening of the methionine ligation when the cytochrome is in its oxidized form, which is reflected in an ability to bind small exogenous ligands, such as cyanide. 136 Removing the disulfide by mutations was found to significantly perturb the conformational stability and further weaken the methionine ligation. As a result, the potential of the heme dropped dramatically (more than 300 mV) and the reduced cytochrome showed a capability to bind carbon monoxide, all of which imply change in the heme ligation pattern 135 . In other Cytochromes c1, such as mitochondrial Cytc1, the disulfide is not present. Instead, the βXM structural motif (where β stands for the β-branched amino acid located one residue away from the methionine axial ligand) appears to restrict the conformational flexibility of the domain of the methionine axial ligand to the point that the binding of external ligands is not observed. 136 Some bacterial Cytsc1 have both the disufide and the βXM motif. 135,137 Similarly to the heme c1, 2Fe2S is attached to ISP-HD and situated between the two loops of the protein backbone, that are hooked by a disulfide bridge formed between two cysteine residues. 138 The cluster is coordinated by the thiolate side chains of the two conserved cysteine residues, acting as ligands for one Fe ion, and Nδ atoms of the two conserved histidine residues, acting as ligands for second Fe ion (Fig. 8 D) [START_REF] Berry | X-Ray Structure of Rhodobacter Capsulatus Cytochrome Bc1: Comparison with Its Mitochondrial and Chloroplast Counterparts[END_REF]138 . This type of [2Fe-2S] cluster coordination by two Cys and two His residues is almost exclusive to ISP of Cytbc and aromatic ring-hydroxylating monooxygenases and dioxygenases. 139,140 Iron atoms in all the hemes of Cytbc1 are hexa-coordinated and have strong axial ligands (with bis-His and His-Met ligation pattern for hemes b and heme c1, respectively). Therefore, they all exist in low spin states. In the reduced (ferrous) state, the hemes exhibit partially overlapping α/β absorbance bands in the visible region of the UV-VIS spectrum (Fig. 9 A), with α-band maxima at 560-562 and 552-553 nm for hemes b and c1, respectively [141][142][143][144] . The spectral resolution of heme bH and heme bL by optical spectroscopy is difficult. In the bacterial Cytbc1 a deconvolution of the spectra obtained upon redox titration implicates a dominant contribution from the heme bH at 562 nm and a significantly smaller contribution from heme bL, which has a split α-peak spectrum with maxima at 564-566 and ~558 nm. [START_REF] Crofts | The Role of the Quinone Pool in the Cyclic Electron-Transfer Chain of Rhodopseudomonas Sphaeroides: A Modified Q-Cycle Mechanism[END_REF]145,146 On the other hand, the heme c1, due to its much higher redox midpoint potential and α-band maximum at a lower wavelength, can be easily isolated spectrally from hemes b in the partially-reduced enzyme (Fig. 9 A, in orange). 141 In their oxidized (ferric) state, hemes b and c1 are paramagnetic (S = ½) 147 , thus detectable by electron paramagnetic resonance (EPR) spectroscopy at low temperatures (<20 K). All three hemes fall into the HALS (Highly Axial/Anisotropic Low Spin) hemes category, with only their gz (often called gmax) transition being observable by EPR. 132 In general, the bis-His coordinated hemes b exhibit a HALS signal when the imidazole rings of the two His residues are nearly perpendicular to each other. 148 In the case of hemes bL and bH the angles between the two imidazole ring planes are ~84˚ and ~58˚, respectively 132 , which translates into the two separate EPR transitions at g = 3.78 and g = 3.44, respectively (Fig. 9 B) 149,150 . Thus, EPR, unlike optical spectroscopy, offers a full spectral separation of the two hemes b. It is of note that the gz transition of heme bH is sensitive to Qi site occupancy: when the site is occupied by antimycin A (a specific Qi site inhibitor) or semiquinone radical (SQi), the gz value is shifted to g = 3.47 or 3.42, respectively (Fig. 9 B) 149,151 . In R. capsulatus Cytbc1, the gz transition of heme c1 occurs at g = 3.16. 152 The exact g-values for gz transitions of each heme may differ among organisms. Some of them were included in Table 1 It is difficult to use optical spectroscopy in studies on the 2Fe2S, as it has no prominent spectrum 157 and its transitions are dominated by the spectra of hemes. The one-electron reduced form

([2Fe-2S] 1+
) is paramagnetic (due to its ground state with S = ½ resulting from the antiferromagnetic spin coupling between Fe 3+ (S = 5 /2) and Fe 2+ (S = 2) ions) and can only be detected by EPR at temperatures <50K. The fully oxidized ([2Fe-2S] 2+ ) cluster is in the EPR-silent (S = 0) ground state (the spins of the two Fe 3+ ions are spin-coupled). 140,158 The EPR spectrum of the reduced cluster ([2Fe-2S] 1+ ) exhibits a unique rhombic symmetry (Fig. 9 C), with transitions at gz = 2.03, gy = 1.90 and gx between 1.78 and 1.76, resulting in the average g value of approximately 1.9. 157 In bacterial Cytbc1, the shape and the position of the gx transition was found to be sensitive to the occupant of the Qo site (whether it is occupied by substrate, or inhibitor or remains empty), the redox state of quinone (whether quinone or quinol are present) and the size of the quinone pool/potential number or quinone molecules at the Qo site (see section 7.2). For those reasons, EPR analysis of the cluster has proven to be a convenient spectroscopic tool for monitoring the status of the Qo site. [START_REF] Darrouzet | Probing the Role of the Fe-S Subunit Hinge Region during Qo Site Catalysis in Rhodobacter Capsulatus Bc1 Complex[END_REF]159 Midpoint redox potentials (Em) of hemes b, heme c1 and 2Fe2S may vary in different systems, depending on the quinone derivatives employed by the organism to serve as electron/proton carriers.

In organisms using menaquinones, they are usually lower than in organisms that employ ubiquinones. 160 However, a general rule can be drawn, that the hemes b have much lower Em's than 2Fe2S and heme c1. For this reason, the chain of cofactors built by hemes b is referred to as a lowpotential chain, while that formed by the 2Fe2S and heme c1 -high-potential chain. The large gap in potential between components of the high-and low-potential chains is crucial for electron bifurcation and efficiency of the catalytic cycle (see section 7.4). Among the hemes b, heme bL has lower Em (-290 to -50 mV) than heme bH (-160 to 100 mV). [START_REF] Xia | Structural Analysis of Cytochrome Bc1 Complexes: Implications to the Mechanism of Function[END_REF]160 This difference in Em's of the hemes b is evolutionary conserved. 149,160 It is generally assumed to facilitate cross-membrane electron transfer and assist in overcoming the barrier of membrane potential. 161,162 However, such a function becomes less obvious in light of recent studies demonstrating that the difference in Em's of the hemes b is not required to support the in vivo and in vitro functionality of the enzyme. 149 Interestingly, Em of heme bH depends on the occupant (and the redox state) of the Qi site. When the Qi site is occupied by Q, heme bH has a higher potential than when the site is occupied by SQi. This results in a biphasic titration of the heme. 163 However, when the Qi site is occupied by an inhibitor, the bH titrates as a single species. 163,164 The 2Fe2S and the heme c1 usually have close values of Em, both falling in range of 100 to 350 mV. [START_REF] Xia | Structural Analysis of Cytochrome Bc1 Complexes: Implications to the Mechanism of Function[END_REF]160 Some of the known values of Em for each of cofactors in various organisms were included in Table 2.

Cytochrome b6f -specific structural and functional elements

The discussion of structure-function of Cytb6f is based on a high resolution (2.50 Å) crystal structure (PDB ID: 4OGQ) of the complex. The complex consists of a 250 kDa hetero-oligomeric intra-membrane lipo-protein complex containing, per monomer, thirteen transmembrane α-helices and seven prosthetic groups, the latter consisting of five hemes, one chlorophyll a molecule and one β-carotene. In addition, each monomer contains 23 lipid binding sites, the function of which involves a new area of research into the structure-function of intra-membrane proteins, both for Cytb6f and the entire field of membrane proteins.

Comparison of Cytochromes f and c1. In contrast to the conserved nature of Cytb, for the Cytc1 and Cytf, except for the single transmembrane helix (TMH) which binds the subunit to the membrane, the structures are different. The only conserved sequence segment is the CXXCH motif, responsible for covalent binding of the c-type heme. The p side heme binding domain has a completely different secondary structure, β sheet and α-helical, respectively in Cytf and Cytc1 171,172 . In fact, all three of the p side electron transfer proteins which are part of Cytb6f, or interact with it, the ISP, Cytf, and PC, which mediate electron transfer between the quinol hydrogen donor and the P700 PSI reaction center are predominantly in the β-conformation. Both Cytf and Cytc1 have a relatively positive potential, + 370 mV and + 260 mV, respectively, sufficient to provide an oxidant for the ISP.

Subunit composition of Cytochrome b6f

Crystal structures have been obtained for the Cytb6f from the green alga, C. reinhardtii [START_REF] Stroebel | An Atypical Haem in the Cytochrome B6f Complex[END_REF] and cyanobacteria [START_REF] Kurisu | Structure of the Cytochrome b6f Complex of Oxygenic Photosynthesis: Tuning the Cavity[END_REF][START_REF] Agarwal | Role of Domain Swapping in the Hetero-Oligomeric Cytochrome B6 f Lipoprotein Complex[END_REF][65][START_REF] Hasan | Quinone-Dependent Proton Transfer Pathways in the Photosynthetic Cytochrome B6f Complex[END_REF][START_REF] Yan | Intraprotein Transfer of the Quinone Analogue Inhibitor 2,5-Dibromo-3-Methyl-6-Isopropyl-p-Benzoquinone in the Cytochrome B6f Complex[END_REF]173,174 .

The crystal structures of the mitochondrial and bacterial Cytbc1 contain 11 and 3 or 4 polypeptide subunits, respectively, compared to 8 in the Cytb6f from cyanobacteria [START_REF] Kurisu | Structure of the Cytochrome b6f Complex of Oxygenic Photosynthesis: Tuning the Cavity[END_REF]174 and the green alga, C. reinhardtii [START_REF] Stroebel | An Atypical Haem in the Cytochrome B6f Complex[END_REF] . This subunit count refers to those which remain after the purification and Subunits containing redox prosthetic groups with a high degree of conservation between Cytb6f and Cytbc1 are: (i) Cytb in the Cytbc1, which is equivalent in many structure-function aspects to Cytb6 and subunit IV in the Cytb6f 175 ; and (ii) the high potential (Em,7 = + 300 mV) 2Fe2S, often called the "Rieske" protein because of the origin of its discovery 176 . The protein subunit containing the covalently bound c-type cytochrome, Cytf and Cytc1, respectively, is not conserved between

Cytb6f and Cytbc1 complexes.

The Four Small Peripheral Subunits, Pet G, L, M, and N. A unique lattice of four short (3.3 -4.1 kDa), single TMH "hydrophobic sticks," denoted as PetG, PetL, PetM, and PetN (Table 3), provides a hydrophobic lattice, or "picket fence," around each monomer, a structure which is unique among all known integral membrane protein structures. Of these four subunits, PetM 177 , along with the ISP, are the only subunits among the thirteen in the complex that are nuclear-encoded. Specific functions of these four subunits are not defined, although PetL interacts sterically with the TMH of the ISP and may provide a constraint on the membrane-spanning orientation of the latter. The position of much of the lipid in the complex (Fig. 12) between the Pet picket fence and the conserved Cytb6-subIV core of the complex suggests a "boundary lipid" function and that the "picket fence" may have been added to the core of the complex relatively late in the evolution of the complex.

3.6.2 Structure and spectral properties of hemes b, f, cn and 2Fe2S cluster.

High resolution structures of a C-terminal soluble domain of the 250 residue extrinsic Cterminal domain of Cytf 171,172 , and of the N-terminal soluble domain of the ISP 178 , were obtained prior to their determination in the intact multi-subunit complex. The crystallized complex is a symmetric (C2 symmetry) dimer that consists of eight transmembrane polypeptide subunits in plants, green algae, and cyanobacteria [START_REF] Kurisu | Structure of the Cytochrome b6f Complex of Oxygenic Photosynthesis: Tuning the Cavity[END_REF] . The analogous Cytbc1 consists of eleven subunits in the yeast, PDB 1KYO [START_REF] Hunte | Structure at 2.3 Å Resolution of the Cytochrome Bc1 Complex from the Yeast Saccharomyces Cerevisiae Co-Crystallized with an Antibody Fv Fragment[END_REF] and bovine, PDB 1QCK, 1BE3 60 complex, and three in the complex from the photosynthetic bacterium, Rhodobacter sphaeroides [START_REF] Esser | Inhibitor-Complexed Structures of the Cytochrome Bc1 from the Photosynthetic Bacterium Rhodobacter Sphaeroides[END_REF] . In all cases, the dimeric structure of the complex is required for a physiologically significant electron transfer activity, 250 -400 electrons transferred (Cytf/sec) - 1 179-181 . This is possibly because of a defined inter-monomer quinol entry pathway.

A ribbon diagram of Cytb6f (PDB 2ZT9, 4H44, 4OGQ) derived from crystal structure analysis of the cyanobacterium Nostoc sp. PCC 7120 65,[START_REF] Hasan | Quinone-Dependent Proton Transfer Pathways in the Photosynthetic Cytochrome B6f Complex[END_REF]174 , is shown in 2 views differing by a 180° rotation in order to display more clearly the arrangement of the 8 subunits that span the membrane with 13 Heme cn is unique as it lacks an amino acid axial ligand, and the central Fe-atom is penta-coordinated. The sole ligand of heme cn, is provided by H2O or OH -on the heme bn side. Fe of heme cn separated by 4.0 Å from a propionate oxygen of heme bn, which results in electronic coupling, a high spin g =12 EPR signal [START_REF] Zatsman | Heme-Heme Interactions in the Cytochrome B6f Complex: EPR Spectroscopy and Correlation with Structure[END_REF]185 , and an oxidase-like reaction with nitric F and G helices of subunit IV (cyan), using its phytyl tail, functions as a gate for quinol/quinone traffic in the Qp-portal.

Drawing by S. Saif Hasan

Unique Chlorophyll a and β-Carotene. The presence of a single chlorophyll a 182,187,188 and βcarotene 183 in each monomer of Cytb6f is enigmatic. The β-carotene is separated by 14 Å from the chlorophyll [START_REF] Stroebel | An Atypical Haem in the Cytochrome B6f Complex[END_REF][START_REF] Kurisu | Structure of the Cytochrome b6f Complex of Oxygenic Photosynthesis: Tuning the Cavity[END_REF] , too large to allow β-carotene to quench a chlorophyll excited triplet state 189 . (ii) The β-carotene protrudes by approximately 11 Å from the complex between the TMH of Pet G and Pet M.

Transmembrane Hemes, bn and bp. The four TMH of the Cytb6 are connected by loops at the p and n side membrane interfaces. SubIV (~17 kDa) has three TMH (E-G) that form a p side saddle around the four helix bundle of Cytb6. Helix E of SubIV is located in proximity to the A-and Bhelices of Cytb6 while the F and G-TMH span the four helix bundle, close, respectively, to the C, D and B-TMH (Fig. 12). The C-terminus of the E-TMH is separated from the N-terminus of the F-TMH by a distance of ~40 Å, which is bridged by the p side ef-loop. This seven TMH assembly forms the conserved core of Cytb6f, which is bounded in each monomer by the four subunit hydrophobic "picket fence".

The arrangement of the two pairs of transmembrane b-hemes, bp and bn, with inter-heme edgeedge and center-center (Fe-Fe) distances (in parentheses) is shown (Fig. 10), along with the heterogeneity of the inter-heme dielectric constants 184 , and the complex of heme bn with a skeleton of the covalently bound c-type heme cn [START_REF] Zatsman | Heme-Heme Interactions in the Cytochrome B6f Complex: EPR Spectroscopy and Correlation with Structure[END_REF]185,186,190,191 . The heme bn-cn complex, a unique feature of the Cytb6f compared to the Cytbc1, in which the close proximity of the two hemes results in sharing of the 3d electronic shells is displayed in a g = 12 EPR signal [START_REF] Zatsman | Heme-Heme Interactions in the Cytochrome B6f Complex: EPR Spectroscopy and Correlation with Structure[END_REF]185 .

Hemes, bn and bp, Oxidation-Reduction Potentials. Determination of the oxidation-reduction potentials of these hemes in Cytbc1 are consistent in showing a separation of 100-150 mV between the Em's of the two hemes, which are thus often labeled bH and bL (high and low potential hemes).

For the two hemes in the Cytb6f, there is disagreement as to whether the Em's of the two hemes are 191 or are not [START_REF] Furbacher | On the Question of Interheme Electron Transfer in the Chloroplast Cytochrome B6f in Situ[END_REF]192 separable in a redox titration of thylakoid membranes. A simulation shows that a midpoint potential difference of 50 mV or less of a one electron titration cannot be resolved. For the isolated complex, the midpoint potentials of the two hemes in the Cytb6f are separated by approximately 90 mV.

Reducibility of b Hemes; Heterogeneity of Internal Dielectric Constants. Excitonically split circular dichroism (CD) spectra of the Cytbc1 193 and the Cytb6f 184,194 arise from interactions of the reduced hemes. Determination of the time course of heme reduction and the onset of the split CD spectra for the isolated complex shows that the heme pair that is preferentially reduced is the intramonomer pair, bp and bn. This result contrasts with that which is expected. If heme bn, whatever its exact redox potential, has a more positive potential in isolated Cytb6f than heme bp, the two hemes bn should be preferentially reduced under equilibrium conditions. The straight-forward explanation is that the protein medium between hemes bp and bn is more polarizable, i. e., has a higher dielectric constant, than the medium between the two hemes bn 184 implying that the dielectric constant in the complex is heterogeneous and anisotropic. The unique heme cn at the n side quinone (Qn) binding site of Cytb6f 173,191 , located in close ( 4Å) proximity to heme bn, constitutes a major structure difference between Cytbc1 and Cytb6f complexes. Consequently, it would also imply a difference, compared to the mitochondrial and bacterial Cytbc1, in the details of the involvement of heme bn in the transmembrane electron transport pathway [n.b., heme cn is designated heme ci in the C. reinhardtii crystal structure, which first emphasized its unique existence in the structure of Cytb6f 41 ].

Heme cn has no axial ligand on the heme surface facing the intermonomer cavity. As shown by EPR studies which document its open axial ligand position 186 , and co-crystallization of the Cytb6f complex with quinone analog inhibitors 173 , it has been inferred that heme cn serves as the n side quinone binding site (Qn site). The Qn site is located closer to the membrane-water interface in Cytb6f complex than in the respiratory Cytbc1, as noted in the original crystal structure [START_REF] Stroebel | An Atypical Haem in the Cytochrome B6f Complex[END_REF] . This surfaceproximal location of the Qn site implies that a short pathway may suffice for proton conduction to the n side bound quinone from the n side aqueous phase. The crystal structure of Cytb6f from the filamentous cyanobacterium Nostoc PCC 7120, revealed a unique anhydrous Asp20→Arg207 (Cytb6) pathway for proton conduction from the n side aqueous phase to the Qn site for reduction and quinone protonation [START_REF] Hasan | Quinone-Dependent Proton Transfer Pathways in the Photosynthetic Cytochrome B6f Complex[END_REF] .

ISP Electron Transfer; a p Side Conformation/Mobility Problem. One remaining structurefunction problem is that the closest approach distance of the Cytf heme to 2Fe2S in Cytb6f is 28 Å, which is much too large to support physiologically meaningful millisecond electron transfer rates 195,196 . How, then does 2Fe2S of ISP transfer electrons to Cytf? A major gap in understanding of the p side electron transfer reactions for the Cytb6f results from the absence of the structure data that would allow kinetically competent electron transfer from 2Fe-2S to the heme of Cytf. Given the documented conformation change determined from crystal structure data for the avian mitochondrial Cytbc1 (see section 3.4 for details), electron transfer from 2Fe2S to the Cytf heme must involve a rotationaltranslational conformational change in the ISP [START_REF] Hasan | Quinone-Dependent Proton Transfer Pathways in the Photosynthetic Cytochrome B6f Complex[END_REF] .

Lipid content and other subunits

Lipids in the Cytb6f Lipoprotein Complex. (i)'Boundary lipids' define a domain structure.

Because the conserved Cytb6-SubIV core of the complex is separated by a lipid layer from the "picket fence", it is implied, as noted above, that the "picket fence," was added to the core structure at a later stage in the evolution of the Cytb6f, perhaps to facilitate interaction with the reaction center complexes. As noted above, the β-carotene, which protrudes from the surface of the picket fence, may act as a "latch" to facilitate super-complex formation with the PSI reaction center complex 197 .

Lipid Content

The dominant lipid species in plant Cytb6f are monogalactosyl-diacylglycerol (MGDG), digalactosyl-diacylglycerol (DGDG), phosphatidyl-glycerol (PG) and sulfoquinovosyl diacylglycerol (SQDG). The galactolipids (MGDG, DGDG and SQDG) and phospholipids dilinolenoyl-,phosphatidyl glycerol (DLPG), 1,2-dioleoylphosphatidylglycerol (DOPG) and 1, 2 dioleoylsn-glycerol-3-phosphatidylcholine DOPC) stabilize the Cytb6f to a varying extent 198 .

PGRL Components. A 35.7 kDa (pK = 5.2) protein, "PGRL1," is present at unknown stoichiometry in the PSI super-complex C. reinhardtii 197 , and at a stoichiometry comparable to, although smaller (0.5:1), that of ISP in Arabidopsis thaliana, and has been inferred to be the ferredoxin-quinone reductase 199 . The absence of the protein results in a partial decrease of the chlorophyll fluorescence yield that results from reduction of the quinone pool. The redox function is believed to be derived from the six cysteine residues in the protein, perhaps in conjunction with iron that is present in the preparation. PGRL1 is also inferred to be able to bind to the Cytb6f 199 , although it has not been detected in mass spectroscopic analysis of isolated Cytb6f 200 . In addition, it is noted:

(a) the fluorescence yield effects ascribed to PGRL1 are perhaps not quantitative indicators of an obligatory function in the ETC, as they involve changes of a factor of 2-3 on a time scale of 10 seconds, approximately a factor of 1000 times slower than the rate-limiting step of electron transport in this region of the ETC; (b) the rate of P700 reduction, a standard kinetically competent assay for cyclic electron transport, in the presence and absence of the peptide has not been reported. (c) Details of the chemical nature and quantitative properties of the hexaCys-Fe redox moiety in PGRL1, proposed to serve as the redox group responsible for FQR activity, are not presently available. The significance of the PGRL1 components may need to be re-evaluated in light of the recent information on their very diminished stoichiometry relative to the known electron transport components 201

Q cycle in cytochrome b6f: mechanism and controversies

The observation of oxidant-induced reduction 202 in Cytbc1 (see details in section 7), and measurement of a greater than unity ratio of coupled protons translocated to electrons transferred, H + /e > 1, was important in the formulation of the 'modified Q cycle' mechanism [START_REF] Crofts | The Role of the Quinone Pool in the Cyclic Electron-Transfer Chain of Rhodopseudomonas Sphaeroides: A Modified Q-Cycle Mechanism[END_REF] , whose general formulation and application to photosynthetic membranes [START_REF] Mitchell | Possible Molecular Mechanisms of the Protonmotive Function of Cytochrome Systems[END_REF] Support for the application of Q cycle model to Cytbc1 in the purple photosynthetic bacteria has been well documented and discussed extensively in reviews [START_REF] Berry | Structure and Function of Cytochrome Bc Complexes[END_REF][START_REF] Crofts | The Cytochrome Bc1 Complex: Function in the Context of Structure[END_REF][START_REF] Sarewicz | Electronic Connection between the Quinone and Cytochrome c Redox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling[END_REF][START_REF] Crofts | The Role of the Quinone Pool in the Cyclic Electron-Transfer Chain of Rhodopseudomonas Sphaeroides: A Modified Q-Cycle Mechanism[END_REF]117,118,[206][207][208] . Studies on function and, lately also the structure of the related Cytb6f functioning in chloroplasts, green algae, and cyanobacteria, have been reviewed in the last decade [START_REF] Cramer | Structure-Function of the Cytochrome b6f Lipoprotein Complex[END_REF][START_REF] Mulkidjanian | Activated Q-Cycle as a Common Mechanism for Cytochrome Bc1 and Cytochrome b6f Complexes[END_REF][START_REF] Hasan | Transmembrane Signaling and Assembly of the Cytochrome B6f-Lipidic Charge Transfer Complex[END_REF]184,[209][210][211][212][213][214][215][216] .

While there is a general consensus on the occurrence of the "modified Q cycle" in the case of Cytbc1,(see section 3.2 for details) experimental evidences exist that are not completely in agreement with this hypothesis in the case of Cytb6f. In the following, we summarize this evidence, compare contrasting results, and summarize different interpretations of the results.

Arguments which imply that the PQ/PQH2 cycle in Cytb6f is different from the ''modified Q cycle''

proposed for regeneration of UQH2 through function of Cytbc1 in the respiratory chain and photosynthetic bacteria are: i) the presence of an additional heme cn, which could act as a PQ reductase. Based on its redox properties and location within the complex, this heme could catalyze the injection of an electron into the quinone binding side using stromal reductants as a donor (i.e. the presence of ferredoxin: NADP + reductase (FNR) bound to purified plant (spinach) Cytb6f, see below). The electronic coupling between hemes bn and cn, would imply that PQ reduction need not proceed through a semiquinone intermediate but perhaps cooperatively through hemes bn and cn, providing protection against the formation of superoxide and other reactive oxygen species. This mechanism is not compatible with the modified Q cycle mechanism, but, as introduced in section 3.2, it is actually consistent with the original mechanism of the Q cycle as proposed by Mitchell (see Mulkidjanian [START_REF] Mulkidjanian | Activated Q-Cycle as a Common Mechanism for Cytochrome Bc1 and Cytochrome b6f Complexes[END_REF] for a discussion) (see also Fig. 4).

The possibility of direct injection of stromal electron in the b hemes could also account for another observation that is not entirely compatible with the modified Q cycle in Cytb6f:

ii) the 'slow' electrochromic phase attributed to oxidation of the PSQ in the Qp site and transfer of its electron across part of the low dielectric membrane to reduce the intra-membrane b hemes is also observed when the hemes were chemically reduced and therefore not able to function as an electron acceptor 192 . In principle, injecting 1 electron into the stromal site via the heme cn would allow oxidation of one b heme, generating one PQH2 and regenerating the Q cycle (the so called 'activated'

Q cycle mechanism, [START_REF] Mulkidjanian | Activated Q-Cycle as a Common Mechanism for Cytochrome Bc1 and Cytochrome b6f Complexes[END_REF] ). Note, however, that another explanation has been proposed by Joliot and

Joliot, who suggest that the slow electrochromic phase observed in reducing condition could reflect the transmembrane movement of a charged SQ, (the SQ cycle hypothesis, 217 , see however 218 for a different view). This mechanism may represent an adaptation to reducing conditions when no Q is available at the Qn site. Note, however, that some inhibitors of Cytb6f (NQNO, stigmatellin and MOAstilbene) act on the n side at the side of heme cn that faces the quinone-exchange cavity, as shown in crystal structure 173 and prevent oxidant-induced reduction in Cytb6f, in agreement with the modified Q cycle hypothesis.

iii) the initial slope of the 'slow' electrochromic phase shows a pronounced isotopic effect, being slowed four-fold by a H2O/D2O substitution, in contrast to the redox-reactions of Cytf and Cytb6 that were only slightly affected 219 . These findings have led to the hypothesis that proton pumping across Cytb6f could be triggered by the oxidation of PQH2 220 , an idea that is not conceived in the frame of the 'modified' Q cycle, but that is supported by the analysis of Cytb6f mutants of Chlamydomonas 221 .

iv) one component of the 'driving force' for the Q cycle in mitochondria and the purple photosynthetic bacteria is the pronounced difference (> 100 mV) in the redox potentials of the two b-type hemes, (i.

e., bL and bH) which span the membrane. However, it has not been possible, in redox titrations done in situ, i. e., in thylakoid membranes to define, through anaerobic redox titrations a difference in midpoint redox potentials of the two b hemes, b p and bn [START_REF] Furbacher | On the Question of Interheme Electron Transfer in the Chloroplast Cytochrome B6f in Situ[END_REF]192,222 , More recently, however, Alric and colleagues used a different approach for redox titrations and found Em differences between the two b hemes of isolated Cytb6f from Chlamydomonas (-130 mV and -35 mV) 191 , which are closer to those predicted by the 'modified' Q cycle. Thus, an Em difference is seen for the isolated Cytb6f complex, but this difference is not clear in titrations done in situ, in membrane preparations.

v) studies on the kinetics of heme b reduction show that an intramonomer pair (hemes b n and b p ) is reduced first, rather than the two hemes b n being preferentially reduced as implied by the Q cycle 184 .

Moreover, reduction of the two b hemes is not observed with repetitive laser flashes [START_REF] Furbacher | On the Question of Interheme Electron Transfer in the Chloroplast Cytochrome B6f in Situ[END_REF] .

vi) The existence of CET in oxygenic photosynthesis could help to maintain a high H + /e -ratio, as required for proper CO2 assimilation, thereby making the occurrence of a Q cycle less stringent in photosynthesis than in respiration. One of the main purposes of the Q cycle is to couple electron and proton transfer generating a H + /e -ratio = 2 for proton translocation across the complex. Thus, the transfer through the high potential chain of the Cytb6f of the 4 electrons arising from water splitting and oxygen evolution can result in the translocation of 8 protons into the chloroplast lumen which, together with the 4 translocated protons released into the lumen with each O2 molecule, would provide 12 of the 14 H + needed to drive a full rotation of the rotatory c-ring of the chloroplast ATP synthase and therefore the synthesis of the three ATP molecules needed to fix a CO2 molecule. In plants, the additional 2 H + could be generated by CET, assuming that it runs at a prescribed rate of 25 % of the linear non-cyclic pathway. Applying the same reasoning to the cyanobacterium, Spirulina platensis,

where the ATP synthase c-ring contains 15 subunits, implies that CET would have to run at 3/8 or 37.5 % of the rate of the linear pathway to generate the additional three H + needed to drive a complete rotation. However, no such percentage of CET is observed in most photosynthetic organisms, at least under steady state conditions (see section 10). This suggests that CET alone, is not sufficient to provide the 'extra' H + for ATP synthesis. Consistent with this conclusion, Kramer and colleagues measured the H + /e -ratio illumination in intact tobacco leaves and found that it was constant under low to saturating illumination. Therefore, they inferred that this ratio was maintained by a continuously engaged, proton-pumping Q cycle at Cytb6f 223

Although, the Q cycle mechanism has a readily conceived role in contributing to the transmembrane proton flux required to provide ATP levels commensurate with the need for CO2 assimilation, it may be suggested that the Q cycle mechanism applied to Cyt b6f is not obligatory (see however Cape et al,224 for a different conclusion). An alternative mechanism is a membrane Bohr effect, a mechanism which is prominent in the description of the mechanism of proton pumping in the classical bacteriorhodopsin system 225 , and which has been applied to the problem of proton translocation in the mitochondrial cytochrome c oxidase 226 .

An additional electron path related with cyclic electron transfer in photosynthesis

Electrons generated at the PSI reducing side can be re-injected into its donor side via the CET pathway. Discovered by Arnon in the 50's 227 , this process is now considered as a relevant mechanism to counterbalance over-reduction of the PSI acceptor side 228 and to inject 'extra' protons into the thylakoid lumen, to adjust the ATP/NADPH ratio for CO2 assimilation. Although the ratio of ATP/NADPH generated by LET is still uncertain 229 , it could be insufficient to fuel CO2 accumulation in chloroplasts and its assimilation via the Calvin, Benson and Bassham cycle (see Allen 2002 230 for a discussion). In viridiplantae (including green algae and higher plants) CET could be the main route to optimise this process, by bypassing NADPH production while permitting the formation of the pmf, and therefore ATP synthesis.

Two main pathways have been proposed for CET (see section 10 for further details). The first one involves the activity of a chloroplast NAD(P)H dehydrogenase (NDH) complex (review in Peltier et al. 231 ). In plants, this enzyme has similar characteristics to the mitochondrial complex I, while mainly sharing features with bacterial complex one in algae 232 . The plant complex would mainly use Fd as an electron donor, while the algal counterpart (Nda2 complex) uses NADPH as a source of electrons in the green alga Chlamydomonas reinhardtii 231 .

The second CET pathway would correspond to the so called Ferredoxin-Quinone Reductase complex (FQR), the existence of which was proposed by Bendall and coworkers in the 90's 233 , based on the effect of antimycin -a putative inhibitor of this complex -on photosynthetic electron flow.

An alternate target of antimycin in the chloroplast has been proposed by Sugimoto and colleagues as PGR5, a thylakoid protein 234 previously identified as a component of the CET pathway by a genetic screening or the Arabidopsis thaliana 235 . The FQR complex itself has been putatively identified as a membrane complex 199 , containing PGR5 as well as PGRL1, another protein previously invoked as an essential component of the CET machinery in plants 236 . However, see the critique of these studies in section 3.6.4.

Alternatively, the FQR may correspond to Cytb6f itself, mediating electron flow from reduced Fd to the PQ(H2) pool through the additional c heme in the stromal pocket [START_REF] Stroebel | An Atypical Haem in the Cytochrome B6f Complex[END_REF][START_REF] Kurisu | Structure of the Cytochrome b6f Complex of Oxygenic Photosynthesis: Tuning the Cavity[END_REF] . This pprocess could take place thanks to the interaction between the cytochrome complex and the Ferredoxin-NADP Reductase (FNR) enzyme (see section 4.3).

Characteristics of substrates for cytochrome bc1 and b6f

As a main function of Cytbc is the generation of proton motive force utilizing energy associated with electron transfer from low-potential electron donors (membranous pool of quinone derivatives) to water soluble c-type cytochrome (c, c2, c6) or plastocyanin, it is worth introducing basic information on the substrates that are used by these enzymes. A brief summary on structure and redox properties of quinones is especially important as proton translocation and electronic bifurcation is largely associated with quinone chemistry.

Basic redox properties of Quinones

A role of quinones in electron transfer between membranous respiratory complexes was first proposed by Crane et al. in 1957 237,238 . It was shown that extraction of relatively low-weight molecules -ubiquinones from the membrane completely abolished the process of electron transfer within the mitochondrial respiratory chain 239 . In biology, the most widespread quinones belong to a family of 1,4-benzoquinone derivatives. While in eukaryotic cells quinones are generally restricted to ubiquinones (in mitochondria) and plastoquinones (in chloroplasts), prokaryotic organisms can use several other quinone derivatives as membranous electron carriers. Besides ubiquinones and plastoquinones that are commonly found in bacteria or cyanobacteria, respectively, there is a relatively large group encompassing naphthoquinone derivatives -menaquinones 240 . In some bacteria there are numerous, less common quinones that are based on other chemical structures of the redox-active rings. These are not discussed in this review. Interested readers are referred to a comprehensive review on biological quinones 241 .

Despite differences in chemical structure of the redox-active quinone rings, a common feature shared between of mena-ubi-and plastoquinones is a relatively long hydrocarbon chain consisting of several isoprenoid fragments. This imposes a high level of hydrophobicity on natural quinones.

The number of isoprenoid fragments in the chains is usually given by a number put after the name of the respective quinone 241 . For example, UQ-10 means that the ubiquinone posses the tail built of 10 isoprene molecules. The extent of hydrophobicity of quinones is expressed by a large partition coefficient (log10P) for organic/water mixtures such as octanol and water 242 . Quinol forms are generally slightly more hydrophilic than the respective quinone forms. In case of ubiquinone-1 or plastoquinone-1, log10P exceeds 3 and further increase in the length of isoprenoid tail increases the hydrophobicity, forcing quinone molecules to be constrained to a lipid environment 242 . The natural quinones engaged in electron transfer usually have the hydrophobic tail built of 7 -10 isoprenoid moieties, depending on the organism and very rarely exceeds 10. In plastoquinones the number of isoprenoids is usually 9, while in mammalian mitochondria ubiquinones usually have 9 or 10. For menaquinones, the length of the chain most often varies between 7 and 8 241 . Comparison of the chemical structure of menaquinone-7, ubiquinone-10 and plastoquinone-9 is presented in figure 13. Another common feature that makes quinones important members of nearly all enzymatic processes of electron exchange between the membranous enzymes is their ability to undergo reversible two-electron, two-proton reactions of oxidation or reduction without damaging the chemical structure of the redox-active ring 243 . This reaction can be generally described as:

Q + 2H + + 2e -= QH2 (eq. 1)
where Q stands for quinone (fully oxidized form) and QH2 -for quinol or hydroquinone (fully reduced form)

The complete oxidation/reduction reaction of eq. 1 can be divided into two, one-electron steps involving a semiquinone (SQ) intermediate.

Q + e -= SQ -(eq. 2)

SQ -+ e -+ 2H + = QH2 (eq. 3)

Equations 2 and 3 assume that reduction of quinone leads to the semiquinone anion, and further reduction of SQ -is coupled to the protonation of the reduced quinone. This particular proton/electron sequence is supported by the fact, that pK of neutral semiquinone (SQH) is significantly lower than the first pK (pK1) of the first proton dissociation of QH2 244 .

The average redox midpoint potential (Em) of Q/QH2 couple is equal to the arithmetic mean of redox midpoint potentials of the Q/SQ -and SQ -/QH2 couples 245 .

Em (Q/QH2) = 0.5(E1 + E2) (eq. 4)
where: E1 and E2 represent Em values of Q/SQ -and SQ -/QH2 couples, respectively.

The average redox potential of Q/QH2 couple depends on the type of the redox-active ring of a particular quinone molecule and it generally decreases upon substituting the 2,3 and 5,6 positions of the ring with a methyl group 246 . Conversely, when the ring is substituted with a group that has high electronegativity such as chlorine or bromine, the Em of Q/QH2 rises considerably [246][247][248] . For example, when one considers a simple case of 1,4-benzoquinone, its average redox midpoint potential at pH 7 As the redox reactions of quinones involve 3 electron and 3 protonation states, the theoretical number of possible states is 9. However, from all these possible states (electrons and protons), QH + (protonated quinone), QH2 2+ (doubly-protonated quinone) and SQH2 + (doubly-protonated semiquinone) are not possible since their expected pK values are less than 0 244,249 . The typical pK1 and pK2 values for the first deprotonation of QH2, leading to QH -, and the second deprotonation, leading to Q 2-are separated by 2, thus pK2 = pK1+2. Interestingly, pK1 and pK2 are inversely correlated with the redox midpoint potential of Q/SQ -couples and for mena-ubi-and plastoquinone pK1 is in the range 9-11 246 . For semiquinone, the pK value also depends on the Em of Q/SQ -couple with typical values falling in the range 4 -5 244 .

Stability and reactivity of semiquinone radical with molecular oxygen

A potential danger associated with quinone redox reactions, not only those catalyzed by enzymes, is the possibility of reaction of SQ with molecular oxygen 248,250-254

SQ -+ O2 = Q + O2 - (eq. 5)
Although this reaction is reversible, occurrence of superoxide dismutation or superoxide scavenging by, for example Cytc or PC, will shift the equilibrium to the right side 246 . A superoxide release during the enzymatic catalysis by Cytbc1 or Cytb6f is considered deleterious and leads to a decrease in energetic efficiency of the enzyme, as generation of superoxide decouples the oxidation of QH2 at the Qo/Qp site with Q reduction at the Qi/Qn site. Thus, this side reaction decreases the number of protons translocated across the membrane during the catalytic cycle. However, in living cells some portion of O2 -can be scavenged by electron donation from the radical to the oxidized Cytc 255 or PC 256 with a relatively large second order rate constant. Such scavenging of superoxide can be considered as a protection and partial remedy to energy-wasting side reactions, as the electrons that leak to oxygen can be subsequently used to generate pmf at the level of cytochrome c oxidase (CcO) (complex IV) 257 .

The second-order reaction rate constant for the reaction of SQ -with O2 -depends on the Em value of the Q/SQ -couple and generally increases when the potential of the Em of the Q/SQ -couple decreases. When Em(Q/SQ -) is more negative than -200 mV, the reaction of SQ -with dioxygen becomes diffusion-limited 246 .

The mechanism of reduction of O2 to O2 -by the SQ -radical is generally viewed as electron transfer process that follows the Marcus theory of electron transfer 252 . An alternative mechanism proposes a chemical reaction of addition of oxygen to the SQ -ring followed by release of the superoxide anion, which after protonation changes to the neutral, peroxyl radical 253 .

SQ -+ O2 → SQ-OO -+ H + → Q + HOO* (eq. 6)

Valgimigli et al. showed evidence that reaction 6 is significantly slower in solutions in which a semiquinone can form H bonds with molecules of the solvent 253 . Therefore, it can be supposed that formation of a hydrogen bond to the SQ -may be protective against superoxide formation in catalytic sites of enzymes (see section 7.5).

Stability constant of SQ -.

Under equilibrium conditions Q, QH2 and SQ are linked by a reaction of comproportionation (or in reverse direction by disproportionation) 246 :

Q + QH2 = 2 SQ -+ 2H + (eq. 7)
The equilibrium constant of this reaction defines the stability constant (Ks) of SQ -which depends on the difference in Em values of Q/SQ -(E1) and SQ -/QH2 (E2) couples [258][259][260] :

𝐾 𝑠 = [𝑆𝑄 -] 2 [𝐻 + ] 2 [𝑄][𝑄𝐻 2 ] = 10 (𝐸 𝑚 (𝑄 𝑆𝑄 ⁄ )-𝐸 𝑚 (𝑆𝑄 𝑄𝐻 2 ⁄ )) 59.1 ⁄ = 10 (𝐸 1 -𝐸 2 ) 59.1 ⁄ (eq. 8)
The stability constant for SQ defines the equilibrium between concentrations of Q/SQ -/QH2 in this triad. It means that in equimolar mixtures of Q and QH2 some SQ -is also present but its relative amount depends on the equilibrium constant Ks. For some chlorine substituted quinones Ks can be higher than 1 247 . However, for biologically important quinone derivatives, the Em of the Q/SQ -couple is more negative than SQ -/QH2, thus the exponent in the equation 8 is always less than zero (and therefore Ks < 1). Any further increase in the split between the Em values of these two couples (E1 -E2) leads to a decrease in the equilibrium concentration of SQ -in solutions containing a mixture of Q and QH2 forms. This, on one hand, increases the rate of reaction of SQ -with O2 but on the other hand, decreases the probability of superoxide generation as the concentration of SQ -decreases.

Despite the fact that Ks for SQ -is used in chemistry of quinones under equilibrium conditions, it is also widely used to describe the properties of semiquinones that are generated at catalytic sites of enzymes such as the Qo/Qp and Qi/Qn site of Cytbc during catalysis [261][262][263][264] .

The concentration of quinones within the membrane is usually expressed as the number of molecules (Q and QH2) vs the number of CcO molecules. In mammal and plant mitochondria the number of quinones per CcO is around 6-8, while it is much larger in yeast mitochondria (~ 38 per CcO). In purple bacteria the estimated number of quinone molecules per reaction center (RC) is also quite large -in the order of 15 -30. The number of quinones in humans depends significantly on the tissue and the age. The content of Q in mitochondria increases during the first 20 years and after that time gradually decreases to the level even lower than after birth (see discussion in ref. [START_REF] Sarewicz | Electronic Connection between the Quinone and Cytochrome c Redox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling[END_REF] ).

A significant content of the quinone molecules in the membrane may suggest that the comproportionation reactions should be very efficient leading to generation of a significant amount of SQ, which after dismutation would lead to the process of electron self exchange:

Q (A) + Q (B) H2 = Q (A) H2 + Q (A) (eq. 9)
However, such a reaction is very unlikely in membranes as it requires at least two steps that involve reactions of protonation and deprotonation 243,265 , which is difficult in the lipid environment.

Chloroplast photoactive vs non photoactive plastoquinone pools.

The concepts discussed above apply to quinol or quinone (Q(H2)) in bioenergetic membranes in ideal conditions. However, due to crowding of both the photosynthetic and respiratory membranes, differences exist between Q(H2) located in different lipid environments. In the case of photosynthetic membranes (the thylakoids) the proportion of Q(H2) that participates in electron transport (i.e. that feeds the Cytb6f complex) does not correspond to the entire population of PQ(H2). This fraction (the photoactive PQ(H2) pool) makes up a relatively small percentage of the entire PQ(H2) pool, because 60-70% of the total PQ(H2) is non-photoactive, being stored inside thylakoid-associated lipid droplets known as plastoglobules [266][267][268] . There is increasing evidence that the size of this photoactive PQ(H2)

pool is tightly regulated in response to environmental cues, for example changes in light intensity 269,270 . This homeostasis is necessary because the PQ(H2) pool fulfills multiple functions in vivo.

Firstly, it shuttles electrons within the crowded thylakoid membranes, 271 probably via percolation through the lipid environment between other membrane complexes 272,273 . Moreover, its redox state controls physiological processes in response to environmental changes, such as changes in the PSII and PSI antenna size via state transitions 274 ), as well as gene expression and pigment biosynthesis 275 .

Recent results suggest that the amount of PQ(H2) active in electron transport is mainly regulated through exchanges between the photoactive pool (in the thylakoids) and the nonphotoactive pool (in the plastoglobules). Homeostasis of photoactive PQ(H2) abundance seems to be under the control of ABC1 (Activity of Cytbc1) atypical kinases 276 . Knocking out a member of this family (pgr6, 277 , where the ABC1K1 kinase is inactivated (abc1k1) prevents changes in the size of the photoactive PQ(H2) pool, that would otherwise occur in response to high light, facilitating increased electron flow and replacing photo oxidized plastoquinones. Thereby, abck1 mutants display decreased electron flow and photoacclimation responses in high light 278 . ABC1K1 acts in tandem with ABC1K3, another member of the ABC1 family, and the push pull relationship between these two kinases provides a fine tuning mechanism to control the size of the PQ(H2) pool in response to high light 279 .

This tight control of the size of the photoactive PQ(H2) pool in vivo likely explains the phenotype of the menD1 mutant of Chlamydomoans reinhardtii. This mutant is deficient in MenD, the enzyme catalyzing the first step of phylloquinone biosynthesis 280 . Therefore, the phylloquinones playing the role of electron acceptors within PSI are replaced by PQ in this mutant, making PSI less active. However, the menD1 mutant has another phenotype -its photoactive PQ(H2) pool is decreased by 20-30%. This can be explained by assuming that the size of the photoactive PQ(H2) pool is constant between the WT and the mutant. Therefore, the overall number of diffusing PQ(H2) molecules would decrease from around 6 per Cytb6f in the WT to around 4 per Cytb6f complex in menD1 280 . Based on this result, it is tempting to propose that the mechanism by which ABC1K1/ABC1K3 maintains a constant photoactive PQ(H2) pool size depends on "quantification" of the total number of PQ(H2) molecules available per electron flow chain. If this were the case, the size of the pool would be unresponsive in menD1, as the ABC1K1/ABC1K3 machinery will not deliver two additional PQ(H2) per chain to the thylakoid membrane from the plastoglobuli to replace the PSI bound PQ.

Cytochrome c and plastocyanin as electron acceptors

Electron donation from both Cytbc1 and Cytb6f to soluble proteins at the p side has been extensively studied. These molecular events have often served as pioneering models for electron transfer between different proteins, and reflect the development of techniques and theories of how such processes occur. In both cases, a single electron passes from a cytochrome in the complex (Cytc1 in Cytbc1 or Cytf in Cytb6f) to a small, soluble redox protein that is specifically, but transiently bound.

Once reduced, the electron carrier protein must rapidly dissociate (so as not to block the catalytic cycle), and diffuse through the p side space to another large membrane bound protein complex. In mitochondrial respiration this is cytochrome c oxidase (CcO, complex IV) 281 , while in plant photosynthesis this is photosystem I 282 . Protein acceptors vary between Cytc1 and Cytf. In the case of the respiratory complex the acceptor is a c-type heme containing protein, cytochrome c (Cytc) 283 , perhaps one of the most studied of all redox proteins. Cytbc1 in purple bacteria uses a homologue of Cytc, soluble Cytc2, and/or membrane-anchored Cytcy 284,285 as an electron acceptor, although this organism is photosynthetic and the final electron acceptor for the soluble electron carrier is a photosynthetic reaction center 286 . In the case of Cytb6f, the dominant electron acceptor is a coppercontaining protein called plastocyanin (PC) Interestingly, some prokaryotic photosynthetic organisms retain a cytochrome, Cytc6 as their Cytf electron acceptor, and in primitive oxygen evolving cyanobacteria this can be the only electron acceptor from Cytf. Where both genes are present, Cytc6 replaces PC under conditions of limiting copper 290 . Cytc6 also occurs in some algae where it is also upregulated to act as an electron acceptor of the Cytb6f under copper limiting growth conditions 291 . A Cytc6 homologue gene is present in plant genomes 292 . However, despite initial reports, the surface charge distribution on higher plant Cytc precludes its function as an effective in vivo electron acceptor from Cytf 293 .

The basic pattern of interaction is thought to be the same for electron transfer processes between Cytbc1: Cytc and Cytb6f:PC. The redox centers are oriented for optimal electron transfer by long distance charge effects in an encounter complex, before formation of a transiently stabilized productive complex, dominated by hydrophobic interactions. Mismatched surfaces between the partners, along with the retention of some water molecules at the interface, prevents interaction being so strong that it inhibits dissociation following the electron transfer event. The following sub-sections will describe the soluble electron carrier proteins in more detail, and outline the mechanisms by which soluble electron carriers interact with, and oxidize the Cytbc1 and Cytb6f complexes.

Redox properties and Interaction of Cytochromes c with Cytochrome bc1

Cytochrome c is a small globular heme protein (90-120 amino acids, 10-13 kDa) which in bioenergetic systems serves as a water-soluble electron carrier. This definition encompasses the representatives found in eukaryotic (mitochondrial Cytc, yeast iso-Cytc) and prokaryotic (Cytc2 present in several bacteria species) respiratory chains. A more distinct relative -Cytc6 -is also a member of this category. However, due to its occurrence in plant-related systems, Cytc6 is discussed in the chapter devoted to plastocyanin. For the scope of this review we will briefly go through the most important properties of Cytc with respect to its function and research in bioenergetic systems.

More detailed information about the c-type family of cytochromes can be found in excellent reviews on cytochromes 294,295 . In the Cytc molecule the heme is bound via two thioether linkages to the peptide. The covalent attachment of heme is a distinctive property of all c-type family cytochromes 296 . The bond is formed between a cysteine residue (C) and the penultimate carbon of a vinyl sidegroup of the heme moiety. Comparison analyses revealed a specific heme binding motif -CXXCH (present also in Cytc1 and Cytf, see sections3. 5.2 and 3.6.2, respectively). The cysteine residues (C) involved in thioether bonds are separated by any two residues (XX). This four amino acid subsequence is immediately followed by a histidine residue, which is an axial ligand to the heme iron.

The second axial ligand is the sulphur atom of a methionine residue which together with four pyrrole nitrogen atoms of porphyrin completes the octahedral ligand geometry of the heme iron.

Redox properties of Cytc are related to the ability of heme iron to undergo reversible oxidation and reduction of ferrous (Fe 2+ ) and ferric (Fe 3+ ) states, respectively. The presence of a methionine ligand significantly elevates the redox potential of Cytc compared to bis-His coordination 297 . Thus among biological compounds the redox midpoint potential of Cytc, and its counterparts, is considered high -in the range of +260 mV for horse heart Cytc 298 and bacterial Cytc2 +360 mV 299 . This is consistent with a location of Cytc in electron transport chains as an acceptor of electrons from quinol oxidising membrane complexes (i.e. Cytc -Cytbc1; Cytc2 -Cytbc1; Cytc6 -Cytb6f) [START_REF] Crofts | The Cytochrome Bc1 Complex: Function in the Context of Structure[END_REF][START_REF] Sarewicz | Electronic Connection between the Quinone and Cytochrome c Redox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling[END_REF]211 .

Upon reduction Cytc donates electrons to a terminal oxidase of the mitochondrial electron transport chain -CcO. In related systems cytochrome c-type electron carriers are oxidised by photosynthetic complexes of high oxidising power i.e. the bacterial photosynthetic reaction centre in case of Cytc2 300 or PSI for Cytc6 in the case of cyanobacteria and some algae 301 . Beyond its primary role as a carrier shuttling electrons between membrane complexes, mitochondrial Cytc, is also involved in the process of cellular apoptosis in higher organisms 302 .

The amino acid sequence of Cytc varies between different organism. The degree of identity spans quite high values (70%) among vertebrates 303 to low values (20%) when mitochondrial and prokaryotic Cytc are compared 304 . Nevertheless general structural features are evolutionarily conserved -the number of helices (5) and their general spatial arrangement 305 . The heme is buried in a hydrophobic pocket within the apoprotein with only one edge being exposed to the surface of the protein. Such an asymmetric cover insulates most of the heme moiety from the water environment and only exposes the heme to the area where the binding occurs with physiological partners. Cytc, as for other hemeproteins, absorbs strongly in the visible spectrum of electromagnetic radiation -in the range of 500-600 nm and the Soret band (around 400 nm). Reduced Cytc (Fe 2+ ) shows a sharp peak at 550-556 nm (α band) and a slightly broader peak around 520 nm (β band). A unique feature of ferriheme c (Fe 3+ ) is the presence of a weak absorbance band around 700 nm due to interaction with the sulfur of the methionine ligand. The peak in the Soret band (γ band) of Cytc shifts slightly when the redox state of heme c changes. The Soret peak is often used as a mark of the protein condition because when the protein denatures, significant changes in the γ band peak are observed. This was exploited in studies of Cytc folding stages and its stability 306,307 . The heme c also possesses peaks in the UV region around 320 nm and 280 nm (δ and ε band, respectively) 308 , which are, however of little use due to overlapping signals from aromatic amino acids residues.

Octahedral heme iron in Cytc is in a low-spin configuration, hence, the reduced Cytc (Fe 2+ ) is diamagnetic (S = 0) while oxidized (Fe 3+ ) is paramagnetic (S = 1/2). Oxidized Cytc molecules from different organisms are the source of the EPR spectrum with significant g-factor anisotropy that usually exhibit rhombic symmetry with well resolved g-tensor principal values 309 . In some cases the EPR spectrum of Cytc is a HALS type 310 . These differences originate from structural changes in the mutual orientation of the His and Met ligands. However, no simple rules have been found so far which allow prediction of the type of EPR spectrum 148 . Such peculiarities are assigned to rather weak interaction with Met that under some circumstances can be disrupted. It was shown that pH changes, denaturation stress, or interaction with lipids (cardiolipin) can even lead to the breakage of the coordination bond changing heme c from hexa-to pentacoordinated high-spin geometry [311][312][313] .

Several studies involving measurement of the Cytc reduction rate by Cytbc1 and kinetics of electron transfer showed sensitivity to ionic strength [314][315][316][317][318][319] . This led to the notion that electrostatics is a key player in a "find-and-bind" game between the two protein contenders.

Chemical modification and site directed mutagenetic studies allowed identification of the crucial amino acid residues responsible for Cytc -Cytbc1 interaction. Indeed, it was established that the interaction is possible due to electrically charged surfaces. The surface of the Cytc molecule around the heme pocket has a net positive charge due to the presence of basic amino acid residues (mostly lysine residues), while the binding domain on the Cytc1 surface includes complementary (negative) charges of acidic residues [320][321][322][323][324][325] . These long range interactions are thought to orient the Cytc molecule in an encounter complex which enables maintenance of an electron-transferproductive orientation at the early stages of interaction. Additionally the Cytc molecule itself possesses negatively charged residues on the side opposite to the interaction domain. This spatial separation of charges makes the Cytc molecule an electric dipole. It was shown that the presence of a dipole moment further facilitates the orientation of Cytc in the Cytc -Cytbc1 complex that is proper for physiologically efficient electron transfer 326 .

Following initial orientation, the proteins form a productive complex, knowledge of which is based on the structure of co-crystalized yeast proteins, either both in the oxidised, or both in the reduced state [START_REF] Solmaz | Structure of Complex III with Bound Cytochrome C in Reduced State and Definition of a Minimal Core Interface for Electron Transfer[END_REF]327 . Only subtle differences in structure are observed between the different redox states. In these complexes, the oppositely charged side-chain pairs on Cytc1 and Cytc do not directly interact, being 4 to 9.6 Å apart and therefore beyond the Debye length in physiological salt concentrations. Rather, the interface is dominated by non-polar interactions. The core surface area of interaction is 880 Å 2 around the heme clefts, and contains a core of 4 pairs of amino acids in nonpolar interactions, including a cation-π interaction between a Cytc1 Phe residue and a Cytc Arg residue. The interface contains 30 water molecules, only 2 of which coordinate H bonds between the proteins. This highly solvated surface aids rapid dissociation of the complex. The two heme cofactors have an inter-planar angle of 55° and are very close -only 4.1 Å between the closest thioester bonded carbons of the tetrapyrrole rings, and 17.4 Å between the Fe centers. This short distance means that, in principal, electron transfer could already occur when Cytc is approaching the Cytbc1. It is possible that many other, less stable, productive complexes are also possible aside from those in the crystallized structure. A molecular dynamics approach has attempted to rationalize the apparent importance of charged side chains in mutational studies with the dominance of non-polar interactions in the crystal structures 328 . This analysis indicates that many of the lysine side chains on Cytc, which surround the hydrophobic interface, are highly dynamic, and capable of forming H-bonds or even salt bridges with Cytc1. Based on this, the authors suggest a more dynamic mixture of interaction modes between Cytc and Cytc1.

No significant structural changes seem to occur between non-complexed and complexed proteins in the crystal structures, although one loop region that is poorly resolved in the crystal structure does change orientation in the molecular dynamics experiments 328 . This in combination with the small surface area of interaction and short distance between the cofactors probably contributes to the rapid rate of electron transfer by the complex. Interestingly, only one Cytc1 site in the dimer is occupied by Cytc in the crystal structures (Fig. 14 A), and this coincides with an increase in Qi site occupancy in the same monomer, indicating binding of the substrates could be coordinated. Indeed, a change in the conformation of ISP-HD head group is observed on Cytc binding to the Cytbc1. The molecular dynamics studies on Cytc1 -Cytc interaction show that Cytc binding to one monomer induces structural changes at the Cytc binding site of Cytc1 in the other monomer 328 , perhaps explaining this. Co-crystalisation of yeast Cytc1 with a second yeast isoform of Cytc (Cytc isoform 2), showed a nearly identical structural arrangement, although some of the charged pairs vary slightly [START_REF] Solmaz | Structure of Complex III with Bound Cytochrome C in Reduced State and Definition of a Minimal Core Interface for Electron Transfer[END_REF] .

Interestingly, an inspection of the electrostatic surface potential of crystal structures with different ISP-HD positions indicates that a negatively-charged area accessible to Cytc is significantly larger when ISP-HD is in the b-position compared to the case when it is in the c-position (Fig. 14 B). This could potentially influence the molecular organization of Cytc near the binding domain.

The idea of one Cytc per dimer stoichiometry attracted some attention and brought some productive scientific effort. On one hand this idea suggested that binding of Cytc to the one monomer disables the binding site in the other monomer as explored in context of half-of-the-site activity model of Cytbc1 regulation. This proposed that during enzyme turnover only one monomer of Cytbc1 is active at a time 329 . On the other hand it provoked other authors to investigate the stoichiometry issue by other experimental approaches. Titration data obtained by plasmon resonance spectroscopy suggested that Cytc2 interacts with the Cytbc1 dimer in a biphasic manner which was interpreted as a modulating effect of ISP-HD on the binding 330 . The data obtained by NMR and ITC for Cytc interaction with the soluble Cytc1 head domain in low ionic strength (both in plant and bovine system) proteins revealed two binding sites of different affinity on Cytc1 331 . The authors proposed that Cytc1 exposes a proximal site at which electron transfer takes place, and the distal site, which is too far for electron transfer, but maintains another molecule prior to electron transfer. In this way a subpool of Cytc is trapped close to the surface of the Cytbc1 -CcO supercomplex. This restricts diffusion of Cytc in the vicinity of the Cytbc1 -CcO complex leading to the enhancement of electron flow rate between the two complexes. The Cytc then does not need to return to the intermembrane bulk phase.

A somewhat similar finding was revealed by experiments in which titration of Cytbc1 binding sites was done by means of spin-labelling and pulse EPR 332 . Titration curves obtained at low ionic strength (no NaCl in buffer) were explained by a multiple-site-binding model. When electrostatic interactions are strong, Cytc1 attracts many Cytc molecules which organize in a molecular cloud near the Cytc1 binding surface (Fig. 14 C).

The putative presence and a role of such supramolecular structures in the Cytc pool are intriguing. However as the authors state in both cases (NMR, ITC) and (pulse EPR) the titration curves at higher ionic strength (25-50 mM NaCl) could be readily explained with one binding site per Cytc1 332,333 . This means that binding of Cytc to the proposed additional sites under physiological conditions is much weaker than binding to the primary (electron transfer relevant) site on Cytc1.

Hence in order for these additional collective interactions in the Cytc subpool to be physiologically relevant, other factors including macromolecular crowding, may be involved. This matter remains to be determined.

All available titration data report dissociation constants in the range of several to tens of μM, indicating that Cytc -Cytbc1 affinity is not very strong [330][331][332] . What is even more striking is that EPR data indicates that when the ionic strength is increased to the physiological levels (100-150 mM NaCl) binding of Cytc to the primary interaction site is not even detectable 334 . Even though the stationary concentration of stable long-lived Cytc -Cytbc1 complex is low, the proteins do interact because the electron transfer between them is not stopped 314 . Therefore this interaction is transient and the life-time of the complex is much shorter than the timescale of electron transfer. This would also explain the discrepancy between the measured rate of Cytc -Cytc1 electron transfer that turned out to be several orders of magnitude lower 314 than the calculated electron transfer rate based on crystallographic data 327 . This supports a diffusion-coupled (in contrast to diffusion limited) mechanism of electron transfer between Cytc and Cytbc1, in which the proteins constantly collide and several collisions are needed before one ET event occurs (Fig. 14 C) 332,334 . Therefore, the dynamic aspect of the Cytc -Cytbc1 interaction has tremendous impact on the flow of electrons from Cytbc1

to Cytc pool and it cannot be neglected.

Redox properties and Interaction of Plastocyanin with Cytochrome b6f

A structure of co-crystalized Cytf and PC is lacking, and so our understanding of their interaction is based on a combination of mutagenesis studies, nuclear magnetic resonance (NMR) and molecular dynamics. These have recently been augmented with atomic force microscopy (AFM) and 2D infra-red spectroscopy (2D-IR) experiments, meaning that although we lack a perfect snap-shot of any one interaction mode, we do have an excellent understanding of the dynamic events that occur during oxidation of Cytb6f.

While the C-teminal domain of Cytf forms a membrane spanning domain, the soluble, Nterminal portion contains the heme group. The heme lies below the surface for interaction with PC or Cytc6 171 . This is composed of a hydrophobic surface with a ridge of associated charged residues. The hydrophobic surface is highly conserved between Cytf proteins from different species, while there is great variation in the ridge residues, and even their charge, between different groups of photosynthetic organism [335][336][337] . In higher plants and algae, the ridge is functionalized by basic residues, and mutation of these disrupts interaction with PC 171,[338][339][340] . The charged residues on the Cytf ridge in cyanobacteria are by contrast usually acidic 341,342 .

The secondary structure of PC is fundamentally different from Cytc. It is an 11 kDa protein composed of eight β strands and a small α-helix [343][344][345][346] . It contains a single copper center, coordinated by one cysteine, one methionine and two histidine residues. There are two areas on the surface of PC involved in association with Cytf 171,342,[347][348][349][350] . The first (site 1) is a hydrophobic patch over the copper-His ligand, while the second (site 2) is functionalized by charged residues complementary to those on the Cytf. These therefore vary between photosynthetic organisms, being predominantly acidic in plants and algae, but basic in cyanobacteria. Long distance attraction between the charged residues on the surface of Cytf and PC are proposed to orient the molecules for optimal electron transfer, prior to formation of the catalytic complex. This is supported by the observation that low ionic strength results in a strongly bound complex incapable of turnover, while high ionic strength disrupts complex formation 351 . In addition, AFM experiments, with the Cytb6f complex tethered to the surface and PC attached to the tip, indicate that ionic strength strongly impacts both frequency of initial interactions and unbinding forces 352 . Despite this evidence supporting the importance of electrostatics for Cytf: PC interaction in vitro, the only in vivo analysis to date, on the alga Chlamydomonas reinhardtii, indicates that mutation of the basic ridge residues on Cytf has little impact on its oxidation rate 339,340 .

While a recent molecular dynamics study supported a role for the charged amino acid side chains at site 2 in the initial complex formed between higher plant Cytf and PC 353 , it also found no evidence for electrostatic pre-orientation in the cyanobacterium Nostoc. Moreover, there is little impact of electrostatics on formation of the Cytf:PC complex from the thermophilic cyanobacterium Phormidium laminosum, where non-polar interactions appear to both orient the complex and stabilise it 342 . Recent 2D-IR experiments on cyanobacterial (Nostoc) PC:Cytf interaction used site specific labelling of PC with cyanophenylalanine probes to measure very fast changes in conformation and orientation 354 . This indicated that a wide variety of initial encounter complexes are formed, with rapid (1-2 ps) changes in orientation. It should be noted that this experiment was performed with the proteins in redox states opposite to that expected in electron flow (oxidized Cytf and reduced PC).

Following correct orientation of the electron transfer partners, interaction occurs mainly through non-polar interactions, with exclusion of some water from the interface. Chemical shift perturbation experiments indicate that in plants and cyanobacteria, the surface area of interaction is around 600-850 Å 2 , with the Cu-His ligand close to the Fe coordinating Tyr reside in Cytf 349,350,355,356 .

Different plants appear to have the PC rotated slightly relative to Cytf, although the distance between the two redox centers remains around 11-14 Å. Although the surface charge distributions are reversed in cyanobacterial complexes, the orientation of PC relative to Cytf is conserved between higher plants and the cyanobacterium Nostoc 347 . However, NMR studies 347 and modelling approaches 353,357 indicate that other cyanobacteria show different patterns of interaction: in Phormidium laminosum, where Cytf lacks the charged ridge, computer modeling indicates that PC is highly mobile, with only the site 1 hydrophobic patch interacting. This also seems to be the case for Prochlorothrix hollandica, where NMR studies show that although electrostatics make a modest contribution, site 1 dominates the interaction, although PC is oriented slightly differently 358 . On binding to Cytf, X-ray absorption spectroscopy indicates that the geometry of the co-ordination sphere around the Cu ligand changes dramatically 359 . This may be responsible for the -30 mV negative shift in PC redox potential of PC on Cytf binding 360 , which counterintuitively makes electron transfer to PC less energetically favourable. Nevertheless, electron transfer is rapid, on the order of 104 ket s -1 361,362 . It appears that the relative charge of the interaction partners also plays a large role in interaction, presumably in terms of the electrostatics of initial interaction. AFM experiments show a 5 fold increase in interaction frequency between the two proteins when redox states are opposite (irrespective of whether Cytf or PC is reduced or oxidized) 352 . This suggests that the rate of PC dissociation might be enhanced by rapid re-reduction of Cytf.

Cytb6f also uses the Cytc homologue, Cytc6 as an electron acceptor in cyanobacteria and algae grown under copper deficiency condition 290 . It is reported to show a faster rate of electron transport between Cytf and terminal oxygenases than does PC 363 . Intriguingly, Cytc6 is also the principle electron acceptor in the heterocysts of the cyanobacterium Anabaena, even when Cu is replete 363 . These cells are specialised for N-fixation, a process dependent on an O2 sensitive enzyme (nitrogenase), and so PSII activity is very low, and the environment anaerobic 364 . In heterocysts, the majority of electron transport is therefore thought to result in oxygen reduction or in cycling between PSI and Cytb6f via PQH2 and Cytc6. Anabaena could no longer grow under diazotrophic conditions (obligatory N-fixing), when the gene for Cytc6 was knocked out 363 .

Cytc6 is a 10 kDa single c-heme type cytochrome bound by a typical CXXCH motif, where the His and a Met act as axial coordinators 365 . The protein has a midpoint potential of +335 mV 366 , close to that of PC. In cyanobacteria, Cytc6 has a hydrophobic surface area around the heme, and a positively charged surface patch of amino acid side chains analogous to sites 1 and 2 in PC 367 . Although modelling of the Chlamydomonas Cytf:Cytc6 complex indicates that a charged patch on the Cytc6 is important, many orientations are possible for the encounter complex 368 . A combination of NMR and molecular dynamics 367,369 has shown that the heme edge region (where the cofactor breaks the surface of the protein) is critical for interaction with Cytf, and long distance charge interactions approximately orient the two proteins in an encounter complex, as for PC. Many orientations of Cytc6 result in cofactor-cofactor distances that are close enough for electron transfer, and thus no specific interaction complex is expected 369 .

Our knowledge of the interactions between Cytbc1 and Cytb6f carriers and their p side electron carrier is not equivalent -in the case of the respiratory complex, co-crystalization has given a wealth of molecular detail about one particular productive complex. The absence of such a structure for the photosynthetic complex has lead to the innovative use of NMR and molecular dynamics, along with other recent techniques to provide abundant information on dynamic events. It is to be hoped that we will reach an equivalence of knowledge between these two fascinating systems soon, so that more detailed comparisons can be made.

4.3

FNR/Fd interactions with cytochrome b6f

Potential n side donors

In the initial model of the Q cycle proposed by Mitchell, one of the electrons reducing the heme bH site (heme bn) in the Cytbc1 complex came from a soluble n side electron donor [START_REF] Mitchell | Protonmotive Redox Mechanism of the Cytochrome B-C1 Complex in the Respiratory Chain: Protonmotive Ubiquinone Cycle[END_REF] . He revised this idea not long after proposing the classical model of the Q cycle [START_REF] Mitchell | The Protonmotive Q Cycle: A General Formulation[END_REF] , and more recent studies have given us a much firmer grasp on the classical Q cycle mechanism (see 216,259 for detailed reviews).

However, solution of the crystal structure of Cytb6f complexes [START_REF] Stroebel | An Atypical Haem in the Cytochrome B6f Complex[END_REF][START_REF] Kurisu | Structure of the Cytochrome b6f Complex of Oxygenic Photosynthesis: Tuning the Cavity[END_REF] prompted reassessment of whether an n side soluble electron donor might contribute to a modified Q cycle. The discovery of an additional heme group, named heme ci, or heme cn, 6 Å closer than heme bn to the n side solute interface and in contact with the Qn site has re-ignited the debate as to whether the Cytb6f might indeed receive electrons from a soluble, n side source. By contrast, the Cytbc1 lacks heme cn, and it is reasonable to assume that such a mechanism is precluded. This may be a reflection of the different bioenergetic environments of the two complexes, where the Cytbc1 can rely on consistent availability of quinols, while the Cytb6f is maintaining a proton gradient in the face of variable quinone reduction by PSII, due to fluctuations in light intensity.

So, where could such n side soluble reductant come from? In photosynthetic systems, the final steps in electron transport involve the donation of electrons from photoexcited PSI to the soluble, single electron carrier protein ferredoxin (Fd), and subsequent oxidation of Fd by the Fd:NADP(H) reductase (FNR) 370 . In turn FNR transfers these electrons over a flavin moiety (flavin adenine dinucleotide, FAD) to the 2 electron carrier NADP + , making NADPH (see 371,372 for a detailed description of the reaction mechanism). Both Fd and NADPH are used as electron donors by multiple enzymes, supplying the cytosol (bacteria) or stroma (chloroplasts) with reducing equivalents.

Crucially, the FAD in FNR enables the relatively safe storage of a single electron from Fd, before a second reduced Fd can donate the second electron necessary for NADP + reduction. The injection of these electrons back into the quinone pool from soluble, n side carriers in CET therefore depends upon events involving Fd and FNR.

The two dominant mechanisms of CET have been historically separated on the basis of their sensitivity to the inhibitor antimycin A. Curiously, despite the sensitivity of the Cytbc1 to this inhibitor, the Cytb6f was until recently thought to be insensitive 373 , and linear electron transport to NADPH was not found to be impeded by antimycin A. For many years it proved extremely difficult to identify the proteins involved in quinone reduction by the antimycin A sensitive CET pathway.

Three independent mechanisms have been proposed: 1) direct electron donation from Fd to the Cytb6f [START_REF] Mulkidjanian | Activated Q-Cycle as a Common Mechanism for Cytochrome Bc1 and Cytochrome b6f Complexes[END_REF]374,375 ; 2) electron donation from FNR to the Cytb6f 180,376 ; 3) electron donation from Fd via Cytb6f associated Pgr5 and PgrL1 pathways 199 . It remains a possibility that all three of these pathways occur, depending on the bioenergetic situation. Although robustly debated 234,[377][378][379][380] , it has been the consensus among many researchers that a pathway involving Pgr5/PgrL1 catalyses the bulk of CET in most algae and higher plants 197,[381][382][383][384][385] . However, results in Arabidopsis 386 and the green algae

Chlammydomonas reinhardtii 387 lead the authors to propose that the role of these proteins is regulatory, rather than mechanistic 374,377,386,387 . Moreover, the PgrL1 protein appears to be significantly sub-stoichiometric to the Cytb6f, and would therefore only be able to drive electron flux into a small proportion of Cytb6f complexes present in the membrane 201 . Finally, it has recently been reported that Antimycin A can indeed act as an inhibitor of the Qi site on Cyt b6f in conditions of highly reduced PQ, dependent on the presence of the STT7 kinase and pgr5. 388 Given the controversy, it is timely to discuss how the relationship between these different candidates and the Cytb6f might help to resolve this question.

Direct electron donation from Fd to cytochrome b6f

As mentioned previously, the original Q-cycle proposed by Mitchell involved a single electron from the soluble n side [START_REF] Mitchell | The Protonmotive Q Cycle: A General Formulation[END_REF] , and if this were to be the case for the Cytb6f, Fd would be an obvious candidate for such a donor. The advantages of such a system in amplifying the proton motive force (pmf) are evident, with additional electrons from the n side enabling immediate quinone reduction at the Qn site on oxidation of a single quinone at the Qp site, and also providing the means to close "incomplete" catalytic cycles in the Cytb6f that would otherwise leave Cytb6 in a reduced state [START_REF] Mulkidjanian | Activated Q-Cycle as a Common Mechanism for Cytochrome Bc1 and Cytochrome b6f Complexes[END_REF] . It is possible that this could be automatically poised by the redox state of the quinone and stromal electron carriers, but seems more likely that some regulation would be required, particularly in the dark, when reduced Fd is required for multiple other metabolic reactions 389 and continuous input of n side electrons to the Cytb6f undesirable.

Nearly all photosynthetic organisms possess a suite of Fd proteins that are presumed, and in some cases proven, to have variable affinity for different electron acceptors [390][391][392][393][394][395] . Such a system means that, depending on the relative abundance of these Fd iso-proteins, electron flux into different pathways could either be increased or decreased. There is abundant evidence that Fd iso-proteins specific for CET exist (see 396 for a full discussion). For example, maize plants perform an adapted photosynthetic pathway known as C4, in which one cell type has a greatly increased ATP demand and low PSII activity -a situation that demands high CET. A specific Fd iso-protein (FdII) is exclusively present in this ATP demanding cell type 397 and when this is used to replace the native Fd in cyanobacteria it prompts massive CET 398 . Pea plants also possess an Fd iso-protein that is expressed only in times of high ATP demand [399][400][401] and when this is over-expressed in tobacco it also results in elevated CET 402 . At this point it is not clear whether these CET specific Fds operate in the antimycin A sensitive or insensitive pathways.

After the characterisation of Fd-dependent CET by Arnon and colleagues 403 , the possibility of direct electron donation from Fd to the Cytb6f was investigated in isolated spinach thylakoids 404 .

The authors showed that although Cytb-563 (heme bn) is only slowly reduced by dithionite, addition of hydrophobic quinone mediators accelerated this reduction, while Fd had no effect. Moreover, the addition of NADPH and Fd in the dark had little effect on the redox state of the cytochrome. In these experiments, rapid re-reduction of the oxidized P700 center of PSI was measured in the presence of Fd and NADPH, indicating that Fd-CET was operating very efficiently. This result indicates that, in this system at least, Fd is a poor direct electron donor to the Cytb6f. It remains possible, that direct electron donation from Fd to heme cn is downregulated in the dark adapted membranes used in this experiment, or that it might not result in reduction of the heme bn. Interestingly, the broken chloroplast assay, used extensively to characterize the pgr5 and pgrL1 mutant Arabidopsis plants 235,236 does not work if attempted on washed thylakoid membranes (personal observation, GH). The possibility therefore also remains that components only weakly associated with the Cytb6f complex are required to regulate direct Fd: Cytb6f interaction, and the obvious candidates for such proteins are pgr5 and pgrL1, although documentation of the role of these proteins in competent electron transport reactions is minimal and their presence sub-stoichiometric. 201 If this is not the case then further experiments are necessary to identify 1) the interaction partners and 2) their mechanism of function.

As part of a possible model involving direct electron donation from Fd to the Cytb6f 374 , one can propose a docking model of Fd binding to the n side of the Cytb6f. Although binding sites are notoriously difficult to predict, interactions between Fd and Fd-dependent enzymes are characterized by charge interactions between the proteins, whose purpose is to optimally orient the redox centers for electron transfer [405][406][407] . For example, Figure 15 shows the Fd-binding sites (highlighted in black)

on two classical Fd-dependent enzymes (C, nitrite reductase; D, FNR). There is an obvious ring of basic charges around the active center to which Fd can dock. When the n-facing surface of the Cytb6f is examined, such a patch does appear to exist on the C. reinhardtii complex, used in the model proposed by Nawrocki et al. 374 , highlighted with a hashed circle (Figure 15 B). However, such a mechanism would be expected to be universal throughout CET performing organisms, and when the same surface of the cyanobacterial M. Laminosus complex is examined (Figure 15 A) the location of such a binding site is much less clear, although co-evolution of Fd iso-proteins with the Cytb6f complex and auxiliary subunits could result in differences in Fd-Cytb6f interactions.

Direct electron donation from FNR to the cytochrome b6f

Involvement of the FNR enzyme in re-injection of electrons into the quinone pool from the n side of the membrane has long been debated, and there is plenty of circumstantial evidence to suggest that this is the case. For example, CET appears inhibited in cyanobacteria when FNR is no longer tethered to the membrane 408 , FNR is more strongly membrane bound in higher plant cells performing enhanced CET 409 and plants with diminished FNR content also show perturbed CET 410 . Moreover, FNR has been consistently co-localised with PgrL1 Mutant studies on FNR would provide a definitive answer, but are lacking due to the difficulty in fully knocking out the gene for a protein that is essential for photo-autotrophic growth. Plants with minimal FNR downregulate the entire electron transport chain to compensate, leaving an extremely severe phenotype 420 , making interpretation of CET measurements problematic.

The discovery that FNR is co-purified with the Cytb6f complex 180,421,422 prompted the suggestion that FNR could act as a conduit of electrons from Fd into the Cytb6f, potentially via the heme cn 180,376 . As with Fd, the absence of information about where and how FNR docks to the Cytb6f is currently the major impediment to our understanding of a functional mechanism of electron transfer from FNR to the complex. It seems clear that the PgrL1 and Pgr5 proteins are involved in FNR recruitment to the membrane (and therefore probably the Cytb6f), at least in algae 411 , but in higher plants FNR is recruited to the membrane by two tether proteins, Tic62 423 and TROL 424 . Based on blue native PAGE analysis, it has been suggested that these are the only sites of FNR interaction 424,425 .

However, this is likely a function of detergent disruption of interactions between FNR in complex with its tether proteins, and other thylakoid membrane components such as the Cytb6f. Indeed, in a blue native PAGE experiment using different solubilisation conditions, FNR co-migrated with several complexes, including the Cytb6f 426 . It remains unknown whether the tethering proteins Tic62 and TROL also play a role in FNR association with the Cytb6f, or whether release of FNR from these tethers (which happens in response to the generation of ΔpH across the membrane) might prompt association with the Cytb6f. Interestingly, only FNR iso-proteins capable of binding to the TROL tether were found co-purified with the Cytb6f from maize 422 .

If FNR acts as a conduit of electrons from Fd into the heme cn, it prompts some energetic questions. The FAD moiety of FNR can be reduced by one electron from Fd to the flavin radical state (FNRox to FNRsq), and a second electron can then fully reduce the FAD (FNRsq to FNRred), with redox midpoint potentials of -338 mV and -312 mV respectively measured at pH 8 427 , or NADPH can directly donate two electrons to fully reduce the FAD (FNRox to FNRred), with a redox midpoint potential around -380 mV 428,429 . Any of these reduced centers would be adequate to reduce heme cn, irrespective of whether the quinone site was occupied or not 430 . So, is the mechanism of FNR to

Cytb6f reduction a single or double electron transfer to the Qn site from FNR? The dependence on Fd of NADPH mediated reduction of Cytb 180 , and the seeming independence of CET on the NADPH binding site of FNR 414,418 implies that single electron reduction and oxidation of the FNR FAD moiety is sufficient for CET. It has also been suggested that rather than having an electron transfer function, FNR could simply act as an Fd recruiting module -providing a site for Fd to bind on the Cytb6f complex before transfer of electrons directly to heme cn or via PgrL1 199 . A number of experiments are required to clarify the role of FNR recruitment to Cytb6f in CET, principally: 1) more information is needed regarding the binding site of FNR on Cytb6f, and its orientation; 2) the capacity for CET has to be correctly measured in membranes or cells where the FNR protein has no capacity to interact with the Cytb6f or is missing. Finally, if electron donation from Fd or FNR directly to Cytb6f is the major route of antimycin A sensitive CET, rates of electron transfer for this complex close to those estimated for the CET pathway -in the range of 10 e -1 s -1 PSI -1 to 130 e -1 s -1 PSI -1 431 -would be the final proof.

Inhibitors

Cytochrome bc1 specific inhibitors

Microorganisms often produce and secrete chemical substances in order to restrain or kill other species or strains. As Cytbc1 is a crucial component of the respiratory chain in many bioenergetic systems, its quinone binding catalytic sites have become a critical target in this chemical warfare. Indeed, many compounds that bind to the catalytic sites and act as Cytbc1 inhibitors can be found in bacteria and fungi. Over the past few decades, these naturally-occurring toxins became popular templates for man-made chemicals, with many proposed functions, from medicinal drugs and treatments for parasitic infections, to fungicides in agriculture.

Depending on the site of action, the Cytbc1 inhibitors are often divided into two groups. The first group is considered to be small, and consist of compounds with the ability to inhibit the Qi site.

The second group consists of the Qo site inhibitors and is considered to be much larger, with numerous synthetic compounds developed over the years of research. Moreover, with recent developments, dual-mode inhibitors, capable of binding to both active sites, have been found or synthetized. It is important to note that this classification also excludes the inhibitors that do not act on the quinonebinding sites, such as cyanide, which has been shown to inhibit bacterial cytochrome bc1 136 or 2,3dimercaptopropanol (also known as BAL or British anti-Lewisite) which is known as a respiratory inhibitor since the early studies on mitochondrial material 432 and later has been shown to destroy the Rieske cluster 433 .

Inhibitors of the Qi site. The best known compound that inhibits the Qi site is antimycin A.

However, this group consists also of naturally occurring ilicicolin H, funiculosin, funiculosin-related compounds, and various synthetic inhibitors (Fig. 16 and17).

Antimycin A (Fig. 16 A) is a depsipeptide with an acyl-(R1) and alkyl-substituted (R2) dilactone ring with an amide linkage to a 3-formamidosalicylate moiety. In fact, antimycin A is a whole family of compounds (about 29) 434 , produced by actinobacteria from the Streptomyces genus, with the first members isolated and identified in 1948. 435,436 Antimycin A1 has a high binding affinity to the Qi site, with a dissociation constant (Kd) of 32 pM in bovine Cytbc1. 437 It has been shown that antimycin is able to displace quinones and even other inhibitors from the active site. [438][439][440] Based on the crystal structures [START_REF] Esser | Inhibitor-Complexed Structures of the Cytochrome Bc1 from the Photosynthetic Bacterium Rhodobacter Sphaeroides[END_REF][START_REF] Gao | Structural Basis for the Quinone Reduction in the Bc1 Complex: A Comparative Analysis of Crystal Structures of Mitochondrial Cytochrome Bc1 with Bound Substrate and Inhibitors at the Qi Site[END_REF]123 it is known that the 3-formamidosalicylate group acts as the toxophore and forms hydrogen bonds with highly conserved residues of Qi (Fig. 16 B), including the His, Asp and Lys, that serve as proton donors in Q reduction 163 , and a conserved Ser. The dilactone ring of antimycin appears to control its solubility and hydrophobicity. 441 There have been attempts to create synthetic analogues (example in Fig. 16 C top) with the dilactone replaced by various other moieties [442][443][444] , with promising in vitro results for biphenyl and biphenyl ether groups. 444 Some of synthetic inhibitors where the 3-formamidosalicylate moiety is replaced with benzazoles and the dilactone rings are replaced with a trifluoromethyl-substituted biphenyl ether moiety (Fig. 16 C, middle and bottom), were shown to be almost as potent as the natural antimycin A in in vitro inhibitor assays. However, they exhibited a negligible in vivo activity because of low uptake into the living cell. 442 Recently UK-2A (Fig. 16 D), which belongs to a group of dilactone compounds isolated from Streptomyces sp. 517-02 445,446 , has been revisited and shown to bind to the Qi site in a similar mode to antimycin A. 447 In contrast to antimycin, UK-2A comprises a 4-methoxypyridin-3-ol moiety as its toxophore. Its inhibitory potency was estimated to be about 3-fold lower than that of antimycin. 448 Funiculosin (Fig. 17 A top) is an N-methyl-4-hydroxy-2-pyridone derivative, substituted with a cyclopentanetetrol moiety. It is a natural compound with antifungal properties, isolated from Penicillium funiculosum. 449,450 Funiculosin was shown to inhibit Cytbc1 by a similar mechanism to antimycin A. 164,451,452 However, there is no structural data on funiculosin binding mode to the Qi site.

It is speculated that it binds in close proximity to heme bH, as a rise in Em of this heme by about 100 mV was observed in the inhibited enzyme. 164 Moreover, studies on yeast mutants of Sc Asp208 revealed that this residue can possibly be involved in funiculosin binding, which distinguishes it from antimycin. 452 Recently, funiculosin analogues were shown to be produced by other microorganisms, for example the compound AS2077715 (Fig. 17 Another 2-pyridone deriviative, Ilicicolin H (Fig. 17 A, lower middle), was isolated from Cylindrocladium ilicicola 454 and Gliocladium roseum. 455 Despite some structural differences to funiculosins, ilicicolin was shown to act in similar way, being a potent and broad-spectrum antifungal agent 456 , inhibiting yeast and fungal Cytbc1 with high selectivity. 439,456 Thus, a 2-pyridone moiety is the suggested toxophore in both funiculosins and ilicicolins. 439 However, a closer examination of the influence of antimycin A, funiculosin and ilicicolin H on Cytbc1 isolated from distinct organisms and yeast Qi-site mutants, reveals some differences in sensitivity between the enzymes, which suggests that there is some variation in the binding mode of these inhibitors. 457 The same 2-pyridone moiety as in funiculosin and ilicicolin H can be found in sambutoxin (Fig. 17 A, bottom) and related compounds isolated from Fusarium sambucinum 458,459 and other strains of Fusarium. 460,461 Sambutoxins have been also shown to be toxic to mitochondrial respiration and specific towards Cytbc1. 459 Although their binding mode was not specified, they can be expected to bind to the Qi site as well. More recently, several synthetic antimalarial compounds comprising a 4-pyridone ring with a biphenyl ether tail were also shown to inhibit the Qi site, eg. GSK932121, GW844520 (Fig. 17 Diuron (Fig. 17 C), 3-(3,4-dichlorophenyl)-1,1-dimethylurea, is a relatively simple synthetic compound which was shown to have strong inhibitory properties in photosynthesis and weak in mitochondrial respiration. 464 No structural data is available for its binding mode. The mechanism of action, through inhibition of the Qi site, is known from genetic analysis of diurone-and antimycinresistant mutations in the yeast model 465,466 and comparative kinetic studies. 467

Cyazofamid (Fig. 17 high selectivity towards oomycetes. [468][469][470] Cyazofamid was shown to be almost inactive towards yeast, rat liver and potato tuber Cytbc1 470 and a weak inhibitor of porcine Cytbc1 Qi site when compared to antimycin A. 437 Little is known about the binding mode of these inhibitors to the Qi site.

However, based on computational docking studies, it has been suggested that cyazofamid forms a hydrogen bond to Bt Asp228. 437 Inhibitors of the Qo site. They are commonly divided into two subclasses. The compounds belonging to the first subclass, of which stigmatellin is the best known example, are capable of immobilizing the ISP-HD in a position near the Qo site (in the b-position). The inhibitors belonging to the second subclass, such as myxothiazol or MOA-type compounds, bind deeper in the Qo site cavity and do not fix the ISP-HD position. Therefore, these two subclasses are often refered to as Pf (with f originating from "fixed") and Pm (with m originating from "mobile") inhibitors, respectively.

The influence of these inhibitors on movement of ISP-HD is explained in detail in section 3.4.

Stigmatellin A (Fig. 18 A, upper) is a natural compound isolated from the myxobacterium Stigmatella aurantiaca 471 and the inhibitor of Qo site exhibiting the lowest Kd (<0.01 nM). 472 It consists of a chromone moiety, acting as its toxophore, substituted with a lipophylic alkenyl chain.

Crystallographic studies have revealed that stigmatellin fixes the position of the ISP-HD near the Qo site, by formation of hydrogen bond between carbonyl oxygen of chromone ring and one of the His residues acting as a ligand to the 2Fe2S (Fig. 18 B). [START_REF] Zhang | Electron Transfer by Domain Movement in Cytochrome Bc1[END_REF][START_REF] Esser | Inhibitor-Complexed Structures of the Cytochrome Bc1 from the Photosynthetic Bacterium Rhodobacter Sphaeroides[END_REF]123,473 The second hydrogen bond is formed between the phenolic hydroxyl group of the compound and the carboxylate side chain of a conserved Glu residue (belonging to the PEWY motif) of the Cytb. [START_REF] Esser | Inhibitor-Complexed Structures of the Cytochrome Bc1 from the Photosynthetic Bacterium Rhodobacter Sphaeroides[END_REF]474 The alkenyl tail of stigmatellin is not involved in any specific interactions, but studies on synthetic analogs indicate that modification of this tail can decrease the inhibitory potency of the compound. 472 However, such a modification was also made in the case of tridecyl-stigmatellin (Fig. 18 A, lower), which has the native tail replaced with tridecyl chain but retains its potency. It was developed as a stigmatellin replacement and a potential inhibitor of Cytb6f. 475 Tridecyl-stigmatellin has been shown to bind to the Cytb6f at both active sites [START_REF] Hasan | Quinone-Dependent Proton Transfer Pathways in the Photosynthetic Cytochrome B6f Complex[END_REF] and also to inhibit the Qo site of the bacterial Cytbc1 151,263,472 in similar way to the natural compound. Atovaquone (Fig. 18 C left) is 2-hydroxynaphthoquinone substituted with a cyclohexylchlorophenyl moiety, which was developed as a drug for malaria in 1990s. 476 Among numerous other naphthoquinone derivatives created at the time, it was shown to be the most active against apicomplexan parasites, including several species of Plasmodium. 441,[476][477][478] It is the only drug against Cytbc1 currently in clinical use. 441 Atovaquone inhibits the enzyme of P. falciparum with nanomolar affinity, 476,479,480 and has much lower toxicity to the human counterpart. 480 The results of early studies, based on EPR and redox potentiometry, indicated that atovaquone has a similar binding mode to stigmatellin, and also arrests ISP-HD at b-position. 479 Moreover, based on the complementary molecular modeling studies, atovaquone was suggested to bind within the Qo site, forming a hydrogen bond between its hydroxyl group and the 2Fe2S cluster-ligating His residue, and a water bridge between the carbonyl oxygen at position 1 of naphthoquinone ring and the conserved Glu of PEWY motif. 479 However, crystallographic studies on yeast showed that the only hydrogen bond is formed between the ionized hydroxyl group of the inhibitor and the Nε atom of Sc His181. The Sc Glu272 residue side chain was shown to face the opposite direction with respect to the carbonyl oxygen, and does not appear to be involved in atovaquone binding. 71 DBMIB (Fig. 18 C, right), 2,5-dibromothymoquinone, is a halogenated benzoquinone derivative. Although DBMIB is known more as a Cytb6f inhibitor 110 (see section 5.2), it has been shown to exhibit similar potency against Cytbc1. 481 DBMIB should not be considered as a regular inhibitor competing with QH2, as has been commonly assumed. Rather, it binds to the Qo site in the form of a semiquinone and there forms a spin-coupled state with 2Fe2S, which leads to locking of the ISP-HD at b-position. 482 (see section 7.6). The spin-coupled state can be detected in EPR experiments as the prominent g = 1.94 EPR signal in X-band at low temperatures. [START_REF] Schoepp | The Qo-Site Inhibitor DBMIB Favours the Proximal Position of the Chloroplast Rieske Protein and Induces a pK-Shift of the Redox-Linked Proton[END_REF]108,110,483 UHDBT and HHDBT (Fig. 19 A) are both 6-hydroxy-4,7-benzothiazolediones (HDBT) with undecyl and heptyl tails, respectively. HDBT compounds are synthetic inhibitors capable of binding to the Qo site. [START_REF] Palsdottir | Structure of the Yeast Cytochrome Bc1 Complex with a Hydroxyquinone Anion Qo Site Inhibitor Bound[END_REF]116,484,485 As they comprise the hydroxybenzoquinone moiety, they exist as a mixture of ortho-and para-quinone tautomers (Fig. 19 B). The 6-hydroxy group is ionizable, with a pKa of 6.5, which is reflected in a distinctive color change from yellow to violet when pH changes from acidic to alkaline. 486 These compounds are also redox active and, as is typical for quinones, the Em exhibits a pH dependence with a slope of about 60 mV/pH unit. 486 Thus, HDBTs may be regarded as substrate analogues. However, the Em value of UHDBT was shown to be about -40 mV at pH 7 486 , thus much lower than the ubiquinone pool (about 100 mV). 487,488 Cytochrome bc1 inhibition by UHDBT was found to be pH dependent, with a 10-fold decrease in the binding rate constant at more alkaline pH. 485 This had been initially associated with ionization of the hydroxyl moiety. However, this effect was also noted for 6-bromo substituted UHDBT. 484 As indicated by the results of X-ray crystallography, HDBT compounds immobilize the ISP-HD in the b-position, via formation of a hydrogen bond with one of His ligands of the 2Fe2S cluster. [START_REF] Palsdottir | Structure of the Yeast Cytochrome Bc1 Complex with a Hydroxyquinone Anion Qo Site Inhibitor Bound[END_REF][START_REF] Esser | Crystallographic Studies of Quinol Oxidation Site Inhibitors: A Modified Classification of Inhibitors for the Cytochrome Bc1 Complex[END_REF]474 However, there is no agreement on the detailed mode of hydrogen bonding for HHDBT and UHDBT within the Qo site. Based on the structure of yeast Cytbc1 co-crystalized with HHDBT, hydrogen bonds are proposed to form between:

1) the oxygen from the ionized hydroxyl group of HHDBT and the protonated Nε of Sc His181 of the ISP, 2) the carbonyl O4 atom and the water molecule that is itself bonded to the backbone nitrogen of the Glu within the PEWY motif. [START_REF] Palsdottir | Structure of the Yeast Cytochrome Bc1 Complex with a Hydroxyquinone Anion Qo Site Inhibitor Bound[END_REF] A slightly different model has been proposed in the case of bovine Cytbc1 interaction with UHDBT. It was suggested that the protonated His of ISP interacts with the carbonyl O7, while its deprotonated form interacts with the hydroxyl group of the inhibitor. Moreover, the carbonyl O4 and N3 of UHDBT were suggested to form a hydrogen bond with the hydroxyl group of the conserved Bt Tyr131 of the Cytb ( Sc Tyr132). [START_REF] Esser | Crystallographic Studies of Quinol Oxidation Site Inhibitors: A Modified Classification of Inhibitors for the Cytochrome Bc1 Complex[END_REF] . Nε of the 2Fe2S-ligating His. Moreover, it was suggested that the glycine residue of crocacin can form hydrogen bonds to the backbone nitrogen with the PEWY motif Glu and to the carbonyl group of the conserved Met ( Bt Met138) of Cytb. However, the existence of a hydrogen bond to the Met residue is not supported by the crystal structure. Interestingly, the Z-enamide seems to adopt a hairpin conformation, with an intramolecular hydrogen bond between the two amide groups. [START_REF] Crowley | The Role of Molecular Modeling in the Design of Analogues of the Fungicidal Natural Products Crocacins A and D[END_REF] Because natural crocacins have been shown to exhibit a photostability of minutes under field conditions, [START_REF] Crowley | The Role of Molecular Modeling in the Design of Analogues of the Fungicidal Natural Products Crocacins A and D[END_REF] they are not suitable for use in agriculture, despite their inhibitory potency. However, in recent years, several methods for synthesis of crocacins 491,492 and their simplified, more stable analogues have been developed. 493 Famoxadone, JG144 and fenamidone (Fig. 20 B) are synthetic fungicides. The toxophores of famoxadone and JG144 comprise 1,3-oxazolidine-2,4-dione, and that of fenamidone comprises imidazol-4-one [START_REF] Esser | Surface-Modulated Motion Switch: Capture and Release of Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF]117,494,495 . The famoxadone molecule is chiral, and the S-(-)-enantiomer has been shown to be active against Cytbc1. 496 The crystal structures of famoxadone [START_REF] Esser | Surface-Modulated Motion Switch: Capture and Release of Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF]117,495 and its analogs [START_REF] Esser | Surface-Modulated Motion Switch: Capture and Release of Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF]117,495 indicate that these inhibitors bind to the Qo site in the same fashion, with carbonyl oxygen at ring position 4 forming a hydrogen bond to the backbone nitrogen of the PEWY motif Glu.

This resembles the binding mode of MOA-like inhibitors, but in contrast to those, famoxadone-like inhibitors are capable of immobilizing the ISP-HD near the Qo site, close to b-position (Pf inhibitors subclass). 474 However, famoxadone and its analogs do not form a hydrogen bond with the His of the ISP as in the case of other ISP-fixing compounds. [START_REF] Esser | Surface-Modulated Motion Switch: Capture and Release of Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF]117 Rather, the inhibitor binding is thought to induce conformational changes, which result in formation of a hydrogen bond between the 2Fe2S His ligand and a highly conserved Tyr residue of the ef α-helix of Cytb, thus immobilizing the ISP-HD in the near b-position. 117

Myxothiazol A (Fig. 21 A, top) is a natural compound obtained from the predatory bacterium, Myxococcus fulvus. 497,498 It is the best known representative of the thiazole-based bacterial toxins that possess a β-methoxyacrylamide or β-methoxyacrylate (MOA) toxophores. 474,499,500 Myxothiazol is considered a classic Pm type Qo inhibitor -its binding does not involve the immobilisation of the IPS-HD in the b-position. Based on the structure of the bovine Cytbc1 co-crystallized with myxothiazol [START_REF] Esser | Crystallographic Studies of Quinol Oxidation Site Inhibitors: A Modified Classification of Inhibitors for the Cytochrome Bc1 Complex[END_REF] , it has been proposed that the inhibitor molecule is bound to the Qo pocket, mostly through interactions of the dithiazole moiety with aromatic residues in the binding site and by a characteristic hydrogen bond formed between the amide oxygen atom of myxothiazol and the backbone amide of the conserved Glu in the PEWY motif. Moreover, the amide nitrogen of myxothiazol can form a hydrogen bond with the conserved Tyr of the PEWY motif. It was also suggested that the C18 methyl group of myxothiazol can serve as an unusual hydrogen bond donor to the sulfur atom of Bt Met124.

It is worth mentioning, that myxothiazol-related compounds that demonstrate inhibitory properties towards the Qo site are more abundant. In the case of M. fulvus toxins, myxothiazol Z, which comprises a β-methoxyacrylate moiety, has also been shown to exhibit antifungal properties in vivo. 501 This group also includes the melithiazols (Fig. 21 A, middle) isolated from Melittangium lichenicola and several other strains 502 and cystothiazoles (Fig. 21 A, bottom) from Cystobacter fuscus 503 , which were shown to exhibit antifungal properties in vivo. 502,503 In the enzymatic assays with bovine SMP's, melithiazol A was shown to exert a similar spectroscopic effect on redox cofactors to that of myxothiazol. 502 Therefore, it can be expected that these inhibitors have a similar mode of binding to the Qo site.

Another group of myxobacterial compounds, cyrmenins (Fig. 21 B), were obtained from

Cystobacter armeniaca and Archangium gephyra. Cyrmenins are distinct from myxothiazol-related compunds, and comprise a MOA moiety substituted at the α-position. 504 Cyrmenin B1 has been shown to inhibit Cytbc1, but the binding mode was not investigated in details. 505 The presence of the MOA toxophore suggests that cyrmenin targets the Qo site as well. Strobilurins (Fig. 22) are currently recognized as a large group of synthetic Pm class Cytbc1 inhibitors with various structures and toxophore moieties. However, such generalization can be confusing, and in fact they were all designed using natural fungal poliketydes as a template. These compounds were isolated from several species of large, wood-decaying Basidiomycetes, initially named strobilurins, eg. strobilurin A (also known as mucidin 506 ), (Fig. 22 A left) from Strobiluris tenacellus 507 and oudemansin A (Fig. 22 A, right) from Oudemansiella mucida 508 . Later an Ascomycete, Bolinea lutea was also reported to produce strobilurins and their derivatives 509 . Natural strobilurins possess the β-methoxyacrylate (MOA) toxophore 499,510 (Fig. 22, 1 in red frames), which targets the heme bL proximal part of the Qo site 511,512 . In contrast to myxothiazol-like inhibitors, the β-methoxyacrylate moiety of strobilurins is substituted with a tail at the α-position, not the β-position.

Due to this difference, their distinct origin, and their separate paths of biosynthesis 513 , myxothiazol and other myxobacterial compounds should not be referred to as "strobilurins". The presence of MOA in both groups of compounds should be rather taken as a biochemical example of evolutionary convergence. Natural strobilurins exhibit a low photostability 510 . Therefore, various synthetic compounds (Fig. 22 B) with differently substituted MOA toxophores (Fig. 22, 1 in red frames) have been created, such as MOA-stilbene [START_REF] Brandt | Analysis of Inhibitor Binding to the Mitochondrial Cytochrome c Reductase by Fluorescence Quench Titration. Evidence for a "catalytic Switch" at the Qo Center[END_REF] , azoxystrobin [START_REF] Esser | Crystallographic Studies of Quinol Oxidation Site Inhibitors: A Modified Classification of Inhibitors for the Cytochrome Bc1 Complex[END_REF] or picoxystrobin 510,514 . Several deviations from classic MOA toxophore were developed (Fig. 22 B, 2-8 in red frames), such as oximino-acetate

(2) e.g. kresoxim-methyl 510,514 and trifloxystrobin 510,514 , oximino-acetamide (3) e.g.

dimoxystrobin 510 , methyl N-methoxycarbamate (4) e.g. pyraclostrobin 514,515 , triazolinone 516 (5) e.g. JZZ 117 , tetrazolinone ( 6) eg. metyltetraprole 517 , dihydro-dioxazine ( 7) eg. fluoxastrobin 510 and the methyl-carbamate ( 8) e.g. pyribencarb. 518 Such diversity of synthetic strobilurin analogues is mostly a result of "patent wars" between agrochemical companies, driven by a large demand from the global fungicidal market. 514 Despite apparent differences in structure, man-made strobilurins are expected to share the same binding mode to the Qo site as the classic MOA inhibitors. This has been partially confirmed by the results of X-ray crystallographic studies on Cytbc1 with bound MOAstilbene [START_REF] Esser | Crystallographic Studies of Quinol Oxidation Site Inhibitors: A Modified Classification of Inhibitors for the Cytochrome Bc1 Complex[END_REF][START_REF] Kim | Inhibitor Binding Changes Domain Mobility in the Iron-Sulfur Protein of the Mitochondrial Bc1 Complex from Bovine Heart[END_REF] , azoxystrobin [START_REF] Esser | Crystallographic Studies of Quinol Oxidation Site Inhibitors: A Modified Classification of Inhibitors for the Cytochrome Bc1 Complex[END_REF] , trifloxystrobin 474 , iodinated derivative of kresoxim-methyl 474 , and JZZ (PDB ID:3L73) 519 , showing that the toxophores of these inhibitors superimpose within the binding site, with formation of a hydrogen bond to the backbone amide of the conserved PEWY motif Glu residue. Among the synthetic strobilurin-like inhibitors mentioned here, pyribencarb has been suggested as a novel, "benzylcarbamate" type of Qo inhibitor. 518 Indeed, it has been shown to possess a higher selectivity than other MOA analogues. However, neither its methyl-carbamate toxophore nor binding mode are as unique as has been claimed because methyl-carbamate moiety is present in previously described natural crocacins, and also in macrolides isolated from sponges, such as neopeltolide (Fig. 23 A, upper) from unidentified Daedalopelta sp. 520,521 and leucascandrolide A (Fig. 23 A, lower) from Leucascandra caveolata. 522 Enzymatic assays confirmed that these compounds also exhibit inhibitory properties towards Cytbc1. 523 Moreover, the recent molecular modeling docking studies revealed that pyribencarb, crocacin and neopeltolide all bind to the Qo site through their methyl-carbamate moieties, in a way similar to the MOA moiety of azoxystrobin, forming a hydrogen bond to the backbone amide group of Bt Glu271. 524 Pyrimorph (Fig. 23 B) is a synthetic morpholine derivative, already in use as a fungicide against pathogens of plants. [525][526][527] It has been shown to exhibit inhibitory activity towards multiple targets, including cellulose synthase A 526 and Cytbc1. 527 Results of enzymatic assays indicate that pyrimorph acts as a non-competitive inhibitor of the Qo site. Moreover, complementary molecular modeling studies showed that pyrimorph docks in the central cavity between Cytbc1 monomers, at the entrance of the Qo site, and thus blocks access of the substrate. The binding mode of pyrimorph has no resemblance to any other Qo site inhibitor. It was found that it does not form hydrogen bonds within the Qo site, but instead it is held in place by hydrophobic and van der Waals interactions. 527

Thus, pyrimorph should be considered a unique inhibitor type, distinct from MOA-related compounds. Dual-mode inhibitors. Some inhibitors are capable of binding to both Qi and Qo catalytic sites, therefore they should be considered as a third, dual-mode class of Cytbc1 inhibitors. They are often quinone analogues or compounds semi-isosteric with quinones, and therefore able to mimic the natural substrate within both sites.

The 2-alkyl derivatives of 1-hydroxyquinoline-4-one, such as HQNO, NQNO or HDQ (Fig. 24 A) are usually considered Qi site inhibitors, with a mechanism of action similar to that of antimycin A. 111,438,528 Some early reports suggested that HQNO and NQNO bound to Cytbc1 can be displaced by both antimycin A and UHDBT. 529,530 This effect was explained as a possible result of allosteric interactions between the two binding sites. 530 However, more recent crystallographic analysis has shown that NQNO is indeed capable of binding to both Qi and Qo sites. It has been proposed that in solution, NQNO most likely exists as a mixture of two tautomers (Fig. 24 B), which is likely a determinant factor in this dual mode of binding. At the Qi site, the NQNO tautomer with its hydroxyl group protonated (Fig. 24 B, left) can form a hydrogen bond to Bt Ser205, while the form with N-oxide protonated (Fig. 24 B, right) can hydrogen bond with Bt Asp228. In the structural model obtained in these studies, the Bt His201 imidazole ring was not in the correct orientation for hydrogen bonding. At the Qo site, the N-oxide protonated form of NQNO can form two hydrogen bonds. The N-oxide oxygen forms a water-mediated hydrogen bond with Bt Glu271 of the PEWY motif, and the 4-carbonyl oxygen forms a hydrogen bond with the His ( Bt His161) ligand of 2Fe2S cluster, immobilizing the ISP-HD in b-position. [START_REF] Gao | Structural Basis for the Quinone Reduction in the Bc1 Complex: A Comparative Analysis of Crystal Structures of Mitochondrial Cytochrome Bc1 with Bound Substrate and Inhibitors at the Qi Site[END_REF] However, there is no data to confirm the dual-mode binding for other 1hydroxyquinoline-4-ones. Recent studies suggest that HQNO induces changes in the environment of the Qo site, but not due to binding in the site. 111

ELQ-400 (Fig. 24 D, upper) is a 4(1H)-quinolone, one among a large number of derivatives of the antimalarial drug, endochin (Fig. 24 D, lower) 441,531 . Most of ELQ's (endochin-like quinolones) are expected to inhibit the Qi site of apicomplexan Cytbc1. 441,531,532 Thus, it was proposed that they be used in combination with the Qo site-targeting atovaquone. 532 However, it has recently been suggested that the binding mode of ELQ-400 is more complex, with the inhibitor targeting both active sites 533 . This proposal was strongly supported by mutational analysis and molecular modeling studies. At the Qi site, ELQ-400 was predicted to bind in a similar manner to the substrate or pyridone inhibitors (mentioned earlier). At the Qo site, it was predicted to bind similarly to atovaquone or stigmatellin, with a hydrogen bond formed by the NH group of the quinolone to the His ligand of 2Fe2S cluster. 533 Ametoctradin (Fig. 24 C) is a triazolopyrimidine derivative, acting as a highly selective inhibitor against the Cytbc1 of oomycetes. 469,534,535 It has been shown to be active against mammalian (porcine) 468 , but inactive towards yeast 535 Cytbc1. Ametoctradin is commonly used in agriculture and available under trade name Initium®. 534 No cross-resistance of ametoctradin with MOA-like inhibitors has been observed, so it is speculated to bind in the Qo site in a manner similar to stigmatellin. 535 However, the molecular modeling docking studies have shown that although ametoctradin binds in roughly the same part of Qo pocket as stigmatellin, and forms a hydrogen bond to the carboxylate side chain of the conserved PEWY motif Glu residue, the distance to the 2Fe2Sligating His residue is too far to expect formation of a strong hydrogen bond. 468 In addition there is no experimental indication that ISP-HD is immobilized by ametoctradin in the b-position, as in the presence of stigmatellin. More recent studies suggest that ametoctradin is a dual-mode inhibitor, targeting both the Qi and Qo site. 536 This model seems to be supported by the observation that ametoctradin is inactive towards the wild-type strain of Saccharomyces cerevisiae and active towards a mutant with an altered Qi site. Based on this finding it was suggested that, like NQNO, ametoctradin can bind to both active sites.

Ascochlorin (Fig. 24 E) is an isoprenoid compound, which was independently isolated from four distinct fungal strains (Ascochyta viciae 537,538 , Cylindrocladium ilicicola 454 , Fusarium sp. LL-Z 1272 539 and Acremonium luzulae 540 ) and described by four different research groups at about the same time. Because of this remarkable coincidence, it can be found in the literature and databases under three different names: ascochlorin, LL-Z 1272γ and ilicicolin D 541 . In the recent comprehensive biochemical, spectroscopic and crystallographic studies, ascochlorin has been shown to bind to both Qi and Qo sites of bacterial, yeast, mammalian and avian Cytbc1 542 . At the Qi site the position of the aromatic ring of bound ascochlorin overlaps with the position of the bound ubiquinone aromatic ring.

Moreover, at the Qi site, it has been proposed that ascochlorin forms a halide bond between the Cl atom and the conserved His (the proton donor in Q reduction) and a hydrogen bond between its hydroxyl group in the para position to Cl and the conserved Asp (the second proton donor in Q reduction). At the Qo site, ascochlorin occupies a position similar to stigmatellin. A hydrogen bond, is formed, possibly from both the hydroxyl group and the C=O group to the His ligand of 2Fe2S cluster, immobilizing ISP-HD at the b-position. On the other side of the aromatic ring, the Cl atom is suggested to form a halide bond with Glu in the conserved PEWY motif. However, as it has been pointed out that a mixture of conformations may exist, this remains uncertain. The dual mode of action can be explained in terms of ascochlorin and related compounds being in general, ubiquinone analogs, as they can mimic natural substrates also in other ubiquionol oxidoreductases. 543 The inhibitors of Cytbc1 already comprise numerous groups of compounds, both natural and synthetic, with new ones being discovered or developed almost every year. 524,544 Many were omitted in this section, as little is known about their mechanism of action, and any attempt to deduce or model it would go beyond the scope of this review. Some recent discoveries in the field are rather unexpected, for example the recent findings with karrikinolide (Fig. 25, upper), a naturally occurring byproduct from the combustion of plant material, which earlier had been shown to stimulate plant seed germination. 545 Karrikinolide itself exhibits almost negligible inhibitory properties towards Cytbc1. However, some of its synthetic derivatives, such a 4-n-butylphenyl-substituted variant (Fig.

Cytochrome b6f-specific inhibitors

Several inhibitors have been employed to characterize the functional and structural features of the Cytb6f. These compounds act on both the Qp and the Qn sites similarly to the situation described above for the Cytbc1. Stigmatellin (Fig. 18A ) is a potent antibiotic obtained from the myxobacterium Stigmatella aurantiaca, which inhibits both the CytbcI and Cytb6f. 552 This compound binds to the PQH2 oxidising site, as shown by crystallographic studies in cyanobacteria and green algae [START_REF] Stroebel | An Atypical Haem in the Cytochrome B6f Complex[END_REF][START_REF] Kurisu | Structure of the Cytochrome b6f Complex of Oxygenic Photosynthesis: Tuning the Cavity[END_REF] , although not in the same position in the two organisms. Stigmatellin interacts with the 2Fe2S, raising its midpoint potential (in isolated Cytb6f) from 320 to about 460 mV 553 . This increase in potential is accompanied by changes in the EPR spectrum of the 2Fe2S cluster. In vivo, its action mechanism is similar to that DNP-INT. 551 Under single turnover flash illumination, stigmatellin reduces, in parallel, the rate of Cytf and of Cytb6 reduction in a concentration-dependent manner.

Inhibitors of the

In Cytbc1, stigmatellin has been extensively used to probe conformational changes related to the moment of ISP-HD from a proximal site, i. e. close to the quinol, to a distal site, close to Cytc (reviewed in Berry et al. [START_REF] Berry | Structure and Function of Cytochrome Bc Complexes[END_REF] ). This movement is required to facilitate electron flow in the high potential chain, and to promote electron bifurcation, as predicted by the Q cycle mechanism [START_REF] Berry | Structure and Function of Cytochrome Bc Complexes[END_REF] . In Cytb6f, the transition between these two positions has not been resolved by X-ray crystallographycally in the same clear cut manner as in Cytbc1. Nonetheless, electron crystallography 554 has underlined movements of the ISP-HD in the Cytb6f, via the comparison of projection maps of thin threedimensional crystals of Cytb6f prepared with or without stigmatellin. 2D crystallography has also suggested possible conformational changes in the transmembrane region of the complex, upon stigmatellin binding.

Unlike other inhibitors of Cytb6f, stigmatellin binds not only to the luminal but also to the stromal side of the Cytb6f [START_REF] Hasan | Quinone-Dependent Proton Transfer Pathways in the Photosynthetic Cytochrome B6f Complex[END_REF]173 . The Qn site has a lower affinity for stigmatellin than does the p-side lumenal site. In the Qn site stigmatellin faces the quinone exchange cavity, being in interaction with Arg 207 of the Cytb6 and the supplementary heme cn. (see also section 3.6.2).

DBMIB (2,5- ). However, crystallographic data unambiguously showed that only one molecule of DBMIB can bind to the PQH2 oxidation site. [START_REF] Yan | Intraprotein Transfer of the Quinone Analogue Inhibitor 2,5-Dibromo-3-Methyl-6-Isopropyl-p-Benzoquinone in the Cytochrome B6f Complex[END_REF] To reconcile these findings, Roberts and coworkers, repeated the EPR spectra with oriented Cytb6f 110 , and found that DBMIB affects the orientation of the ISP-HD within Cytb6f depending on the stoichiometry of the inhibitor at the Qp/Qo site. With a single DBMIB, the EPR signatures of the ISP-HD are consistent with this protein being in the proximal position (close to the Qp/Qo pocket), similar to the orientation observed in the X-ray crystal structure of the Cytb6f in the presence of DBMIB [START_REF] Yan | Intraprotein Transfer of the Quinone Analogue Inhibitor 2,5-Dibromo-3-Methyl-6-Isopropyl-p-Benzoquinone in the Cytochrome B6f Complex[END_REF] . With ≥ 2 equiv. of DBMIB bound, the ISP-HD would be in a position resembling the one observed in the Chlamydomonas reinhardtii, Cytb6f in the presence of tridecylstigmatellin (TDS) [START_REF] Stroebel | An Atypical Haem in the Cytochrome B6f Complex[END_REF] , suggesting that the low-affinity DBMIB site is at the distal niche. This conclusion is consistent with earlier EPR measurements of isolated Cytb6f poisoned with DBMIB [START_REF] Schoepp | The Qo-Site Inhibitor DBMIB Favours the Proximal Position of the Chloroplast Rieske Protein and Induces a pK-Shift of the Redox-Linked Proton[END_REF] , and can explain the peculiar mechanism of inhibition of PQH2 oxidation by this compound observed in vitro and in vivo.

Early studies by Rich and colleagues 557 showed that reduced DBMIB (DBMIBH2) does not block the Cytb6f activity during its first turnover. Thus, DBMIB would bind the complex in its reduced form but only become inhibitory upon its oxidation. The inhibitory complex would thus involve a semiquinone or a quinone form of DBMIB. This hypothesis was subsequently confirmed in vivo 551 .

The Besides being extensively used to characterize electron flow in the photosynthetic chain, DBMIB has also been largely employed to study cell biological responses in photosynthetic organisms. Combining DBMIB with the PSII specific inhibitor DCMU allows the PQ(H2) pool to be either reduced (DBMIB) or oxidised (DCMU) in the light. Based on this approach, it was proposed that the redox state of the PQ(H2) pool regulates the transcription of a subset of chloroplast genes to adjust the relative stoichiometric amounts of the two reaction centres (PSII and PSI) (see, e.g. reviews by [558][559][560] ). These findings have led to the concept of redox mediated plastid retrograde signalling that orchestrates the expression of genes by the plastid and nuclear genomes, a concept that is still discussed (review in Pfannschidt 2010 and Chan et al., 2016 561,562 ).

Ions. Divalent cations also inhibit the catalytic cycle of the Cytb6f in concentration dependent manner. Inhibition was first reported for Cu 2+ 112 and then for Zn 2+ 108 . Both metals were proposed to hamper the movement of the ISP-HD. Based on EPR data, it was proposed that binding of the two ions in proximity of His143 in the ISP would induce the inhibition. Later crystallographic studies with Cd 2+ poisoned Cytb6f 173 provided a structural confirmation of this hypothesis, showing the existence of two Cd 2+ binding sites, the strongest one being mediated by Cd-H143 interactions.

Inhibitors of the Qn site. Typical inhibitors of this site are: N(H)QNO, MOA stilbene and, to a lower extent, stigmatellin.

NQNO (2-n-nonyl-4-hydroxy-quinoline N-oxide) was first investigated by Jones and

Whitmarsh 563 in vitro and by Joliot and Joliot 564 in Chlorella sorokiniana cells. Jones and Whitmarsh 565 used single turnover and steady state absorption spectroscopy to show that NQNO binds to the stromal quinone site, thereby inhibiting the oxidation of the Cytb6 heme, and slowing down electron transfer between the two b-hemes. Based on the effect of NQNO on the ECS kinetics, the authors concluded that: i) the slow phase of the ECS under single turnover flash (phase b, see above) is due to electron transfer between the two b hemes, followed by a reaction associated with PQ reduction;

ii) the two quinone binding sites would be separated by 70% of the dielectrically weighted distance across the membrane.

Joliot and Joliot further explored the relationship between this inhibitor, redox changes of Cytb6 and changes in the kinetics and amplitude of phase b of the ECS. Based on the consequences of NQNO poisoning of phase b, the authors proposed that reduction of the quinone occurring at the stromal site (Qn) would be coupled to protonation, implying that this site is connected to the outer face of the thylakoid by a proton channel. Recent crystallographic studies have provided a molecular interpretation for these findings, showing that at least two residues (Asp 20 and Arg 207) form a H + wire connecting the stroma to the quinone binding pocket [START_REF] Hasan | Quinone-Dependent Proton Transfer Pathways in the Photosynthetic Cytochrome B6f Complex[END_REF] .

NQNO binds in close proximity to the heme cn, acting as a direct axial ligand of the heme (Yamashita et al., 2007). This interaction explains why NQNO binding to the Cytb6f leads to a strong modification of the properties of heme cn 191 . This heme titrates as a one-electron Nernst curve with a Em value of +100 mV in unpoisoned complexes. Its reduced minus oxidized spectrum displays a broad absorbance increase peaking at approximately 425 nm. Upon binding of NQNO two heme cn titrations waves are visible: one with an Em value similar to that observed in non-inhibited enzyme and the other with a midpoint shift by about -225 mV 191 . Moreover, the Soret spectrum of the heme is shifted by 1 nm to longer wavelengths upon NQNO binding 173

MOA stilbene (Fig. 22, 1 in red frames) This inhibitor was first studied by Rich and colleagues 566 , who showed that at variance with Cytbc1, MOA-stibene does not affect quinol oxidation but instead quinone reduction. Its binding to Cytb6f induces a red-shift of the Soret and visible absorbance bands of the b hemes, enhancing their 'oxidant-induced reduction' and slowing down their subsequent dark reoxidation at the same time. Its effect is therefore similar to that of NQNO.

Later, Kramer and Crofts 218 confirmed these results, showing that MOA-stilbene increases the reduction of the high potential form of Cytb6 by 45% ca, and reduced the amplitude of the ECS signal by about 25%, again in agreement with results obtained with NQNO.

Stigmatellin (Fig. 18 A). As mentioned above, this inhibitor also binds to the Qn site with very similar features as NQNO. However, due to its higher affinity for the Qp site, this inhibitor cannot be used to test the mechanism of electron and proton transfer to PQ.

Thermodynamic background of cytochrome bc catalysis

This section covers the thermodynamic principles behind operation of Cyt-bc, using the reactions characteristic for Cytbc1. If the Cytb6f were to operate according to a "modified Q cycle", as described for Cytbc1, then the equations below would be equally applicable to both complexes, simply by exchanging the parameters for PC/Cytc6 and PQ(H2) with those of Cytc and UQ(H2) respectively. However, as described in detail in section 3.6.5, significant doubts remain about the operation of a Q cycle at Cytb6f, with the possibility that additional electrons may be added to the Qn side from soluble components. In this case, the stoichiometry of the equations below will clearly be altered for the Cytb6f

In aqueous solutions, the quinone-dependent enzymatic reaction of Cytbc1 can be summarized as [START_REF] Berry | Structure and Function of Cytochrome Bc Complexes[END_REF]567 :

2 Cytc 3+ + QH2 = 2 Cytc 2+ + Q + 2 H + (eq. 10)
where Cytc 3+ and Cytc 2+ stand for ferri-and ferro-Cytc, respectively.

In this case, the change in ΔG associated with reaction of electron transfer from quinone molecules to Cytc molecules after mixing UQH2 derivatives with Cytc can be estimated using the relationship 568 : ΔG = -nFΔEh (eq. 11)

where ΔEh is the difference in the actual redox potential between the couples of the donor (Eh(D) of UQ/UQH2) and acceptor (Eh(A) of Cytc 3+ /Cytc 2+ ) and n is the number of electrons in the reaction (here n = 2) and F is the Faraday constant.

ΔEh = Eh(A) -Eh(D) (eq. 12)

The corresponding Eh(A) and Eh(D) can be calculated using the Nernst equation which takes into account the redox midpoint potentials at pH 7 (Em7 Q and Em7 Cyt ) and actual concentrations of reduced and oxidized form of UQ/UQH2 and Cytc 3+ /Cytc 2+ , the temperature (T) and the gas constant (R). 568 .

Eh7(A) = Em7 Cyt + RT/nF ln([Cytc 3+ ]/[Cytc 2+ ]) (eq. 13) Eh7(D) = Em7 Q + RT/nF ln([UQ]/[UQH2]) (eq. 14)
During a typical measurement of Cytbc1 turnover rate, the substrates are added to the solutions in total concentrations that should exceed Michaelis-Menten constants, while proportions of oxidized to reduced substrates are approximately 0.05 and 0. For comparison, the ΔG of oxidation of 1 mol of NADH in complex I and reduction of O2 in complex IV in the mitochondrial respiratory chain is approximately -224 kJ/mol 162 .

However, for the enzyme operating in living cells, eq. 11 is too simple by far and ignores an important fact that Cytc reduction by Cytbc1 is associated with the coupling of two oppositely directed redox reactions taking place at the Qo/Qp and Qi/Qn catalytic sites. Moreover, these reactions are coupled to the release of protons to the p side, and uptake of protons from the n side of membrane.

Therefore, the simple eq. 10 should be split into two equations in order to account for this spatial separation of the two different quinone redox reactions in Cytbc1:

QH2 (p) + Cytc 3+ + Q (n) = Q (p) + Cytc 2+ + SQ -(n) + 2H +(p) (eq. 16) QH2 (p) + Cytc 3+ + SQ -(n) + 2H +(n) = Q (p) + Cytc 2+ + QH2 -(n) + 2H +(p) (eq. 17)
Superscripts (p) and (n) denotes that the respective reaction takes place at the Qo/Qp and Qi/Qn site, respectively.

These reactions lead to proton transfer across the membrane in which Cytbc is embedded, i.e.

4 protons are released (from Qo/Qp site) to the positive side (p side), while 2 protons are taken (at the Qi/Qn site) from the n side of the membrane. In this way the proton transfer catalyzed by the enzyme contributes to an increase of the transmembrane electrochemical potential gradient (Δp) which is used to drive the energetically uphill reaction of ADP to ATP conversion in cells 162 . However, it is worth mentioning here that the protons are transferred across the membrane by coupling deprotonation of QH2 at p side with protonation of Q at n side, according to Mitchelian redox loop mechanisms. This is different form other complexes such as CcO for example which catalyzes active pumping of protons through specific proton channels.

Proton motive force is a value expressed as the sum of two components: difference in proton concentration between two sides of membrane (ΔpH) and the electric potential difference (Δψ)

between the two sides of membrane. However, for simplicity Δp is commonly converted to equivalent differences in electric potential expressed in millivolts.

In mitochondria, ΔpH is usually small and thus Δp is largely dominated by Δψ, which is on the order of 150 -200 mV 162 , depending on the actual state of mitochondria (ADP/ATP ratio, oxygen tension and the activity of decoupling proteins). The architecture of Cytbc imposes an additional energetic barrier to electron transfer from the Qo/Qp site through heme bL/bp and bH/bn to the Qi/Qn site as the electrons must be transferred against Δψ, when the enzyme is embedded in the coupled membrane 161,[571][572][573] . Therefore equattion 15 must take into account this additional energetic barrier: ΔG = -nF(ΔEh -Δψ) (eq. 18)

Under conditions in which the Q(H2) pool and Cytc/PC pool are half reduced, the driving force is significantly lowered. As a result, increasing Δψ influences the ET process hence increasing the probability of reduction of heme bL/bp at the expense of heme bH/bn. Under extreme conditions, Δψ can be high enough to impair the QH2 oxidation at the Qo/Qp site leading to an increase in superoxide generation during the catalysis 572 .

7 Mechanistic insights into the catalytic Qo / Qp site

Overview of the structure of the Qo site

Cytb of Cytbc1 is built of 8 transmembrane helices arranged in two helical bundles with the first bundle (helices A to E) incorporating both hemes b (bL and bH). The second bundle (helices F to H) is located in the vicinity of heme bL and together with the large helix-connecting loops at the intermembrane (periplasmic) side of the membrane, forms the Qo site binding pocket [START_REF] Berry | Unanswered Questions about the Structure of Cytochrome Bc1 Complexes[END_REF][START_REF] Xia | Structural Analysis of Cytochrome Bc1 Complexes: Implications to the Mechanism of Function[END_REF][START_REF] Gao | Structural Basis for the Quinone Reduction in the Bc1 Complex: A Comparative Analysis of Crystal Structures of Mitochondrial Cytochrome Bc1 with Bound Substrate and Inhibitors at the Qi Site[END_REF]132 . This pocket is highly hydrophobic and contains many highly conserved residues. The major loop regions that participate in formation of the Qo site include the two helices cd1 and cd2 of the CD loop and the ef helix of the EF loop. The helices cd1 and cd2 cap the pocket from the intermembrane (periplasmic)

space where Cytb interacts with the ISP-HD. Transient binding of the ISP-HD to Cytb brings the 2Fe2S cluster into proximity with the substrate occupying the Qo site pocket, which means that in this pocket the substrate can be flanked by two redox active cofactors: the 2Fe2S and heme bL. This corresponds to the state when the two cofactors required for electron bifurcation are present close enough to the substrate bound at the Qo site for fast electron transfer to occur (the exact distance is not known but approximated from the crystal position of the Qo site inhibitors, see below). Due to the large-scale oscillatory movement of the ISP-HD between the Cytb and Cytc1, there are states of the Qo site with 2Fe2S is not present at the site and therefore unable to interact with the bound substrate (precluding the bifurcation reaction). The movement itself is an inherent part of the catalytic cycle as it connects the Qo site with heme c of Cytc1: transient binding of the ISP-HD away from Cytb to Cytc1 brings the 2Fe2S and heme c1 close enough for fast electron transfer between these two cofactors.

Quinone binding to the Qo site

Binding of the QH2 molecule to the Qo site is an obvious requirement for initiating the catalytic QH2 oxidation. Despite many years of study the way in which Q or QH2 are bound to this site is still not fully understood. A major part of what is known about these interactions comes from measuring site-directed mutants of bacterial Cytbc1, usually of conserved residues at the Qo site, in spectroscopic studies, mainly EPR spectroscopy and X-ray crystallography. The latter method has always been considered as the most promising tool for detection of specific chemical interactions between substrates and the protein. However, soon after solving the first structures of Cytbc1, it became clear that in the case of this protein (as in some other quinone-binding proteins) the crystallography failed to show natural substrate bound at the Qo site and neither Q nor QH2 has been resolved within this site [START_REF] Berry | Unanswered Questions about the Structure of Cytochrome Bc1 Complexes[END_REF] . It has been proposed that this failure is due to the Q molecules at the Qo site being in a very dynamic state. However, a simple loss of the substrate during the isolation and crystallization of the protein also cannot be ruled out, in particular in the light of spectroscopic observations that samples of isolated Cytbc1 tend to have an empty Qo site [START_REF] Xia | Crystal Structure of the Cytochrome Bc1 Complex from Bovine Heart Mitochondria[END_REF]127,260 In the absence of structures containing natural substrate at the Qo site, structures obtained with many different site-specific inhibitors have served as a starting point for the construction of models for Q binding to Qo. Several of these proposals, including the most recent ones, considered specific interactions derived after substituting the chromane ring of stigmatellin with the natural quinone ring [START_REF] Crofts | Mechanism of Ubiquinol Oxidation by the Bc1 Complex: Different Domains of the Quinol Binding Pocket and Their Role in the Mechanism and Binding of Inhibitors[END_REF][START_REF] Postila | Key Role of Water in Proton Transfer at the Qo-Site of the Cytochrome Bc1 Complex Predicted by Atomistic Molecular Dynamics Simulations[END_REF][START_REF] Barragan | Identification of Ubiquinol Binding Motifs at the Qo-Site of the Cytochrome Bc1 Complex[END_REF]574,575 .

In studying the binding of QH2 and Q to the Qo site, EPR spectroscopy of the reduced 2Fe2S has been one of the most widely used methods. It has benefited from the observation that, for Cytbc1, the continuous wave EPR (CW-EPR) spectra of 2Fe2S is highly sensitive to variation in the type of molecule occupying the Qo site and its redox state, providing a convenient means to monitor the status of the site in the native membranes. This approach seems to also be applicable for detection of PQ or PQH2 bound to the Qp site of Cytb6f. 576 From the very beginning it become apparent that CW-EPR spectra of 2Fe2S could not be simulated by modeling a single EPR component for the 2Fe2S 159 nor by using a statistical theory of g strain that has previously worked for other iron-sulfur clusters 577 .

The sensitivity of the CW-EPR spectra of the reduced 2Fe2S to the occupant of the Qo site is best documented for bacterial Cytbc1. It was observed that the shape of the spectrum differed depending whether Cytbc1 was embedded in native membranes or isolated and solubilized in detergent micelles 127 . It was also observed that the shape of the spectrum in the membranes was sensitive to the total quinone content in the membrane 109,260,578,579 as well as being a convenient tool for discriminating between Q or QH2 occupancy of the Qo site 580 .

In bacterial chromatophores, the most characteristic spectral feature of 2Fe2S is the gx transition which equals 1.800 when UQ bound at the Qo site interacts with the cluster 579 . Among all the transitions of the 2Fe2S spectrum, the gx component appears the most influenced by the occupant of the Qo site. When the site contains UQH2, gx broadens significantly and shifts to approximately 1.777 109,578,579 . Other transitions (gz, gy) are also relatively narrow when UQ is bound to the Qo site suggesting a relative small g-strain of the cluster [START_REF] Sarewicz | Magnetic Interactions Sense Changes in Distance between Heme bL and the Iron-Sulfur Cluster in Cytochrome Bc1[END_REF]111,113,579,581 . These transitions also broaden, although less significantly comparing to gx, when UQ exchanges with UQH2.

Careful analysis of the gx transition has established that measurements of relative proportions of gx at 1.800 vs gx at 1.777 signal can be used to monitor the redox state of the Q(H2) pool in the membrane. Most probably the gx = 1.800 transition results from interaction of the 2Fe2S environment with the carbonyl group of UQ, while the gx = 1777 results from interaction with the hydroxyl group of UQH2 578 . However, this sensitivity of the 2Fe2S spectrum to UQ or UQH2 is completely abolished when low-molecular-weight alcohols, such as methanol, ethanol, glycerol (a common cryoprotectant), are present 582 . This most likely results from interaction of the hydroxyl group with the Rh H156 ligand of 2Fe2S ( Bt H161). It follows that measurements of the redox state of the Q(H2)

pool by CW-EPR spectra of 2Fe2S (which must be performed at low temperatures) must avoid cryoprotective agents as they usually contain hydroxyl groups 583 . On the other hand, the absolute necessity of using cryportectants in low-temperature pulse EPR measurements compromises sensitivity of the 2Fe2S spectra to UQ and UQH2. Otherwise formation of ice may cause aggregation and denaturation and may significantly increase the efficiency of paramagnetic relaxation distorting the results, which are dependent on spin-lattice relaxation rates.

For the empty Qo site, the gx transition is very broad and its g value approaches 1.765 580 . A similar shape and g value for the gx transition is detected when the Qo site is occupied by the inhibitor myxothiazol or MOA-stilbene 578 .

Interestingly, the experiments on partial extraction of UQ from purple bacterial membranes (chromatophores) revealed that when the ratio of the number of Q molecules per Qo sites is between 2:1 and 1:1, the gx value changes to approximately 1.78 and its linewidth lies in-between gx = 1.800 (for UQ) and 1.777 (for UQH2) 584 . Furthermore, this 1.78 transition could not be reproduced by a linear combination of "UQ gx 1.800" and "empty-site gx 1.765" lines. To explain these observations Ding et al, proposed that there are two niches, strong and weak quinone binding at Qo: Qos and Qow, with dissociation constants (Kd) of approximately ~0.05 and ~1 mM, respectively 260 . Taking these Kd values and assuming that, under normal conditions, the effective concentration of UQ in the membrane is ~30 mM 260 , it was concluded that the Qo site should be occupied by two molecules of UQ at the same time. This lead the authors to formulate a "double-occupancy" model, according to which the Qo site can accommodate 2 UQH2 molecules simultaneously 260,578,580 . Further support for the double occupancy model has come from determination of the stoichiometry of UQH2 binding to Cytbc1 by NMR-based analysis of inhibitor displacements. With this approach, two molecules of UQ(H2) were shown to bind specifically to the Qo site. 585 This observation was incorporated into a model that aimed to explain the mechanism of bifurcation. It was proposed that UQH2 and UQ binds at the same time in the Qo site and undergo a comproportionation reaction according to eq. 7, resulting in formation of two SQ molecules, each donating one electron to the c-or b-chain 585,586 . However, this concept has not been verified with other, more direct studies.

In the opinion of the authors of this review, the double-occupancy model remains problematic and it was previously the subject of debate [START_REF] Crofts | Mechanism of Ubiquinol Oxidation by the Bc1 Complex: Different Domains of the Quinol Binding Pocket and Their Role in the Mechanism and Binding of Inhibitors[END_REF] . When measuring the redox state of the Q(H2) pool in samples poised at ambient redox potentials (Eh) equal to or lower than the Em of the UQ/UQH2 couple, the Qo site would be occupied by UQ at the Qos niche with an increasing population of UQH2 at the Qow niche as Eh shifts negatively. If this happened during redox titration of the membranous UQ(H2) pool, as monitored by EPR measurements of the 2Fe2S spectrum, the gx would likely be different from the observed experimentally linear combination of gx = 1.800 (corresponding to UQ Qos UQ Qow ) and 1.777 (corresponding to UQH2 Qos UQH2 Qow ) transitions. Such a titration should also reveal a separate component corresponding to the unique UQ Qos UQH2 Qow state or respective molecules after comproportionation. Alternatively, the presence of two non-equivalent sites for UQ(H2) at the Qo site would very likely lead to a non-Nernstian shape of the redox titration curve for the UQ/UQH2 couple, detected in the gx transition of 2Fe2S. However, none of these observations can be made, and the titration curve in such experiments closely follows the Nernst equation with n = 2 109,579 . In addition, the Em values of UQ/UQH2 obtained from EPR measurements of the 2Fe2S spectra remain consistent with spectrophotometric measurements of the redox midpoint potential of this couple 587 . This suggests that UQ and UQH2 at the Qo site are in dynamic equilibrium with UQ and UQH2 of the membranous pool. Going further, one may conclude that if changes in EPR spectra follow the redox state of the UQ(H2) pool, UQ is not favorably bound to the site over UQH2 and vice versa.

The first step of the QH2 oxidation at the Qo site involves binding of QH2 and association of the ISP-HD with an oxidized 2Fe2S to the Qo site. Thus, the initial step of the reaction is formation of the enzyme-substrate (ES) complex, which depends on equilibrium constants describing the ratio between the population of Qo sites with QH2 bound and those which are empty (KQH2-Qo).

Additionally, the association constant of ISP-HD containing oxidized 2Fe2S with the Qo (KISPox) site is also a crucial parameter defining the probability of ES complex formation. While direct designation of KQH2-Qo and KISPox is not experimentally possible, the respective values for Cytbc1 were approximated by Crofts et al. [START_REF] Crofts | The Mechanism of Ubihydroquinone Oxidation at the Qo-Site of the Cytochrome Bc1 Complex[END_REF] who considered the following states: From eq. 19 it can be concluded that both QH2 and oxidized 2Fe2S have a tendency to form the ES complex to initiate the first step of electronic bifurcation. Unfortunately, a direct observation of the interaction of QH2 with oxidized 2Fe2S has not yet been made, as such a state can be generated only under non-equilibrium conditions, which would make the 2Fe2S diamagnetic and therefore not detectable by EPR spectroscopy. The EPR spectroscopy performed with cryoreduction of the 2Fe2S cluster in oriented membranes is also not informative. Although it can detect the position of ISP-HD with oxidized 2Fe2S, the Qo site contains only UQ, not QH2 100 .

Crucial amino acid residues involved in the binding and catalysis of QH2 / PQH2 oxidation at the Qo/Qp site

This paragraph focuses on some of the crucial amino acid residues of the Qo site that are thought to be involved in Q(H2) binding, and of the Qp site, involved in PQ(H2) binding. At a general level, understanding how individual structural elements contribute to Q(H2) or PQ(H2) binding is challenging because of the difficulty in defining specific and universal Q(H2) binding motifs in proteins 588 and the possibly related observation that many protein-Q(H2) interactions involve atoms of the main polypeptide chain.

Nevertheless, extensive mutational studies targeting residues of the Qo site has identified those that affect catalysis, although some of these mutations were found to primarily affect the stability and/or assembly of the whole protein complex. It is of note that, despite a large number of mutations that have already been tested in the region of the Qo site, relatively few were able to completely abolish enzymatic activity, implying significant robustness and tolerance for structural change of both Q(H2) binding and Qo site catalysis.

Current models of Q and QH2 binding to the Qo site are derived from crystallographic structures of Cytbc1 with bound inhibitors replacing the natural substrate (structures with the substrate have not been solved yet). The models consider both Pm and Pf inhibitors, i.e. inhibitors exerting different effects on the motion of ISP-HD as well as those occupying different niches of the Qo pocket, (see section 5 for details). The latter difference is visualized by a comparison of the structures shown in Fig. 27, which compare binding of myxothiazol (representative of Pm group) at a position closer to heme bL and further from ISP-HD with binding of stigmatellin (representative of Pf inhibitors) -at a position closer to ISP-HD and further from heme bL. The two most prominent, and thus most frequently discussed, candidates for residues that directly participate in binding substrate are histidine Bt H161 ( Rh H156) -one of the ligands of the 2Fe2S and glutamate Bt 271 ( Rh E295) from conserved PEWY motif of Cytb. A binding mode for substrate involving these two residues is thought to be equivalent to the configuration of stigmatellin in the crystal structure. Stigmatellin forms two strong H bonds in the Qo site (Fig. 27 B): one between the >C=O group of the inhibitor and Nτ atom of Bt H161 of ISP-HD and second between -OH group and carboxylic group of Bt E271 residue 123 . Interestingly, myxothiazol crystalised in the Qo site does not form specific hydrogen bonds to either of these groups. A comparison of the two structures reveals variable positions for the Bt E271 side chain: it rotates further toward the chromane ring of stigmatellin in order to create a hydrogen bond with the -OH group at the C8 atom of the ring. It is highly unlikely that Q or QH2 molecules at the Qo site simultaneously form H bonds to Bt H161 and Bt E271 as stigmatellin does. Therefore it was proposed that during the oxidation QH2 undergoes a rapid movement between the two niches within the Qo site filled by stigmatellin and myxothiazol [START_REF] Crofts | The Mechanism of Ubihydroquinone Oxidation at the Qo-Site of the Cytochrome Bc1 Complex[END_REF]575,589,590 . According to this mechanism, QH2 binds to the Qo via Bt H161 ( Rh H156) and the first electron transfer to the oxidized 2Fe2S takes place. The first proton is released and then the neutral SQH moves to form a hydrogen bond to Bt E271. The Glu residue promotes deprotonation of SQH, which then donates an electron to heme bL. After formation of Q, it shifts back to the position that allows interaction with Bt H161 and then it can be replaced with another QH2. This mechanism has not been proven yet and it is a still a matter of debate 259 . While the SQ moving between the two niches should be trappable, the experiments generally fail to detect such an intermediate of reaction (see section 7.6). Importantly, it remains an open question as to whether stigmatellin is actually a good model for Q(H2) binding. The spatial separation of the two -OH groups differs between the QH2 and the chromane ring of stigmatellin, with consequences for the capacity to form H bonds.

Comparing structures crystallized with different inhibitors, it seems that binding of UHDBT [START_REF] Esser | Crystallographic Studies of Quinol Oxidation Site Inhibitors: A Modified Classification of Inhibitors for the Cytochrome Bc1 Complex[END_REF] or atovaquone [START_REF] Birth | Structural Analysis of Atovaquone-Inhibited Cytochrome Bc1 Complex Reveals the Molecular Basis of Antimalarial Drug Action[END_REF] at the Qo site might more closely reflect binding of QH2 as the structures of these inhibitors are more similar to natural quinones, (menaquinone and sulfolobus-or caldariellaquinone for example 591 ), than the chromane ring of stigmatellin.

The idea that Bt H161 might participate in binding of Q and QH2 to the Qo was proposed after ENDOR measurements revealed that the 2Fe2S is coordinated by 2 Cys and 2 His side chains 592 which was later confirmed by crystallographic structures 593 . Electrochemical studies revealed that the Em value of 2Fe2S is pH dependent, and therefore dependency requires two different pK values 594 .

It was estimated that the pKa for oxidized 2Fe2S is ~7.5 and increases to more than 10 upon reduction of the 2Fe2S. This suggests that the protonation of Bt H161 is tightly coupled to the redox state of the 2Fe2S. Such an increase in pK means that the energy of hydrogen bond formation between an occupant of the Qo and Bt H161 is larger when 2Fe2S is reduced compared to when the 2Fe2S is oxidized. Stabilization of the ISP-HD by the reduced 2Fe2S through interaction with Q(H2) occupying the Qo site is also inferred from EPR measurements. Typically, the EPR spectra of the reduced 2Fe2S in chromatophore membranes reflect close interactions with the Q(H2), revealing that the reduced state of 2Fe2S promotes ISP-HD occupancy of a position close to the Qo site (see section 3.4) On the other hand, experiments in which Cytbc1 containing oxidized 2Fe2S was frozen before the cluster was reduced for detection by EPR showed that the ISP-HD with oxidized cluster tends to occupy a position remote from the Qo site. 100 The 2Fe2S coordinating Bt His161 is well established as a crucial amino acid residue for the activity of all Cyt-bc. Its role in formation of initial ES complex is supported by many biochemical and computational studies. However, all mutational studies aimed at replacing this residue result in loss of the whole ISP subunit 138 , making definitive proof elusive.

The majority of the models describing formation of the initial ES complex assume that B His161 must be deprotonated before binding the QH2, whereas Q can bind to the Qo site when it is already protonated [595][596][597] . In some models, this His residue has therefore been proposed as the first proton acceptor during the oxidation of QH2. However, MD simulations indicate that QH2 can also form a stable H bond to the His, even when it has already been protonated. [START_REF] Postila | Key Role of Water in Proton Transfer at the Qo-Site of the Cytochrome Bc1 Complex Predicted by Atomistic Molecular Dynamics Simulations[END_REF][START_REF] Barragan | Identification of Ubiquinol Binding Motifs at the Qo-Site of the Cytochrome Bc1 Complex[END_REF] The role and significance of the conserved PEWY domain Glu residue in binding substrate and catalytic reaction are less understood [START_REF] Crofts | The Mechanism of Ubihydroquinone Oxidation at the Qo-Site of the Cytochrome Bc1 Complex[END_REF]118,598 . In several models Bt E271 is considered to directly accept the second proton from QH2 upon its oxidation. However, mutational studies of this residue in the bacterial enzyme revealed only a modest impact on turnover rate and no significant changes in the estimated binding affinity of QH2 to the Qo site 579 . On the other hand, mutation of the same residue in yeast ( Sc E272Q or Sc E272D) induced stigmatellin resistance 598 . These two observations suggest that Bt E271 may not be directly involved in either binding of Q or QH2 to the Qo site nor be critically required for proton uptake from QH2 during the bifurcation reaction. Rather, it may be residue that increases stability of the water network at the Qo site, which is necessary to create an appropriate environment for efficient and fast proton removal from the Qo site. Clearly, further studies are needed to clarify the role of this residue in binding and/or proton events taking place at the site.

Interesting evolutionary aspects of the PEWY motif are beyond the scope of this review but interested readers will find a discussion on this subject in appropriate reference 118 .

7.3.2

Cytb: Rh Y147 ( Bt Y131) and Cytb: Rh Y302 ( Bt Y278)

The Y147 residue is conserved among all Cytbc1 complexes. Its importance in the activity of the Qo site was postulated before crystallographic structures were available 599 . Mutations of this residue to Phe, Val, Ser or Ala have a dramatic effect on the activity of bacterial Cytbc1 599 . This significant impairment of catalysis is not associated with a change in the redox potentials of heme bL nor 2Fe2S. MD studies imply that Y147 can rotate to form a hydrogen bond between the -OH group at position 4 of UQH2 and the -COO -group of Rh E295 [START_REF] Barragan | Identification of Ubiquinol Binding Motifs at the Qo-Site of the Cytochrome Bc1 Complex[END_REF] , suggesting that this residue might play an important role in formation of the ES complex and the process of proton uptake during the bifurcation reaction. Quantum mechanics calculations performed on MD optimized structures, indicate that when UQH2 interacts with protonated ISP: Rh H156, proton transfer from the hydroxyl group of Rh Y147 to the carboxyl group of Rh E295 takes place, creating a negative Rh Y147 and neutral Rh E295. At the same time, the deprotonated hydroxyl of Rh Y147 forms a hydrogen bond to the O1 atom of the hydroxyl group of UQH2. When similar calculations were performed using the structure in which the hydroxyl O4 atom of UQH2 forms a hydrogen bond to the already protonated Rh H156 of ISP-HD, and the H atom of the ubiquinol -OH group interacts with water molecules, this proton transfer from Rh Y147 to Rh E295 was not observed, despite the fact that Rh Y147 could still perform a relay function between UQH2 and Rh E295 [START_REF] Barragan | Identification of Ubiquinol Binding Motifs at the Qo-Site of the Cytochrome Bc1 Complex[END_REF] . Other MD simulations on the binding of UQH2 to the Qo site did not show involvement of Rh Y147 hydroxyl in interaction with UQH2. Instead, a hydroxyl group at the C1 atom of UQH2 formed a hydrogen bond with a network with water molecules [START_REF] Postila | Key Role of Water in Proton Transfer at the Qo-Site of the Cytochrome Bc1 Complex Predicted by Atomistic Molecular Dynamics Simulations[END_REF] .

Although a possible role of Rh Y147 and Rh E295 in binding and proton uptake from UQH2

during the bifurcation reaction was proposed on the basis of biochemical and mutational studies 590,599 , the concept of H bond network formation between UQH2-Rh Y147-Rh E295 clearly needs further investigation.

An important role for Cytb: Rh Y302 ( Sc Y279) in the binding and oxidation of QH2 by the Qo site emerged from several studies on bacterial and mitochondrial Cytbc1. Mutating this residue to Leu, Gly or Glu in R. sphaeroides Cytbc1, was found to decrease catalytic rates by a factor between 3 -fold to 50 -fold 589,600 and to increase ROS generation 601 . These effects were also reported for Sc Y279C, a mutation associated with mitochondria-related diseases 602 . The gx transition of the EPR spectra of 2Fe2S in this mutant suggested that the Qo site was "partially empty" 600 . Furthermore, the spectrum of 2Fe2S with bound stigmatellin differed from the equivalent spectrum for typical wildtype enzymes, indicating much stronger binding of the chromane than the benzoquinone ring to the Qo site of Cytbc1. Analysis of the structure of yeast Cytbc1 with the inhibitor HHDBT bound suggested that Sc Y279 might contribute to the binding of UQH2 by creation of a H bond to the =O atom of the main chain of Sc C180 in ISP-HD [START_REF] Palsdottir | Structure of the Yeast Cytochrome Bc1 Complex with a Hydroxyquinone Anion Qo Site Inhibitor Bound[END_REF] . Such a hydrogen bond is expected to stabilize UQH2 binding with Sc H181 ( Rh H156 and Bt H161) of the ISP-HD.

In Rhodobacter capsulatus, the Rh Y302C mutation exerted a deleterious effect on stability of 2Fe2S due to disruption of a disulfide bridge in ISP-HD and resulting cross-linking between the Cytb subunit and ISP-HD ( Rh Y302C-Rh C155). This made the 2Fe2S more sensitive to oxidative damage 601,603 .

In Plasmodium falciparum Pf Y268S (equivalent Sc Y279S and 7 Rh Y302S) was identified as a mutation responsible for resistance of the parasite to the anti-malarial drug, atovaquone 604 . This potent inhibitor of the Qo site was found to completely block activity of the yeast Cytbc1 (IC50 = 5 nM) 605 .

Atovaquone arrests the ISP-HD at the Qo site in a similar way to stigmatellin, despite the fact that unlike stigmatellin it does not form a direct H-bond with the conserved PEWY sequence Glu residue [START_REF] Birth | Structural Analysis of Atovaquone-Inhibited Cytochrome Bc1 Complex Reveals the Molecular Basis of Antimalarial Drug Action[END_REF]606 . The crystal structure revealed that Sc Y279 forms hydrogen bonds to Cys180 in the ISP but it also interacts weakly with ionized hydroxyl group at C3. The loss of this Tyr residue in Plasmodium is not lethal to the parasite, but significantly slows its Cytbc1 activity, thus slowing the parasite growth rate 604 .

Other residues involved in substrate binding to the Qo site

Besides the amino acid side chains discussed above, it is expected that the binding of substrate to the Qo site also engages other residues in the site, including atoms of the main polypeptide chain of Cytb and ISP. Indeed, MD simulations investigating possible structures of bound UQH2 and UQ at the Qo site identified the =O atoms of Rh I292 (Cytb) and Rh C155 (ISP) as potential residues participating in binding of UQH2 and UQ through water molecules.

Another residue important for binding of the substrate to the Qo site is conserved Gly Rh G158

( Sc G143 and Bt G142) in the WGA sequence 260,569,607 . Mutagenesis studies have revealed that its replacement with residues that have bulkier side chains, particularly tryptophan, prevents binding of either UQ(H2) or inhibitors such as stigmatellin. This effect is most probably due to a steric hindrance at the Qo site, preventing catalytic activity of the enzyme.

It should be noted that the isoprenoid chain of UQ also constitutes an important part of the Q binding interface. Indeed, several hydrophobic residues of Cytb interact with this tail. In R. capsulatus they are: F144, F166, F298, F337, I162, I169, V333, M336, L165, V161, F298, G141, M140, L201, L178 [START_REF] Postila | Key Role of Water in Proton Transfer at the Qo-Site of the Cytochrome Bc1 Complex Predicted by Atomistic Molecular Dynamics Simulations[END_REF] .

Specific residues involved in PQ/PQH2 binding to the Qp site of Cytochrome b6f

Studies to characterize residues of Cytb6f involved in PQ(H2) binding at the Qp site are more limited than in the case of the Cytbc1 counterpart. These studies have been mainly performed in Chlamydomonas, and have revealed strong similarities between the Qp and Qo sites of Cytb6f and Cytbc1, a concept that was later confirmed by crystallographic studies.

The first characterized mutant (FUD50) bears a 36 base pair duplication in the chloroplast petB gene, leading to a 12 amino acid duplication in the CD loop of Cytb6. This modification decreases the affinity of the Qp site for PQ(H2) 608 , likely by increasing the size of the Qp site itself. Subsequent studies have also focused on this region of the Qp site, revealing the essential role of the PEWY sequence in Q(H2) binding and proton release. Later on, this concept that has been rationalized based on the structures of the Cytb6f in this alga and cyanobacteria [START_REF] Stroebel | An Atypical Haem in the Cytochrome B6f Complex[END_REF][START_REF] Kurisu | Structure of the Cytochrome b6f Complex of Oxygenic Photosynthesis: Tuning the Cavity[END_REF][START_REF] Hasan | Quinone-Dependent Proton Transfer Pathways in the Photosynthetic Cytochrome B6f Complex[END_REF] .

Additional mutants have been made in Chlamydomonas to study the role of the ISP flexible hinge in catalytic turnover of the complex. Six Gly residues in the flexible hinge critical for domain movement in the Cytbc1 (section 3.4) were replaced by alanine residues (6G6A). Moreover, substitutions were created near 2Fe2S (S128 and W163); and seven C-terminal residues were deleted (G171och). The 6G6A and G171och mutations affect highly conserved segments in the chloroplast ISP, but no phenotype was found in these lines. Although extensions of up to five residues or deletion of one residue in the flexible hinge had no significant effect on complex accumulation or electron transfer efficiency, deletion of just three residues (Δ3G) dramatically decreased reaction rates by a factor of ∼10. Overall, these finding indicate that the chloroplast ISP hinge is also very flexible 609 and its movement is not limiting for catalysis, consistent with previous conclusions in the case of Cytbc1 (see section 3.4).

Only one mutant has so far been reported to affect PQ(H2) binding to the Qp site in plants.

This mutant (pgrl1) 610 was obtained by random mutagenesis and bears a point mutation in the ISP (P194L). Although the mutation does not affect photosynthetic electron transport at low light intensities, it drastically affects thermal dissipation of absorbed light energy in high light, suggesting that Pro194 may affect H + /e -coupling in the Cytb6f. Single amino acid substitutions in Cytb6 at the positions D148, A154, and S159 were generated in the cyanobacterium Synechococcus sp. PCC 7002.

Mutant phenotyping revealed increased resistance to DBMIB in the mutants A154G and S159A, increased resistance to stigmatellin in A154G, and induction of myxothiazol sensitivity in the D148G mutant, confirming the role of these residues in PQ(H2) binding and inhibitor sensitivity of the complex 611 .

Catalytic and side reactions at the QH2-oxidation site of Cytochromes bc

The oxidant-induced reduction of b hemes in sub-mitochondrial particles is an intriguing phenomenon first reported by Chance in 1952 during II International Congress of Biochemistry in Paris (see reference 612 and citations therein). This phenomenon was difficult to explain until a concept of a semiquinone-mediated electronic bifurcation was introduced by Wikstrom & Berden in 1972. 202 .

The central role of a semiquinone in the mechanism of bifurcation was further developed by Peter Mitchell in the frames of his original idea of so-called proton-motive Q cycle mechanism. [START_REF] Mitchell | The Protonmotive Q Cycle: A General Formulation[END_REF] This groundbreaking concept of electronic bifurcation, inherent to this mechanism, postulated that QH2 at the Qo/Qp site undergoes two-electron oxidation reaction but routes for these two electrons are obligatorily different and very efficient. This entails the first electron being transferred to the oxidized 2Fe2S center (the first cofactor of the high-potential chain) while the second electron is transferred exclusively to heme bL/bp (the first cofactor of the low-potential chain) see section 3.2.

It is assumed that the mechanism of electron bifurcation is sequential, and involves formation of a transient semiquinone intermediate. While the time between the first electron transfer from QH2 to 2Fe2S and the second electron transfer from SQ to heme bL/bp remains unknown, it seems reasonable to assume that it is not shorter than the time required for electron tunneling from the SQ to heme bL/bp.

According to Mitchell's principles QH2 must donate its two electrons to two different chains of cofactors and the two redox couples involved in the first and the second electron transfer -SQ - /QH2 and Q/SQ -couples, respectively, must therefore possess different redox midpoint potentials [START_REF] Mitchell | The Protonmotive Q Cycle: A General Formulation[END_REF] , as originally discussed by Wikstrom & Berden in 1972. 202 It has been proposed that the Em of the SQ - /QH2 redox couple is very positive, and comparable to the high potential acceptor (2Fe2S and heme c1/f), while the Q/SQ -is expected to be a more reducing couple and therefore able to donate electrons to the low-potential chain (hemes bL/bp and bH/bn). This separation of Em values (the split) of the two redox couples implies that the stability constant for the SQ -intermediate at the Qo/Qp site must be very low and hence, detection of such an intermediate by spectroscopic methods might be difficult, especially under equilibrium conditions 487,488 . Indeed, until recently, the semiquinone intermediate state associated with the operation of the Qo/Qp site could not be experimentally detected (see section 7.6). show exothermic reaction to the heme states for which Ems are not directly measurable by equilibrium redox titration.

Gray horizontal lines shows a possible Em values of hemes bL and bH modified by Coulombic interactions between the hemes (assuming the same energy of interactions ~ 80 mV for bL and bH 133,206 ), that are not accessible for direct measurement. Vertical double arrows denotes the changes in Em depending on the redox state of the coupled heme. Cytc and Fe-S denote oxidized/reduced couples of Cytc2 and 2Fe2S; bL(bH ox ) and bL(bH red ) denote oxidized /reduced couple of heme bL, at a time when heme bH is oxidized and reduced, respectively. bH(bL red ) and bH(bL ox ) redox couple of heme bH at a time when heme bL is reduced or oxidized respectively. Note that this scheme does not necessarily apply for Cytb6f.

7.4.1

Forward, reverse and short-circuits reactions at the Qo/Qp site A simple diagram of Em values taken from Rhodobacter capsulatus Cytbc1 with associated ΔG changes due to electron transfer through the cofactor chains is shown in Fig. 28 As originally proposed by Mitchell, there is a large, albeit unknown, split between Em values (here ~800 mV) of the Q/SQ -and SQ -/QH2 couples, and this split depends on the stability constant of the SQ -. This makes the first electron transfer from QH2 to 2Fe2S essentially endothermic and requires an energy debt for electron transfer to the acceptor of a more negative redox potential (2Fe2S) than the donor (QH2).

This energetic debt is repaid during the second electron transfer from SQ -to heme bL/bp. The forward reactions, meaning the net electron transfer from QH2 to Cytc, is continued as long as there is a difference (driving force) between the Eh of membranous Q/QH2, the p side acceptor pools (Cytc, PC or Cytc6) and all the cofactors of Cyt-bc complexes. Nevertheless, it has been shown by several experiments that under specific conditions Cytbc1 has the capacity to catalyze a fast, reverse reaction of reduction of Q to QH2 at the Qo site. Such a process was observed in in vitro studies of Cytbc1 reconstituted into liposomes 613 and also proposed to occur in vivo to support the growth of the chemolitotrophic bacteria Thiobacillus ferrooxidans using Fe 2+ ions as a source of electrons 614 . This suggests that the reactions at the Qo site are rapidly and efficiently reversible, where "efficient" means that these reactions do not lead to significant energy-wasting side reactions (short-circuits or leaks).

Indeed, light-induced electron transfer measurements using cofactor knockouts of bacterial Cytbc1 to dissect out individual electron transfer steps, demonstrated that the reactions of the Qo site are reversible on a catalytically-relevant timescale (milliseconds) 615 . Fast reversibility of these reactions makes an understanding of the energetic efficiency of electronic bifurcation conceptually challenging.

As long as one forgets about the reversibility, the diagram in Fig. 28 simply explains the energetic efficiency of electron transfer onto two different chains of cofactors. However, the fast reversibility of reactions at the Qo/Qp site means that the enzyme can potentially catalyze energy-wasting short circuit reactions 259,615 . Four possible short-cicuit reactions of the Qo/Qp site are shown in figure 29. The first type of short circuit (SC1) is a reaction in which two electrons from QH2 are transferred to the high potential chain according to the following reaction sequence:

QH2 + 2Fe2S ox = 2H + + 2Fe2S red + SQ - 2Fe2S red + c1 ox /f = 2Fe2S ox + c1 red /f SQ -+ 2Fe2S ox = 2Fe2S red + Q SC1 would be very exothermic since the electron transfer would be associated with a large potential energy change due to the transition from state [b] to [e] in figure 28. Despite the fact that SC1 is thermodynamically very favorable, it is avoided relatively easily, due to the requirement for the movement of ISP-HD (see section 3.4) during transfer of electrons from 2Fe2S to heme c1 or f.

Through this constrained diffusion of ISP-HD, the distance between SQ -and the cluster increases significantly, slowing down any possible second electron transfer from the semiquinone to 2Fe2S. In the absence of ISP-HD in close proximity, the unstable SQ -therefore pushes the second electron onto heme bL/bp. SC1 decreases the proton transfer efficiency across the membrane: it yields +2H + p / 0H + n per oxidation of one QH2, while an energy-conserving Q cycle yields +4H + p / -2H + n per 1 QH2.

In contrast to SC1, the second type of short-circuit reaction (SC2) is extremely difficult to suppress without involvement of any additional mechanisms to lower the probability that it occurs.

SC2 is a mixture of partial reactions of the forward and the reverse catalysis performed by the Qo/Qp site and is a result of the following reactions (for simplicity in the following schemes bL and bH also represent hemes bp and bn, respectively):

QH2 + 2Fe2S ox = 2H + + 2Fe2S red + SQ - bL red + SQ -+ 2H + = bL ox + QH2 SC2 may take place when QH2 undergoes oxidation by 2Fe2S as a part of the chemistry of the normal forward reaction, while heme bL/bp is reduced and unable to accept electron from SQ -. In this situation, heme bL/bp can then donate an electron to SQ -, reforming QH2, as this reaction is also much more energetically favorable than any possible forward electron transfer from heme bL/bp to heme bH/bn (compare the difference in energy associated with

[b] to [a] and [b] to [c] transition in fig. 28).
Energy conversion of this process is compromised as this short circuit gives the same stoichiometry as SC1 (+2H + p / 0H + n per 1 oxidized QH2 ), assuming that every SC2 is preceded by QH2 oxidation.

Conditions in which SC2 could take place are created not only when an inhibitor such as antimycin, blocks electron transfer from heme bH to Q at the Qi/Qn site, but also during reverse electron transfer from QH2 bound at the Qi/Qn site, which is a natural consequence of the physiological reverse catalysis of Cyt-bc. It has been demonstrated (see section 8), that electrons from the Qi site of one monomer can be transferred through the heme bL to the other monomer, creating the possibility that heme bL is already reduced when SQ -is generated during oxidation of QH2 by 2Fe2S at the Qo site.

The third type of short circuit (SC3) is electron transfer from reduced heme bL/bp to Q that is bound concurrently with QH2 to the Qo/Qp site, when the oxidized 2Fe2S is close to the Qo/Qp site.

SC3 consists of two sequential reactions: first uphill (transition form states [c] to [b]), followed by downwhill ET (transition from [b] to [e] in fig. 28) which can be summarized as follows:

bL red + Q = bL ox + SQ - SQ -+ 2Fe2S ox = Q + 2Fe2S red
SC3 is quite similar to SC2 and the proton transfer efficiency is the same. The only difference is the fact that SC3 should be favored over SC2 when concentrations of Q in the Q(H2) pool are higher. However, in the absence of an inhibitor blocking the Qi/Qn site or mutation retarding electron transport from heme bH/bn to the Qi/Qn site, SC3 seems to be less probable than SC2.

The last type of short circuit (SC4) is the two-electron reduction of Q at the Qo/Qp site by the hemes bL/bp and bH/bn of the low-potential chain without involvement of the 2Fe2S. SC4 can be summarized as follows:

Q + bL red = SQ -+ bL ox bH red + bL ox = SQ -+ bH ox + bL red SQ -+ bL red = QH2 + bL ox According to the modified Q cycle [START_REF] Crofts | The Role of the Quinone Pool in the Cyclic Electron-Transfer Chain of Rhodopseudomonas Sphaeroides: A Modified Q-Cycle Mechanism[END_REF] , SC4 is obviously considered as the energy-wasting reaction since it diminishes the number of protons transferred across the membrane. However, calculations of the number of protons that are transferred per oxidized QH2 under condition in which SC4 takes place is more complex than in the case of SC1, SC2 and SC3.

Let us first consider 4 cycles of QH2 oxidation steps according to the energy-conserving Q cycle. If the modified Q cycle is operating, this will lead to 8H + released to the p side, 4H + taken from the n side and the net consumption of 2 QH2 molecules from the Q(H2) pool. The overall stoichiometry of this modified Q cycle is +8H + p/-4H + n and -2QH2/+2Q in the Q(H2) pool.

Now consider a similar sequence of 4 QH2 oxidation reactions but with a single SC4 reaction occurring in the middle of the process: 2 QH2 oxidations -SC4 reaction -another 2 QH2 oxidations.

For clarity let us divide the process into three parts. In the first part, 2 QH2 oxidations yield +4H + p/- 2H + n and -1QH2/+1Q. During the second part SC4 gives -2H + p/+2H + n and +1QH2. During the third part another 2 QH2 oxidation take place yielding the same +4H + p/-2H + n and -1QH2/+1Q as in the first part. Addition of all products from these three parts gives a net stoichiometry of +6H + p /-2H + n and -2QH2/+2Q in the Q(H2) pool, which means that 1 occurrence of SC4 per 4 oxidized QH2 decreases the number of protons released to the p side from 8 to 6. This would give an average +3H + p/-1H + n per 1 QH2 oxidized during this sequence vs +4H + p/-2H + n per 1 QH2 in the Q-cycle.

7.4.2

The possible physiological meaning of short-circuits

In general, short-circuit reactions decrease the efficiency of energy conversion by Cytbc and the probability of their occurrence should be minimized or slowed down by two or three orders of magnitude in comparison to the productive forward and reverse reactions. It must be emphasized however, that it is still possible for some of the SC reactions to be catalyzed under specific conditions, such as severe mutation or presence of inhibitors which preclude normal catalysis. In fact it has been proposed that the reactions of SC2 could be a potential mechanism to support photosynthetic growth of green alga Chlamydomonas reinhardtii strains, in which normal function of Cytb6f is disabled by mutation to replace the heme bH ligand -histidine 202 with glutamine 616 .

The reactions of SC4 could also be potentially considered as desirable for electron recirculation back to the PQ(H2) pool in, for example, photosynthetic CET at the level of Cytb6f (see section 4.3). Assuming alternating reactions of oxidation of PQH2 and reduction of PQ at the Qp site, using electrons delivered through the low-potential chain from the Fd/NADPH + pool, the overall proton pumping efficiency would be significantly increased yielding +6H + p/-4H + n, -1PQH2 in the Q(H2) pool and -2 Fd from the Fd/NADPH + pool. This would contribute to a much higher proton gradient per oxidized PQH2 than +4H + p/-2H + n per 1 PQH2 resulting from the modified Q cycle mechanism. However, a possible involvement of SC4 in the mechanism of CET around Cytb6f and PSI has not been proposed earlier and thus has never been tested experimentally.

Another potential physiological implication of short-circuits relates to the concept of kinetic competition between short-circuits and the leak of electrons (i.e. reactions that may lead to superoxide production). The completion of any type of short-circuit reaction retains electrons within the enzyme, therefore diminishing the probability of a leaks. Thus, this could be considered as a possible means of protection against unwanted release of superoxide, which might be of importance in the context of a postulated signaling role for superoxide release from the Qo/Qp site (see section 7.5).

Short-circuit suppression mechanisms

As indicated in section 7.4.1, understanding the fast reversibility of electron transfer in Cytbc and the efficient suppression of short circuit reactions is conceptually challenging 259,615,617,618 .

Direct electron transfer between the low-and high-potential chains is significantly limited by the 2.3nm distance between 2Fe2S and the ring of heme bL/bp 259 . Such a large spatial separation slows down electron transfer to a timescale of seconds, minimizing the probability of its occurrence on a catalytic time scale. However, the problem appears when a redox active molecule such as quinone (QH2, Q or SQ -) is bound at the Qo/Qp site and cuts the 2.3-nm distance into two much shorter distances, each being well below the 1.4 nm limit [START_REF] Page | Natural Engineering Principles of Electron Tunnelling in Biological Oxidation-Reduction[END_REF] . This means that any type of quinone molecule bound in the site (whether it is the substrate, product or intermediate of the QH2 oxidation) can in principle act as mediator, to significantly enhance electron transfer between the low-and high potential chains.

To explain the general lack of experimentally detected short circuits several "gating" mechanisms were constructed. The problem is however, that in most cases the gating mechanisms are either very effective at suppressing short-circuits, therefore sacrificing reversibility, or if they allow reversibility they are prone to some short circuits 259,615 . It appears that to explain how the Cytbc can accomplish full and rapid reversibility and short circuit suppression while limiting superoxide production, elaborate mechanisms involving more than a single gating factor are needed.

The necessity for elaborate gating is a consequence of the sequential mechanism that is considered to drive the reaction at the Qo/Qp site, which is obliged to form SQ -as a true intermediate of the two-electron reactions of bifurcation. This mechanism assumes formation of SQ -at the Qo/Qp site, albeit at low occupancy 224 . It is generally in line with many observations that the Qo/Qp site can, under specific conditions, generate superoxide and also with recent experiments reporting detection of a SQ -intermediate at this site (see section 7.5 and 7.6 and references there). The sequential mechanism is also in agreement with the chemical properties of quinones and their ability to take part in single-electron redox reactions. However, the major problem with sequential electron transfer is its susceptibility to short-circuiting.

As an attractive alternative and seemingly the simplest solution to the short-circuit problem, a concerted reaction was also proposed for the Qo/Qp site. This reaction, based on a fundamentally different mechanism, assumes that the reaction takes place through a transition state devoid of semiquinone character. In the following sections we will first reflect on the concerted mechanism before discussing the various possible gating mechanisms related to the sequential mechanism of the bifurcation.

Short-circuit-proof and leak-proof concerted reaction

The "concerted" reaction initially proposed for the Qo/Qp site is 'thermodynamic' cooperativity of the bifurcation reaction, in which electron transfer is sequential, but with a transient semiquinone state that never accumulates enough to be observable (i.e. reduction and oxidation of reaction partners may appear concurrent) 155,619,620 . Together with the kinetic resolution of millisecond reversibility of all electron transfer steps within Cytbc1, including the reactions at the Qo/Qp site, the "concerted" reaction was introduced as one of the possible mechanism allowing reversible operation of this site while preventing all short-circuits (the other mechanism introduced in parallel was a "double-gating", as described below) 259,615 . The term "concerted" was regarded as a "kinetic" definition of two events taking place without time for significant atomic rearrangement. This concept recognized that all short-circuits would be efficiently suppressed if QH2 oxidation (forward catalysis)

and Q reduction (reverse catalysis) took place as a virtually single step involving 2 electron and 2 proton transfers. Suppression would then simply be a consequence of forbidding one-electron transfer reactions, of which all short-circuits are composed (see Fig. 29) 155,259,615,619,620 .

It follows that if the concerted reaction is the catalytic mechanism at the Qo/Qp site, the SQ - intermediate would not be formed at all during either QH2 oxidation or Q reduction as its lifetime is shorter than the time needed for nuclei reorganization of molecules taking part in the electron transfer reactions 259 . This mechanism elegantly explains the energetic efficiency of the reversible catalysis of Cytbc1, but it remains hypothetical. While theoretical and experimental descriptions of kinetically concerted two-electron transfer reactions do exist 621,622 , the chemical and physical details of the any concerted reaction at the Qo/Qp site remain unknown, as is the mechanism that would explain how SQ -formation is forbidden.

The concerted mechanism becomes difficult to reconcile with recent experiments reporting the detection of transient SQ -intermediates in the Qo/Qp site (see section 7.6). As, the concerted reaction is by definition short-circuit-proof and leak-proof, it eliminates entirely the possibility of superoxide generation. It follows, that another mechanism of SQ -formation by Cytbc would therefore have to be envisaged to explain this activity of Cyt-bc, such as electron transfer from reduced heme bL/bp to O2, for which there is no evidence.

Gating mechanisms of sequential reaction

i) Catalytic switch and QH2/SQ/Q mobility-driven gating at the Qo site

The "catalytic switch", proposed by Brandt et al. before the discovery of 2Fe2S motion can be considered the first gating model [START_REF] Brandt | Analysis of Inhibitor Binding to the Mitochondrial Cytochrome c Reductase by Fluorescence Quench Titration. Evidence for a "catalytic Switch" at the Qo Center[END_REF][START_REF] Brandt | Significance of the "Rieske" Iron-Sulfur Protein for Formation and Function of the Ubiquinol-Oxidation Pocket of Mitochondrial Cytochrome c Reductase (Bc1 Complex)[END_REF] . This model pointed to a key role of the redox state of the 2Fe2S on the bifurcation reaction. It was formulated based on the difference between the binding constants of two types of inhibitors, stigmatellin and MOA-stilbene to Cytbc1, caused by changes in the reduction state of the 2Fe2S cluster and the discovery that ISP-HD can be found at two different positions. The model assumed that QH2 and Q can interact with oxidized 2Fe2S while SQ -interacts with oxidized heme bL but not with the reduced 2Fe2S cluster. Thus, electron transfer to 2Fe2S or heme bL strictly depends on the redox state of these two cofactors forcing the forward bifurcation reaction at the Qo site [START_REF] Brandt | Analysis of Inhibitor Binding to the Mitochondrial Cytochrome c Reductase by Fluorescence Quench Titration. Evidence for a "catalytic Switch" at the Qo Center[END_REF] .

A somewhat similar model was proposed recently to explain the forward electron transfer in bifurcation through the movement of SQ -at the Qo site, to drive electrons to the correct chain. This model was also based on structural studies of stigmatellin binding to the Qo site [START_REF] Crofts | Mechanism of Ubiquinol Oxidation by the Bc1 Complex: Different Domains of the Quinol Binding Pocket and Their Role in the Mechanism and Binding of Inhibitors[END_REF] . It predicts that reduction of the 2Fe2S cluster and formation of semiquinone leads to disruption of the H bond between Rh H156 ( Bt H161) which favors the movement of ISP-HD to the c-position (see section 3.4

for details) and a rapid movement of the benzoquinone ring toward the Rh E295 ( Bt E271) 119,575 . The latter movement facilitates deprotonation of the semiquinone and therefore increases the rate of electron transfer from SQ -to heme bL by shortening the distance between the reagents [START_REF] Crofts | The Mechanism of Ubihydroquinone Oxidation at the Qo-Site of the Cytochrome Bc1 Complex[END_REF]596 . After the second oxidation, Q returns to the niche that is close to the 2Fe2S cluster. In this way the redox state of Q and heme bL determine the direction of electron transfer at the Qo site. These two mechanisms:

termed "catalytic switch" and "SQ motion" do not protect the enzyme from performing SC2 or SC4.

Furthermore, they do not describe the proton/electron sequence of the reverse direction and, as discussed in 259,615 , they are not easily reversible.

ii) Surface-modulated motion switch of Cytbc1

Another gating mechanism proposes that the movement of ISP-HD after the 2Fe2S cluster is reduced is coupled to the oxidation of heme bL by heme bH [START_REF] Xia | Structural Analysis of Cytochrome Bc1 Complexes: Implications to the Mechanism of Function[END_REF][START_REF] Esser | Surface-Modulated Motion Switch: Capture and Release of Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF][START_REF] Cen | Domain Movement of Iron Sulfur Protein in Cytochrome Bc 1 Complex Is Facilitated by the Electron Transfer from Cytochrome b L to b H[END_REF]623 . The authors proposed that electron transfer from heme bL to heme bH triggers some conformational changes in the region of Cytb 89 responsible for docking the ISP-HD, releasing this domain from the Qo site and then releasing Q and binding QH2. [START_REF] Esser | Surface-Modulated Motion Switch: Capture and Release of Iron-Sulfur Protein in the Cytochrome Bc1 Complex[END_REF] . Although, this mechanism allows the reversible reactions at the Qo site, some short-circuit reactions (SC2, SC3 and SC4) are still possible. Furthermore, this model predicts, that the presence of Q at the Qo site would favour occupation of positions remote from the Qo site by the ISP-HD. However, this seems at odds with the experimental observation that when Q is bound at the Qo site it leads to EPR spectra typical of 2Fe2S interacting with Q for the whole population of the reduced 2Fe2S (see section 3.4 for details).

iii) Proton gated affinity change and proton gated charge-transfer mechanisms

The "proton gated affinity change" mechanism was formulated on the basis of the electrochemical properties of 2Fe2S, in particular the difference between the pK value of the Rh H156 ( Bt H161) for the reduced and oxidized 2Fe2S 574 . According to this mechanism, either QH2 binds to the deprotonated histidine or QH -binds to the protonated histidine. The first electron transfer to 2Fe2S then drastically increases affinity of SQ -for the reduced 2Fe2S, leading to a positive shift in the redox potential of the 2Fe2S in the 2Fe2S red -H + -SQH complex. Such a stabilization should lead to a relatively large occupancy of SQ -at the Qo site. However, when this proposal was framed, experiments had thus far failed to detect any SQ -at the Qo site. This discrepancy was explained on the ground of strong antiferromagnetic coupling between SQ -and 2Fe2S red leading to disappearance of the EPR signal.

Evoking antiferromagnetic coupling as a reason for the failure to detect an EPR signals implies that the energy of such interactions must be very high to depopulate the paramagnetic, excited state at temperatures > 10 K (typical experimental conditions). However, this kind of strong spin-spin exchange is very unlikely for most bonds through which SQ -could interact with 2Fe2S, including a single H bond. Interestingly, experiments performed 16 years after the initial formulation of the "proton gated affinity change" mechanism established that a spin-spin exchange interaction between SQ -and reduced 2Fe2S does occur, but that the energy of such coupling is very low in comparison to the thermal energy of experimental EPR conditions; and that this state can be detected by EPR (this is described in detail in section 7.6).

As described in section 7.2, several experimental results have been interpreted as being indicative of binding two substrate molecules to the Qo site at the same time. This concept was also used to formulate a "proton gated charge-transfer mechanism" in which a QH2/Q pair must first undergo two deprotonation steps followed by electron transfer to 2Fe2S and heme bL 586 . The two proton-gated mechanisms allow reversibility of the Qo site but do not prevent SC2, SC3 nor SC4.

iv) Double-gating mechanism

More recent models of bifurcation take into account the fact that a single gating step is generally not sufficient to suppress all types of short-circuits for reversible sequential bifurcation.

The first model of this type was the "double gating" mechanism, introduced as an alternative to the concerted mechanism in providing a means for short-circuit-proof and efficient reversible operation of the Qo site. It evoked the concept that changes in protonation/deprotonation of water molecules and/or amino acid side chains near the Qo site occur upon changes to the redox state of immediate cofactors, 2Fe2S and heme bL, in order to specifically modulate barriers in the sequential reaction 259,615 . Double gating allows QH2 oxidation only when both 2Fe2S and heme bL are oxidized and allows Q reduction only when both 2Fe2S and heme bL are reduced. This would be accomplished by raising the barrier for SQ -formation when QH2 is bound at the Qo site (involving a SQ -/QH2 couple) when heme bL is reduced and the 2Fe2S cluster is oxidized, or by lowering that barrier when both cofactors are oxidized. This would prevent SC1 and SC2 (Fig. 29). On the other hand, the barrier for SQ formation when Q is bound at the Qo site (involving Q/SQ -couple), would be raised when heme bL is reduced and the 2Fe2S cluster is oxidized, or the same barrier is lowered when both cofactors are reduced. This would prevent SC3 and SC4.

v) Logic-gating mechanism

A "logic-gating" mechanism is quite similar to the "double gating" mechanism. It also evokes a concept of changes in protonation/deprotonation of water molecules and/or amino acid side chains near the Qo site upon changes in the redox state of the immediate cofactors 2Fe2S and heme bL. The model proposes that crucial elements for energy-conserving redox reactions depend on specific Hbond formation in the Qo site 597 . Binding of QH2 requires Rh H156( Bt H161) and Rh E295( Bt E271) to be in deprotonated states, which is possible when 2Fe2S and heme bL are oxidized. Otherwise, reduction of 2Fe2S and heme bL induces protonation of the Nτ atom of the histidine and the carboxyl group of the glutamate, allowing binding of Q to the Qo site. Such a mechanism imposes constraints on the direction of reaction, as QH2 binds predominantly when both 2Fe2S and heme bL are oxidized and Q binds predominantly when both 2Fe2S and bL are reduced. This mechanism, like the double-gating mechanism, protects against all short-circuits while still allowing reversibility 259 . It is also generally consistent with the fact that reduction of 2Fe2S is associated with a shift in the pK value of the 2Fe-2S protein from about 7.6 to more than 10 594 and is in line with the observation that Q bound at the Qo site strongly influences EPR spectra of the 2Fe2S (see section 7.2 for details). In this model a tentative coupling between hemes bL and the Glu residue exists and thus the reduction of heme bL would be associated with protonation of the Glu carboxylic group while oxidation of the heme would induce deprotonation of the same side chain. However, experimental results on a series of mutants that replace Rh E295( Bt E271) with other amino acids revealed only minor effects on the redox midpoint potential of heme bL 579,598 . Also, equilibrium redox titrations of the bacterial chromatophore Q(H2) pool, made by monitoring changes in the gx EPR transition of the 2Fe2S, showed that the reduced cluster is still able to interact with QH2 under conditions in which heme bL remains oxidized. The experimental detection of Q bound to the Qo site under conditions of reduced heme bL is not feasible due to large difference in Em values of the Q/QH2 couple and heme bL.

In this mechanism water dipoles are proposed to be the crucial elements that stabilize Q or QH2 binding when 2Fe2S and heme bL are either both reduced or both oxidized, respectively.

vi) Coulombic gating.

When the rapid reversibility of reactions at the Qo site was established 136 , it became clear that the "SQ -movement" mechanism (see above) does not explain gating, nor does it prevent all types of short-circuit. In an attempt to modify this mechanism to accommodate reversibility and the prevention of all short-circuits "Coulombic gating" was proposed 596 . The crucial element of this model is an assumption that SQ -is attracted by Coulombic interactions, and thus moves toward the oxidized heme bL, so increasing the electron transfer rate. On the other hand, when SQ -is formed while heme bL is reduced, then it is repelled, moving toward the 2Fe2S and decreasing the probability of electron transfer from heme bL to SQ -. Although, this mechanism seems to explain the limited efficiency of short-circuit SC2, it is still not clear how it would allow rapid reversibility of the Qo site.

Current status and emerging concepts of short-circuit suppression. Despite many proposals and the various models outlined above, the exact mechanism of gating and reversibility at the Qo site that minimizes energy-wasting reactions is still not known. It appears that each of the mechanisms presented is able to explain the bifurcation reaction under certain specific conditions, but at the same time they may not be sufficient to explain observations under other conditions, or to provide a detailed molecular basis, or be fully consistent with the ensemble of existing experimental data. Our intention in this review is not to favor any particular mechanism. Rather, in view of their diversity and general complexity, and also given that no obvious elements unifying them into one common mechanism can be proposed, we argue that the bifurcation at Qo site is far from being satisfactorily understood and still deserves extensive experimental and theoretical exploration. This is particularly the case for the Cytb6f, where the situation is further complicated by the possible injection of an additional electrons from the n side through the c-heme to heme bn.

It is worth hypothesizing that the "true" mechanism of energy-conserving reversible bifurcation could be a process to which several of the many different gating mechanisms proposed by different authors could contribute, depending on the starting conditions. We also anticipate that the transition from forward to reverse electron transfer at the Qo site may not be a simple reversal of the proton/electron transfer, and that dynamic events that start the reverse reaction may differ from the final step of forward reaction. It could very well be that the stability constant of the SQ -formed during oxidation of QH2 is different from the stability constant of SQ -formed by reduction of Q. We believe that the key players are likely to be protons, as different proton configurations could favor different stabilities of the SQ -formed during froward and reverse reactions. However, the proton events and paths for uptake and release have not been resolved yet.

Superoxide generation at the Qo/Qp site

The operation of any electron transfer chain (ETC) in the presence of oxygen is connected to the generation of superoxide radical. In vivo, superoxide is quickly converted into H2O2 in a reaction catalyzed by superoxide dismutase. This is presumably why first mentions of ETC-derived radicals were related to H2O2 and not superoxide. Indeed, the first information about reactive oxygen species (ROS) in mitochondria showed mitochondrial levels of H2O2 at 1-2% of total O2 uptake in state 4 of respiration (the state not coupled to ATP synthesis)while the rate of H2O2 generation in state 3 of respiration (the state associated with ADP to ATP conversion) was negligible 624,625 . It was also noticed that antimycin enhances production of H2O2 in intact mitochondria 626,627 .

Semiquinone at the Qo/Qp site as electron donor to molecular oxygen The first reports on superoxide production in antimycin-treated submitochondrial particles to suggest that ROS are Cytbc1-derived appeared in Boveris and Cadenas work in 1975 628-630 . Over the next few years, it was established that the origin of ROS in antimycin-inhibited Cytbc1 of submitochondrial particles is the redox reaction in the UQH2 oxidation site 254,631 . Using Cytbc1 isolated from yeast, it was also shown that inhibition with antimycin A as well as with myxothiazol which binds to the proximal niche of the Qo site, leads to production of ROS at the Qo site 632,633 . The link between superoxide production and the action of Qo/Qp site is consistent with the widely-accepted notion that a highly unstable SQ -at the Qo site (SQo) is a direct one-electron donor to oxygen 224 .

In view of the reversibility of the catalytic reactions in the Qo site, there are two possible reactions that can lead to the formation of semiquinone at this site: one electron oxidation of QH2 by the oxidized 2Fe2S cluster (the "semiforward' reaction) 632,634 or one electron reduction of quinone by heme bL (the "semireverse" reaction, 635,636 . It therefore follows that there are two possible reaction sequences leading to ROS production by the Qo site. These sequences involve either semiforward or semireverse reaction followed by electron transfer from the formed SQ -to molecular oxygen. Both sequences require the initial presence of reduced heme bL which either blocks the heme bL from accepting an electron from SQ -formed via the semiforward reaction or donates an electron back to Q bound at the Qo site, forming SQ -via the semireverse reaction. This requirement is generally consistent with the observation that ROS are detected when electron transfer between heme bL and heme bH is impeded by blocking the Qi site with antimycin A or by an increase in membrane potential 572,637 .

In the currently discussed mechanisms of ROS production by the Qo/Qp site, both reaction sequences (i.e. involving semiforward or semireverse reaction for semiquinone formation) are taken into account. Some models propose that only one from these two sequences is responsible for ROS production, while other models consider a contribution from both. Indeed, it seems plausible that the detected ROS may be a combined product of both mechanisms, but that their relative contributions change depending on the experimental conditions and/or introduced electron transfer barriers within cofactor chains.

The specific rate of superoxide production in Cytb6f, normalized to the rate of electron transport, is more than an order of magnitude greater than that measured in isolated yeast respiratory Cytbc1 complex. [START_REF] Baniulis | Mechanism of Enhanced Superoxide Production in the Cytochrome b6f Complex of Oxygenic Photosynthesis[END_REF] The higher rate of superoxide production in Cytb6f could be a consequence of an increased residence time of plastosemiquinone/plastoquinol in its binding niche near ISP-HD, resulting from (i) occlusion of the quinone portal by the phytyl chain of the unique bound chlorophyll, (ii) an altered environment of the proton-accepting glutamate believed to be a proton acceptor from semiquinone, or (iii) a more negative redox potential of the heme bp on the electrochemically positive side of the complex. [START_REF] Baniulis | Mechanism of Enhanced Superoxide Production in the Cytochrome b6f Complex of Oxygenic Photosynthesis[END_REF] "The semireverse-Rieske off" model of superoxide production

The mechanism involving the semireverse reaction (heme bL to Q electron transfer) is a relatively new phenomenon that emerged from the observation that ROS production exhibits a belllike shape dependence on the redox state of the Q(H2) pool. This means that maximal production of ROS occurred when the pool was partially oxidized, which was taken as an indication that the semireverse reaction, for which the substrate is Q (not QH2), is involved in superoxide generation 120,636 .

A contribution of the semireverse reaction in ROS generation was also established in studies using mutated forms of the bacterial Cytbc1, which introduced limitations in electron transfer at the level of different cofactors within the low and high potential chains 107,120,635 . Furthermore, these studies revealed an important constraint associated with the kinetic effects of the motion of ISP-HD.

In the proposed model, production of radicals depends on the position of the ISP-HD with respect to the Qo site. Mutants with the insertion of a single or two alanine residues (+1Ala, or +2Ala, respectively) at the neck region of ISP, which increases the probability of ISP-HD being located in close proximity to the Qo site [START_REF] Sarewicz | Magnetic Interactions Sense Changes in Distance between Heme bL and the Iron-Sulfur Cluster in Cytochrome Bc1[END_REF]107 , were found to be less prone to ROS production in antimycin A inhibited Cytbc1 107,635 (Fig. 30). To explain this observation, it was proposed that molecular oxygen competes kinetically for electrons with cofactors of the Qo/Qp site. When ISP-HD is present at the Qo/Qp site during SQ -formation, the probability of electron transfer between the 2Fe2S and SQ -is high. The reduced 2Fe2S can then reduce the SQ -, reforming QH2 and completing the reverse reaction, while the oxidized cluster can accept an electron from SQ -and completing short-circuit reaction (SC3 in Fig. 29). However, when ISP-HD occupies positions remote from the Qo/Qp site at the time of SQ - formation, the rate of the electron transfer between SQ -and the 2Fe2S decreases. Under these circumstances, the probability that molecular oxygen outcompetes 2Fe2S for electrons from the SQ - increases, which leads to the release of detectable levels of superoxide from the Qo/Qp site.

Initial studies on the +2Ala mutant were not able to discriminate between the possible contributions of semiforward or semireverse reactions in ROS generation 635 . In addition to the suppression of ROS generation in the 2Ala mutant, this study described another mutant, Rh M183L, that changes one of the axial ligands of heme c1, shifting the Em of this heme by more than -300 mV.

This introduced a large barrier to electron transfer between 2Fe2S cluster and heme c1 without affecting the motion of ISP-HD, causing an increase in ROS production. Given that ROS production is also enhanced when electron transfer barrier is introduced at the level of the low potential chain (addition of antimycin A, a mutation disturbing the oxidation of QH2 at the Qo site, Sc E272Q ( Rh E295Q) 598 or mutations to change the axial ligands of hemes b, Rh H198N and Rh H111N 638 ), it appears that in general any significant asymmetry in the overall rate of electron flow between the high-and low-potential chains might make the enzyme more prone to generate superoxide. One exception to this is if it is caused by arresting the ISP-HD at the Qo/Qp site. Again, these studies could not discriminate between the relative contribution of semiforward or semireverse reactions to ROS generation by the Qo/Qp site.

Such discrimination became possible in subsequent studies with the use of the 1Ala mutant and in combination with Rh M183K (equivalent of Rh M183L described above). These studies clearly pointed toward the semireverse reaction as the dominant contributor in this process 107 . Mutational studies analyzing the molecular effects of mitochondrial disease-related mutations further substantiated the inclusion of both postulates (i.e the one related to involvement of the semireverse reaction in SQ -formation and the other related to the probability of ISP-HD position-dependent interaction of SQ -with oxygen) in what can be referred to as the semireverse-Rieske off model of ROS generation by the Qo/Qp site.

The Rh G167P mutation in Rhodobacter capsulatus is an equivalent of human mitochondrial S151P mutation in Cytb which was found in patients with exercise intolerance 639 . This mutant exhibits enhanced production of ROS even without antimycin A 120 . In contrast to +nAla insertions, Rh G167P influences the motion of ISP-HD in such a way that it tends to occupy positions more remote from the Qo site in comparison to WT (see section 3.4) This effect, in agreement with the semireverse-Rieske off model, increases the probability of SQ reaction with oxygen (Fig. 30). Furthermore, introduction of the +1Ala mutation to the Rh G167P mutant partially compensated the effect of Rh G167P, which resulted in diminished ROS production, again consistent with this model. The Rh G332D mutation, an equivalent of another mitochondrial disease-related mutation G290D

(associated with exercise intolerance in humans 640 ), was also found to influence motion of ISP-HD.

However, this influence is less pronounced compared to Rh G167P, and accordingly the effects on ROS production are more subtle 121 , (Fig. 30).

As introduced in section 7.4.1 the kinetic competition between the leaks of electrons and short-circuit reactions, inherent also to the semireverse-Rieske off model, might be of physiological importance. In general, both reactions lead to a drop in the efficiency of electron bifurcation at the Qo site, but while short-circuits retain electrons within ETC, leaks generate ROS.

Assuming that high production of ROS is deleterious, in some cases short-circuit reactions may play a beneficial role in lowering of ROS generation. For various enzymes involved in bioenergetics it has been postulated that such energetic loss, which minimizes ROS-related damage,

gives the organism a better chance of survival 641 . On the other hand, ROS are also postulated to play a redox signaling role 642 , thus careful balancing of short-circuits and leaks must be considered as one of the possible elements of the redox signaling/control systems.

When disabled, the Qi/Qn site does not completely inhibit the function of the Qo/Qp site, and the enzyme can still sustain electron flux through the ETC. This residual flux is presumably possible via short-circuit reactions and electron leaks. In Cytbc1 that flux is low enough to make the enzyme non-functional. Interestingly, in Cytb6f, a residual flux can sustain the entire photosynthetic chain with the short-circuits that were proposed to act as an "emergency exit" pathway bypassing the Qcycle and making it dispensable 616 .

Other postulated reactions involving molecular oxygen. In addition to the reaction of molecular oxygen with SQ -formed at the Qo/Qp site discussed above, it was also proposed that oxygen might act as a redox mediator between QH2 and heme bL 643 . According to this proposal, heme bL would accept an electron from QH2 via protonated superoxide (HOO) at the Qo site, which would facilitate electron transfer within the low potential chain. This, so far purely hypothetical scenario was introduced to explain the observed enhanced enzymatic activity of Cytbc1 in the presence of oxygen, compared to its activity under anaerobic conditions, MD simulations showed that O2 molecules spontaneously diffuse into the Qo site of Cytbc1 and can react with SQ -while forming superoxide 644 . Molecular oxygen may also occasionally get within 5 Å of the central iron atom of the hemes bL and bH. Its presence near the heme bL raises the question of whether this heme is also an immediate electron donor in the production of superoxide.

Indeed, this hypothesis has never been definitively ruled out, and although much less popular compared to models based on SQ -, can also be found in the literature 643 . It is based on the rationale that the redox potential of heme bL is sufficiently low to make electron transfer to oxygen theoretically possible 645 .

In A separate question that can be found in the literature is whether the Qi/Qn site is also able to produce ROS 646 . Under high concentrations of QH2 and in the presence of an inhibitor of the Qo site, it is possible to generate a stable SQ -intermediate originating from the reverse reaction at the Qi site of Cytbc1 (see section 8.3) 151,647,648 . If Qi/Qn-produced ROS have physiological significance, it should be exposed under such conditions. However, measurements conducted in the presence of stigmatellin -a Qo/Qp site inhibitor, did not show statistically significant production of radicals in such a system 633 . As discussed in section 8 Qi-derived SQ -can be stabilized via hydrogen bonding to prevent potential reaction of this SQ -with molecular oxygen, as was shown for solution-generated SQ -, which was found to be stabilized through hydrogen bonds with the acceptor solvent 253 .

Physiological considerations. The currently available molecular mechanisms of superoxide generation by Cytbc1 are derived mostly from analysis of conditions where electron flow through the cofactor chains was severely impeded by various experimentally imposed barriers (inhibitors, mutations). This raises the question whether the natively operating enzyme contributes to ROS generation in living cells, and if so how it does this. Despite some reports that uninhibited Cytbc1 may produce trace amounts of radicals [START_REF] Baniulis | Mechanism of Enhanced Superoxide Production in the Cytochrome b6f Complex of Oxygenic Photosynthesis[END_REF]572,649 , there are no conclusive data to answer this question in a comprehensive manner. In our view further progress requires development of new spectroscopic approaches that not only allow experiments to be conducted in conditions closer to those encountered by the enzyme in the cells, but also provide high molecular and kinetic resolution of specific electron transfer steps. Nevertheless, several important physiological aspects of cytochrome Cytbc1-related ROS production by ETC are worth discussing.

It is known that production of ROS is dependent on the respiration rate of mitochondria [START_REF] Adam-Vizi | Bioenergetics and the Formation of Mitochondrial Reactive Oxygen Species[END_REF]650 .

Factors enhancing ROS production in mitochondria include high pmf, a high NADH:NAD + ratio, high QH2/Q ratio, low local O2 concentration and nearly inactive ATPase [START_REF] Adam-Vizi | Bioenergetics and the Formation of Mitochondrial Reactive Oxygen Species[END_REF]637,[650][651][652] . As a rather general rule, the more reduced the cofactors of ETC are, the more prone ETC is to radical formation.

Factors lowering production of ROS include high activity of uncoupling proteins which decrease pmf 653 . High respiration rate also lowers the probability of ROS production [START_REF] Adam-Vizi | Bioenergetics and the Formation of Mitochondrial Reactive Oxygen Species[END_REF]650 .

ROS generation is usually expressed in units of s -1 (as the amount of superoxide molecules produced by ETC complex in one second) or as a percentage of ROS production (as % of ETCderived electrons that pass to oxygen forming superoxide). In state 3 of respiration (upon the addition of ADP) when the rate of oxygen consumption is maximal, the fractional ROS production is negligible. However, in state 4 of respiration (when the ADP levels are depleted) and where respiration chain is slowed down, production of ROS is at the level of 1-2 % 624 . Interestingly, expressing production in s -1 shows that the rate of ROS production in state 3 might be even higher than in state 4 654 .

The possible influence of physiological factors on ROS generation by Cytbc1 is perhaps best exemplified by the effects of the membrane potential. In its absence, heme bH is the preferred electron acceptor within the pair of hemes bL -bH 655 . In its presence, the apparent midpoint potentials of these two hemes become similar, leading to a more equal distribution of electrons between them 573 .

Consequently, the reduction level of heme bL increases which, as observed experimentally, is expected to lead to ROS production by the Qo site 573 .

However, a description of dynamic changes in electron distribution between the b hemes appears to be a more complex issue when possible communication between monomers in the dimer occurring at the level of the heme bL-bL bridge, is considered (see section 9). Any significant electron transfer between monomers is expected to decrease the reduction level of the b hemes (including heme bL) which might decrease the levels of ROS released from the Qo/Qp site, in particular if the semireverse-Rieske off mechanism is considered.

The physiological role of superoxide released by the components of ETC remains an open issue, especially in view of the suggestions that it may be one of the components of cellular redox signaling [START_REF] Sena | Physiological Roles of Mitochondrial Reactive Oxygen Species[END_REF] . Among four ETC complexes (Complex I -Complex IV) it is commonly acknowledged that only two of them generate the majority of ROS produced during the operation of the ETC:

Complex I and Complex III (reviewed in 650,656 ). These two complexes release superoxide into different compartments of mitochondrion: Complex I frees superoxide into the matrix, while

Complex III-releases it into the intermembrane space (IMS). ROS in the IMS are more easily accessible to the cytosol compared to ROS in the matrix, as the former need to cross only the outer mitochondrial membrane while the latter have to cross both inner and outer mitochondrial membranes 657 .

It should be emphasized that consideration of the role of ROS in cellular signaling should always account for the fact that the level of ROS released into the cytosol from mitochondria is the net result of ROS production and its scavenging inside the organelles.

Superoxide generated in the matrix is efficiently converted into H2O2 by the highly abundant MnSOD 627 and H2O2 is further consumed by catalase (CAT) and glutathione peroxidase (Gpx).

Estimates indicate that H2O2 is kept at a low level in the matrix, around 5x10 -9 M 658 . Supposedly, matrix-generated superoxide and H2O2 from its dismutation do not leave the matrix 657 and thus any putative signal derived from complex I is probably restricted to this compartment.

ROS signaling communication between the mitochondria and cytosol becomes more plausible for superoxide generated by complex III and released to IMS 642,659 .

In the IMS superoxide can be transformed by CuZnSOD to H2O2 or, theoretically, scavenged by Cytc. The latter reaction, in view of the ratio between superoxide and the high physiological concentration of Cytc in the IMS (estimated to be in the range of 1 mM 660 ), raises concern about whether the lifetime of superoxide released to IMS would be sufficient to carry out any meaningful signaling purposes. However, the probability of superoxide oxidation by Cytc may actually not be that high considering the rather low ratio of Cytc to Cytbc1, (around 2:1 204 , as estimated based on the optical spectrum of reduced mitochondria 254,661 ) and the fact that Cytc pool is almost fully reduced in state 3 or 4 of respiration 662 . While superoxide cannot pass efficiently through the membrane, it can escape mitochondria via voltage-dependent anion channels 663 . After spontaneous or CuZnSODdriven dismutation, superoxide is converted into H2O2, and in this form it can reach cytosol via diffusion much more readily. When overactivity of CuZnSOD in the IMS is observed, this leads to overproduction of H2O2, which in turn may lead to the formation of oxoferryl Cytc. This may result in the oxidation of cardiolipin and turn on early pro-apoptotic processes 664 .

Interestingly, until now no one has succeeded in detecting superoxide evolution from Cytbc1 by spin-trapping. This seems to stem from the methodological difficulty related to the large difference between rate constants for the superoxide reaction with Cytc (k = 2.5x10 5 M -1 s -1 , 665 ) and the EPR spin trap DMPO (k = 10 M -1 s -1 , 666 ). DMPO falls behind oxidized Cytc when competing for superoxide. Cytc is a substrate for Cytbc1 and achieving measurable level of radicals requires measurements to be done in the presence of high micromolar concentrations of Cytc. In order for DMPO to be able to trap superoxide before it reacts with Cytc, it would need to be in a high molar concentration range, which is unattainable due to its limited solubility. electron transfer from heme bL to Q ("semireverse" box), electron transfer from SQ to molecular oxygen ("superoxide production" box). In "forward", and "semireverse" box, white and red rhombus correspond to the redox state of heme bL before the reaction (oxidized and reduced, respectively). The reduced heme bL necessary to start the semireverse reaction can also be a product of electron transfer from heme bH (reaction 2 in SC4 in Fig. 29). Note that the semireverse reaction corresponds to reaction 1 in SC3 and SC4 or reaction 2 in SC2 in Fig. 29 (B) Scheme illustrating that a shift in the equilibrium distribution of the ISP-HD positions from the b-position toward the c-position (from left to right) induced by specific mutations correlates with an increase in the ability of the Qo site to generate ROS.

Semiquinone intermediate at the Qo / Qp site

When discussing the mechanism of QH2 oxidation at the Qo/Qp site, SQ is naturally considered as the transient state of the bifurcation reaction. Over the years the EPR signal related to this state was expected to be typical of a free radical single-line EPR spectrum at a g position close to 2. The very first report of detection of a g = 2 signal assigned to semiquinone at the Qo site (SQo) in submitochondrial particles from 1981 667 was invalidated by studies from 1998 668 . Since that time a notion about SQo being highly unstable and therefore undetectable by EPR has been popular in the literature until 2007, when two independent groups reported on detection of SQo 261,262 In 2013, three additional reports presenting g = 2 EPR signals assigned to SQo were published 263,264,590 All these relatively new reports reopened a discussion on the presence of SQo, its properties and its implications for the mechanism of bifurcation. In general, one would expect the signal to appear under conditions favoring electron transfer reactions at the Qo/Qp site and to be sensitive to all specific inhibitors of this site. It should also have paramagnetic properties related to its interactions with other metal cofactors of the site, the heme bL/bp and/or 2Fe2S (such as enhanced paramagnetic relaxation rates compared to a chemically generated SQ in solution). This, however, has not always appeared to be the case. For this reason, in our view, the issue remains debatable and requires further experimental exploration. A detailed discussion of different spectroscopic properties and controversies surrounding the g = 2 signals assigned as SQo is beyond the scope of this review. The reader is referred to a more detailed review by Pietras et al. 258 .

In one of the reports on SQo from 2013, apart from the single-line signal at g ~2, measured at 200 K, a new, unexpected transition with the most prominent line at g = 1.94 in the region of the spectrum of 2Fe2S was recorded at 20 K 263 (see Fig. 31). The new signal was highly sensitive to the inhibitors of the Qo site and was transient (i.e. was detected only under non-equilibrium conditions before the enzymatic reaction of Cytbc1 with its substrates reached equilibrium). Furthermore it was observed that g=1.94 at the X band (~ 9.5 GHz) shifts to g=1.96 at the Q band (~ 33 GHz). These findings, complemented by the simulation analysis of the EPR spectra led the authors to conclude that the g = 1.94 transition belongs to the spectrum originating from SQo coupled by spin-spin exchange interaction with the reduced 2Fe2S (SQ-2Fe2S). An isotropic exchange constant of the interaction was estimated at 3.5 GHz. This was all consistent with the theory of exchange interactions, which assume that in the case of spin-spin coupling with an exchange constant of several GHz, the position and shape of EPR transitions of a system of coupled paramagnetic centers will depend on the microwave band used to make the measurements 669,670 . It is of note that the spin-spin exchange interaction between SQ and the reduced 2Fe2S has already been put forward as an explanation for the failure to detect the g = 2 radical signal of SQ 574 . However, unlike the original proposal, the more recent data do not result from anti-ferromagnetic coupling of high energy.

Further research showed that a g = 1.94 signal of similar amplitude was observed when the samples were prepared under both aerobic and anaerobic conditions 482,671 . This dismissed concerns raised by others 672 that it might have resulted from oxidative damage to the 2Fe2S. This also pointed towards an interesting possibility that the SQ-2Fe2S state is not reactive toward molecular oxygen (see discussion below).

Upon optimization of reagent concentrations the SQ-2Fe2S signal was detected within the timescale of tens to hundreds of milliseconds, which was comparable to the time of a single turnover of enzyme under the applied conditions (inhibition of the Qi site with antimycin). 671 On the other hand, the time scale of SQ-2Fe2S evolution was slower than the time scale of uninhibited enzyme turnover. Remarkably, so far the SQ-2Fe2S state has been found to coexist exclusively with oxidized heme bL. All together, these results implicated that the semireverse reaction (heme bL/bp to Q electron transfer) at the Qo/Qp site is responsible for initiating formation of the SQ-2Fe2S state. This is partly because the semireverse reaction creates SQo which can undergo spin-spin exchange with reduced 2Fe2S provided that the ISP-HD is docked at the site 263 .

Further studies revealed that the probability of formation of the SQ-2Fe2S state varies in mutants with decreased motion of ISP-HD 671 . It is highest in mutants which have ISP-HD shifted towards the Qo site (+1Ala and +2Ala). In contrast, the SQ-2Fe2S state was not observed in the Rh G167P mutant, which has the oppositely effect to +1Ala or +2Ala and shows impaired docking of ISP-HD into the Qo site 120 ( Rh G167P mutation).

The various combinations of these mutations have shown that direct interaction between the 2Fe2S and SQo is crucial for formation of SQ-2Fe2S state, and even minimal separation of the cluster from SQo induced by mutation breaks the spin-spin interaction.

The same family of mutants also revealed a clear negative correlation between superoxide production and the ability to form the SQ-2Fe2S state. Based on this observation and the non-reactivity of SQ-2Fe2S center with molecular oxygen discussed earlier, it has been suggested that the SQ-2Fe2S state may be involved in protection against massive release of superoxide under conditions when electron flow through the Qo site is impeded 671 . It should be noted that while the lack of the SQ-2Fe2S state is associated with an increase in the production of superoxide by Cytbc1, a high superoxide production may not necessarily imply lack of the SQ-2Fe2S state. This seemingly paradoxical statement is easily explained assuming that the unstable SQo is a common substrate for production of superoxide and generation of the SQ-2Fe2S state. An increase in the probability of formation of SQo will result in both an increase in superoxide production as well as in more efficient generation of SQ-2Fe2S. Since the SQ-2Fe2S state is nonreactive with oxygen its formation can be regarded as an alternative route to leaks of electrons on oxygen thus it reduces the amount of superoxide production.

The SQ-2Fe2S signal was also detected in enzyme operating with native substrates in membranes of photosynthetic bacteria 607 . Remarkably, the signal was present only when lightactivation of the membranes shifted the redox state of the substrate pools, ubiquione and Cytc2 pools, to a more reduced and oxidized state, respectively.

Under these conditions, SQ-2Fe2S titrated at higher ambient redox potentials compared to the Q(H2) pool (see section 4.1), which suggests that the SQ-2Fe2S state is somewhat stabilized at the Qo site. The redox potential at which the SQ-2Fe2S amplitude reaches its maximum increased with decreasing pH indicating that a proton is involved in stabilizing the SQ-2Fe2S state. The interaction of SQo through hydrogen bonding with the 2Fe2S coordinating residue Rh H156, and the role of this interaction in SQo stabilization have been postulated many times 258,574,596 .

Interestingly the SQ-2Fe2S state is not restricted to Cytbc1, but has as it was also observed in isolated spinach Cytb6f exposed to its substrates, PQH2 and PC 482 . In this case however, unlike in Cytbc1, inhibition of the Qn site was not required. This revealed that the probability of SQ-2Fe2S state formation when the enzyme operates without inhibition is higher in the Qp site of Cytb6f compared to the Qo site Cytbc1. The average redox potential of hemes bn and bp is probably more negative in Cytb6f than respective b hemes in Cytbc1, while the redox potential of PQ/PQH2 couple is higher compared to the potential of the respective UQ/UQH2 couple. These differences in the redox potentials of b hemes and quinones would foster the semireverse reactions at the Qp site, and thus increase the probability of the SQ-2Fe2S state formation. In addition, electron transfer through heme cn in Cytb6f

(not present in Cytbc1) might slow electron flow through the low-potential chain, and this could lead to a more favorable semireverse reaction.

The importance of quinone redox potential to SQ-2Fe2S formation is well supported by the observation that the high-potential Q derivative (DBMIB) bound at the Qp site also creates the SQ-2Fe2S state with a g = 1.94 transition. However, in this case the state was already observed under equilibrium conditions, without PC present. In fact, its stability is so high that it inhibits the Qp site.

Indeed, the inhibition of Cytb6f by DBMIB has long been known, as has its effect on the shape of the EPR spectrum in the region of 2Fe2S, [START_REF] Schoepp | The Qo-Site Inhibitor DBMIB Favours the Proximal Position of the Chloroplast Rieske Protein and Induces a pK-Shift of the Redox-Linked Proton[END_REF]108,483 This has been interpreted over the years as a structural alteration of the cluster caused by inhibitor binding 110 . The frequency-dependent shift in the g value of the signal induced by DBMIB (similar to shifts discussed above) dismisses this interpretation, indicating that the signal must reflect a spin-spin exchange interaction between the SQ form of DBMIB and the reduced 2Fe2S 482 (see also section 5).

Considering all the details of the formation and properties of SQ-2Fe2S uncovered to date, an energy diagram was proposed for both Cytbc1 and Cytb6f, in which this state represents a local energetic minimum, placed below the state with the reduced heme bL/bp (given that SQ-2Fe2S is observed along with oxidized heme bL/bp), but above the state with reduced heme bH/bn (Fig. 32). It has been suggested that SQ-2Fe2S is a metastable state, which may play a role as a buffer for electrons, in particular under conditions when electrons cannot freely leave the low-potential chain through the Qi/Qn site. Although the physiological role of this state is not yet known, in view of the observed negative correlation between its presence and superoxide production, and the apparent nonreactivity of this state with molecular oxygen, it was proposed that SQ-2Fe2S may be associated with protection against side reactions at the Qo/Qp site. This concept further assumes that the probability of formation of SQ-2Fe2S might be adjusted to the oxygen tension in the cellular environment and varies between different species. This probability depends on the energy difference between SQ-2Fe2S and the states determined by the Em values of Q/QH2 couple and the Em values of the oxidized/reduced couples of low-potential cofactors of Cyt-bc (Fig. 32). This notion was proposed in view of the observation that Cytb6f, which experiences more than an order of magnitude higher levels of oxygen in chloroplasts than does Cytbc1 in mitochondria, appears to have a greater tendency to reside in this buffered state.

An intriguing hypothesis was put forward for a possible role of the Cytb6f SQ-2Fe2S in regulation of the electron transfer pathways in oxygenic photosynthesis. It was proposed that the SQ-2Fe2S state in Cytb6f might serve as a factor that changes the efficiency of CET vs LET between photosystem I and II. This concept was based on the assumption that transient stabilization of the SQ-2Fe2S temporarily blocks the oxidation of further PQH2 in the Qp site, thus creating conditions in which the probability of delivering the second electron needed to complete the reduction of PQ in the Qn site by Fd/FNR is significantly increased. 482 The Qi site binding pocket is located in the vicinity to heme bH near the n side of the membrane and its entrance opens towards the membrane core allowing substrate and product molecule to enter and leave the site. The pocket is surrounded by residues from transmembrane helices A, D, E, the amphipatic surface helix a, the DE loop and the N-terminal peptide. The crystal structures identified not only the position of site-specific inhibitor (antimycin A) [START_REF] Gao | Structural Basis for the Quinone Reduction in the Bc1 Complex: A Comparative Analysis of Crystal Structures of Mitochondrial Cytochrome Bc1 with Bound Substrate and Inhibitors at the Qi Site[END_REF]123 , but also the position of bound substrate (UQ) [START_REF] Zhang | Electron Transfer by Domain Movement in Cytochrome Bc1[END_REF][START_REF] Esser | Inhibitor-Complexed Structures of the Cytochrome Bc1 from the Photosynthetic Bacterium Rhodobacter Sphaeroides[END_REF][START_REF] Gao | Structural Basis for the Quinone Reduction in the Bc1 Complex: A Comparative Analysis of Crystal Structures of Mitochondrial Cytochrome Bc1 with Bound Substrate and Inhibitors at the Qi Site[END_REF]123,673 . The binding sites of these compounds were found to significantly overlap with little rearrangements in the protein backbone and side-chains, as compared with the rearrangement of the protein around the Qo site upon binding inhibitors. This indicates that the Qi site is structurally more rigid than the Qo site.

The quinone ring bound at the Qi site is nearly perpendicular to the plane of heme bH with the closest distance of 4.2 Å 79 . It appears to be stabilized by both hydrophobic and polar interactions.

The former include interaction with Bt Phe220/ Sc Met221, the latter through formation of hydrogen bonds with three residues: Bt Ser205/ Sc Ser206, Bt Asp228/ Sc Asp229, and Bt His201 [START_REF] Gao | Structural Basis for the Quinone Reduction in the Bc1 Complex: A Comparative Analysis of Crystal Structures of Mitochondrial Cytochrome Bc1 with Bound Substrate and Inhibitors at the Qi Site[END_REF]673 . The His interacts directly with quinone in bovine [START_REF] Gao | Structural Basis for the Quinone Reduction in the Bc1 Complex: A Comparative Analysis of Crystal Structures of Mitochondrial Cytochrome Bc1 with Bound Substrate and Inhibitors at the Qi Site[END_REF] and also in bacterial enzyme (His217) [START_REF] Esser | Inhibitor-Complexed Structures of the Cytochrome Bc1 from the Photosynthetic Bacterium Rhodobacter Sphaeroides[END_REF] , while in yeast ( Sc His202)-it interacts indirectly through a water molecule. 673 As the Asp and His residues interact respectively with O1 and O4 of Q, they are considered to be involved in protonation of Q. In addition to these residues, the crystal structures identified other structural elements that could potentially form proton paths from the protein surface towards the O1 and O4 of Q.

The path toward the O1 (referred to as the D/K path) involves a conserved Lys residue and a bound cardiolipin, which interacts further with a conserved Asp located next to the bound Q (Fig. 33).

The cardiolipin has been suggested to act as a proton attracting antenna, which transfers protons to the nearby Lys 673 . The Lys further passes the protons to the conserved Asp through a string of interconnected water molecules 674 , or alternatively undergoes a rotation towards the Qi site and directly interacts with the Asp, as suggested by MD simulations [START_REF] Gao | Structural Basis for the Quinone Reduction in the Bc1 Complex: A Comparative Analysis of Crystal Structures of Mitochondrial Cytochrome Bc1 with Bound Substrate and Inhibitors at the Qi Site[END_REF]675 .

The path toward the O4 involves a conserved Glu and a conserved Arg that may interact through the water molecules with a conserved His located next to the bound Q 673,674 . Site-directed mutations of this residue fully inactivated bacterial Cytbc1 676 . On the other hand the Asp/Lys path was found to be disabled only when both protonable groups were removed. With just one protonable residue from the Asp/Lys pair, the entrance of protons to the catalytic site was sustained, albeit at lower rates, indicating that protons in the Asp/Lys path can travel through parallel routes, possibly involving water molecules 677 . The Asp/Lys path thus appears to tolerate disruption, as long as all the elements available for functional cooperation secure efficient proton delivery to the catalytic site. In this context, the Qi site is similar to other Q binding sites, such as the QB site of photosynthetic reaction center, for which a multiplicity for proton paths has also been considered 678

Catalytic electron and proton transfers at the Qi site of Cytbc1

The reduction of Q to QH2 at the Qi site involves two sequential single electron transfer steps in which the electrons are delivered from the same cofactor chain [START_REF] Crofts | The Role of the Quinone Pool in the Cyclic Electron-Transfer Chain of Rhodopseudomonas Sphaeroides: A Modified Q-Cycle Mechanism[END_REF] . Because the arrival of the electrons is separated in time, the reaction must involve the formation of an SQ intermediate (SQi) that is stabilized within the Qi binding pocket before the final product (QH2) is formed and leaves the site. Indeed, a stable radical signal originating from the Qi site was recognized in early EPR studies on submitochondrial particles 440 and crude protein extracts 141,679 and has been explored since then as a key mechanistic element of models proposed for that site 151,163,245,647,648,677,[680][681][682][683][684] .

Considering electron transfer, reduced heme bH acts as immediate electron donor to Q and then to SQ formed after the first electron transfer (SQi). These reactions are favored by the generally lower Em of the heme compared to the average Em of the Q(H2) pool (under physiological pH, Em7 of heme bH is around +50 mV, while that of the pool is around +100 mV) 149 . As these reactions leave heme bH in an oxidized form, the SQi associated with the oxidized heme bH (SQi-bH 3+ ) is one of the key intermediate states of the catalytic forward reaction 2 .

At alkaline pH, the Em of heme bH approaches that of the Q(H2) pool and a partial reversal of the reaction can be observed. In this case QH2 bound at the Qi site reduces heme bH, resulting in formation of SQi at this site 163 . As in this reaction, QH2 instead of Q acts as a substrate for the Qi site, and this is favored not only at alkaline pH but also at high QH2 to Q ratios 647,685 . This reaction, unlike the forward reaction, leads to the formation of SQ that is present along with reduced heme bH (SQi-bH 2+ ). For many years, SQi-bH 2+ has been the dominant form of SQi detected and explored experimentally. However, more recent studies indicated that the analysis of both forms of SQi (i.e.

SQi -bH 3+ and SQi -bH 2+ ) is crucial to advance our understanding of the molecular mechanism of the operation of the Qi site (see section 8.3).

In addition to the observation of a stable SQi radical by EPR, the stability of SQi is supported by data from light-induced electron transfer measurements. Under conditions of single-turnover at the Qo site, the offset in the levels of light-induced heme bH reduction observed in the absence and presence of antimycin corresponds to the amount of SQi that is formed in the forward reaction (electron transfer from heme bH to Q). On the other hand, when the Qo site is blocked by an inhibitor, the level of light-induced heme bH reduction corresponds to the amount of SQi formed in the reverse reaction (electron transfer from QH2 to heme bH). The extent of the forward reaction reflects the difference between the Em of heme bH 3+ /bH 2+ and the Em of Q/SQ couples, while the extent of the reverse reaction indicates the difference between the Em of heme bH 3+ /bH 2+ and the Em of SQ/QH2 couples. Further analysis of such extents might provide estimates of Em for Q/SQ and Em for SQ/QH2, which in turn would provide information on the stability constant of SQ (log(Ks) = [Em(Q/SQ) -Em(SQ/QH2)]/60. Using this approach with a specific mutant in which the Em,7 of heme bH was elevated to the level of the Em of the QH2 pool, the stability constant of SQi was estimated to be at the level of 3 × 10 -1 . 149

8.3

Fast-and slow-relaxing SQi as dominant intermediates of forward and reverse reactions at the Qi site, respectively.

Until recently, EPR-based investigation of the SQi intermediate relied on the assumption that the SQi interacts antiferromagnetically with the oxidized heme bH, forming an "EPR-silent exchange couple". Therefore only SQi present when heme bH is also reduced (SQi -bH 2+ ) was thought to be spectroscopically detectable 141,681 . This notion is no longer valid in light of the recent studies that identified two distinct populations of SQi differing in the rates of spin-lattice relaxation (Fig. 34 A) 151,163 . The first population, characterized by a slow relaxation (SQiS), corresponds to the well-known and extensively examined SQi -bH 2+ state, whereas the second population, characterized by a significantly faster relaxation time (SQiF), corresponds to the long-missing SQi -bH 3+ state. The dipolar interaction between SQiF and oxidized heme bH manifests itself in the characteristic properties of SQiF, including fast spin-lattice relaxation leading to dominant homogeneous line broadening 686 and unusual behavior of the signal amplitude that does not follow Curie's law 687 . These findings established that, contrary to long-standing assumptions, the SQi-bH 3+ state is not antiferromagnetic and can be separated spectroscopically (as SQiF) if an appropriate combination of microwave power and temperature is applied during EPR measurements 151 Further studies revealed that SQiS and SQiF are separable in equilibrium redox titrations 163 .

Remarkably, over a pH range from 6 to 8, SQiF represented a large, if not dominant, portion of the whole population of titrated SQi, indicating that early titrations 245 had indeed missed a significant fraction of SQ in this pH range (Fig. 34 B). SQiF was also trapped as a stable intermediate at the Qi site during steady-state turnover 163 , which is consistent with the electron transfer reaction scheme predicting SQi -bH 3+ as the key intermediate state of the catalytic forward reaction (heme bH to Q electron transfer) 2 . On the other hand, SQiS appeared as the dominant intermediate state observed under the conditions favoring the reverse reaction (QH2 to heme bH electron transfer).

Interestingly, when a single electron gets injected into heme bH of one monomer through the reverse reaction at the Qi site, there is a possibility of electron equilibration within all four hemes of the b-chain of the dimer (through the heme bL-bL bridge, see figure 5 and section 9). This way the electron can reach the second Qi site (reducing Q to SQi) which overall will result in a formation of SQiF in both monomers (Fig. 35). The observation that both SQiS and SQiF are observed in the native enzyme with an inactivated Qo site (when only the reverse reaction at Qi is possible) indicates that electron equilibration within the b-chain of the dimer indeed traps some fraction of the molecules of Cytbc1 with the SQi -bH 3+ state at both Qi sites 151 . This conclusion was further supported by the observation that electron redistribution over b hemes /Qi sites taking place within the dimer and monitored by analyzing the change in relative proportions of SQiF / SQiS, is effectively modulated in mutants that significantly raise the redox midpoint potential of either heme bL or heme bH. 149,151 .

8.4

Charge polarization of SQi facilitates electron and proton reactions at the Qi site.

The experimental exploration of both states of SQi (i.e. SQiS and SQiF) complemented by quantum mechanical calculations lead to the formulation of a hypothetical mechanism which places importance on the charge and spin polarization of SQi intermediates in the catalytic reactions at the Qi site 163 . This polarization is imposed by the local electrostatic field induced by the specific distribution of residue charges within the Q-binding pocket. These are mainly His in the vicinity of the O4 atom and the Asp/Lys pair in the vicinity of the O1 atom.

This "charge polarization" mechanism is schematically presented in Fig. 35, right. The evolution of states A to G considers the direction of reduction of Q to QH2. The reaction requires initial entry of Q to the Qi site (states A and B) and heme bH in reduced state (state C). It starts when the O4 atom of the Q present at the site interacts with the protonated His. The electron is then transferred from this heme to Q resulting in the formation of the SQi anion (which is detected by EPR as SQiF when heme bH is oxidized) (state D). The anionic SQi becomes more negative at the O4 atom than at the O1 atom, while the spin density is larger at the C1 side compared to the C4 side (Fig. 35, left). Such polarization and the resulting geometry favor a barrierless exchange of the proton originating from His between the His imidazole ring and the O4 atom of SQi. In this state, the SQi radical is stabilized, and equilibrates between the anionic and neutral forms. This equilibration also takes place after the reduction of heme bH and is associated with the conversion of SQiF to SQiS (conversion of state E to F). This continues until the second electron reduces the neutral SQi to unstable QH -(state F). When this equilibration takes place, the protonation of anionic SQi to the neutral form does not influence the spin density but causes an inversion of the direction of charge polarization: the C1 side becomes more negative than the C4 side. This increases the probability that the second proton will be transferred to the O1 atom. Consequently, the reduction of neutral SQi to QH -is accompanied by the spontaneous transfer of a second proton from the Asp/Lys-path to the O1 atom, completing the reduction of Q to QH2 followed by dissociation of the product (state G). The key feature of the proposed mechanism is an initial response of the radical anion to the local electrostatic field, as it triggers progression of the whole catalytic reaction. At the same time, polarized SQi exchanging a proton in the absence of barrier with His forms a stable intermediate which, during enzymatic turnover, safely "waits" for the second electron without the risk of generating other radicals (such as superoxide).

The charge polarization mechanism sheds new light on the protonation states of SQi described earlier by the pulse spin-echo-based EPR (ESEEM or HYSCORE) 647,648,683 , which must have referred to just the SQiS, as the spin-lattice relaxation of SQiF is fast enough to lead to homogeneous line broadening that precludes the formation of a spin-echo signal. It was proposed that SQi (SQiS in view of the above assumption) is likely to be anionic 648,682 and ligated by a hydrogen bond from the imidazole-ring nitrogen of the His 647,648 . It was also suggested that the redox state of heme bH might influence the protonation state of SQi and the His ligand, rendering SQi deprotonated at the time when heme is reduced 683 . This charge polarization mechanism implicates that SQi exists as neither an anion nor a neutral radical but should rather be treated as a resonant structure in which the proton undergoes exchange between His and SQi. If the frequency of this exchange is much higher than the hyperfine splitting of the radical spectrum caused by the interactions of the proton with unpaired electron spin, then EPR will be unable to detect the presence of protons due to large spectral diffusion effects.

The proposed catalytic role of charge and spin polarization of the SQ is likely to be relevant to other sites catalyzing the reduction/oxidation of Q/QH2 via stable SQ intermediates. Such spin polarization has been noted before for the SQ bound at catalytic sites (including SQi 680 , SQH of quinol bo3 oxidase 688 and SQA of bacterial reaction center 689 ), and could be associated with charge polarization induced by the electrostatic field provided by the catalytic site. Further support for this proposal comes from modeling of the effect of the electrostatic field on spin and charge polarization in diboryl monoradical anions 690 .

Specific residues involved in PQ/PQH2 binding to the Qn site of Cytb6f

Several attempts have been made in the past to study residues important for Q/QH2 binding to the Qn site of Cytb6f via site directed mutagenesis approaches informed by previous results from work on the Cytbc1 complex and by structural features of the Cytb6f. They include residues bG37 and bF41, 691 because substitutions of the corresponding residues in Rhodobacter's Cytb (G48 and A52 respectively) led to impaired redox activity, loss of photosynthetic growth or failure to assemble a functional complex. The F41V mutant of Chlamydomonas displayed a 5-fold decrease in Qn site activity and became sensitive to antimycin, an inhibitor of the Cytbc1 Qi site that is normally not effective against Cytb6f. Conversely, the bF41G and bF44A mutants did not display any phenotype 692 . Substitutions of the Gly37 residue did not rescue phototrophic growth, suggesting that mutation of this residue is detrimental to photosynthesis. The same result was found in the case of a C35V mutant, which was unable to grow phototrophically because this substitution (affecting the residue that covalently bonds heme cn to the Cytb6) prevents the accumulation of the entire complex in thylakoids. 693 Other mutants were generated affecting residues in the proximity of the heme: Leu36, Pro41 of SubIV. No clones were recovered after mutagenesis, indicating that these mutations were also detrimental for photosynthesis. On the other hand, a mutant was obtained after the P40T substitution. This mutant showed a significant slowing down of Cytb6 reoxidation under single turnover flash illumination, consistent with impairment of interaction between the heme and its quinone substrate.

The mutant also displayed a downshift of the cn heme midpoint potential (from +100 mV to -200 mV at pH 7), but conserved WT Cytb6f turnover rates under a continuous light regime. Based on the position of P40 in the Cytb6f structure, it was proposed that this modified accessibility could originate from the ligation of the heme iron by the phenol(ate) side chain introduced by the mutation 430 .

A similar approach has been used in cyanobacteria, where a mutant (R214H) was generated to substitute His for a conserved Arg in the Cytb6 polypeptide of the cyanobacterium Synechococcus sp. SPCC 7002. At high light intensity, the R214H mutant grew ∼2.5-fold more slowly than the WT, due to inhibition of the Cytb6f complex turnover. Under single turnover flash illumination the R214H mutation partially blocked electron transfer to the Qn site, mimicking the effect of the Qn site inhibitor NQNO. Overall, these data define Pro 40 and Arg 214 as key residues for catalysis in the Qn site of the Cytb6f 694 .

Other mutants were generated to modify the environment of the chlorophyll ring that is located close to the Qn site 695 . Residues Val104, Gly136, Tyr124, and Arg125 did not dramatically affect electron transfer in the complex, but strongly affected state transitions (see section 10.3 below).

This indicates that besides catalysis, the reduction of the PQ at the stromal side Qn site is also involved in sensing and signaling, two essential functions of the Cytb6f (see sections 5.2 and 10.3) 696

Inter-monomer electron transfer

Crystallographic structures of Cytbc1 and Cytb6f revealed a high degree of symmetry between monomers including structural arrangement of the redox active cofactors [START_REF] Berry | Unanswered Questions about the Structure of Cytochrome Bc1 Complexes[END_REF][START_REF] Xia | Structural Analysis of Cytochrome Bc1 Complexes: Implications to the Mechanism of Function[END_REF][START_REF] Hasan | Transmembrane Signaling and Assembly of the Cytochrome B6f-Lipidic Charge Transfer Complex[END_REF][START_REF] Berry | X-Ray Structure of Rhodobacter Capsulatus Cytochrome Bc1: Comparison with Its Mitochondrial and Chloroplast Counterparts[END_REF]173 . One of the most intriguing structural features of the dimer is the distance between hemes bL/bp of different monomers, which theoretically seems to be small enough to allow fast (i.e. ~μsec) electron transfer [START_REF] Page | Natural Engineering Principles of Electron Tunnelling in Biological Oxidation-Reduction[END_REF]195,697,698 , in relation to catalytic rates [START_REF] Stroebel | An Atypical Haem in the Cytochrome B6f Complex[END_REF][START_REF] Hunte | Structure at 2.3 Å Resolution of the Cytochrome Bc1 Complex from the Yeast Saccharomyces Cerevisiae Co-Crystallized with an Antibody Fv Fragment[END_REF]615,699 . For this reason, a discussion began about the possible implications of such an electronic connection between monomers for catalysis and opened the door to construction of models extensively utilizing the heme bL-bL bridge [START_REF] Cooley | Protein Conformational Changes Involved in the Cytochrome Bc1 Complex Catalytic Cycle[END_REF][START_REF] Covian | Regulatory Interactions in the Dimeric Cytochrome Bc1 Complex: The Advantages of Being a Twin[END_REF][START_REF] Cooley | Across Membrane Communication between the Qo and Qi Active Sites of Cytochrome Bc1[END_REF]125,329,700,701 .

The fundamental question therefore arises as to whether electron transfer between the bL/bp hemes of the two monomers can be considered competitive to the intra-monomer path: from heme bL/bp, through heme bH/bn to the Qi/Qn site. Alternatively, is this simply a redundancy of the possible electron transfer paths that may be important only under specific and rare conditions 206,571,615 .

The order of the rates for inter-monomer electron transfer can be approximated on the grounds of electron transfer theory 702 and the phenomenological approximation of the Moser-Dutton ruler [START_REF] Page | Natural Engineering Principles of Electron Tunnelling in Biological Oxidation-Reduction[END_REF]195,697,698 :

𝑙𝑜𝑔(𝑘 𝐸𝑇 ) = 13 -(1.2 -0.8𝜌)(𝑅 -3.6) -3.1 (𝛥𝐺+𝜆) 2

𝜆

, where: ρ -is packing density (0 for vacuum, 1 -fully packed matrix), ΔG -Gibbs energy associated with electron transfer from donor to acceptor (in the case of electron transfer from heme bL to bL this is 0) and λ -reorganization energy, R -distance between donor and acceptor (between bL hemerings)

Using average values for electron transfer within a protein matrix -ρ and λ in the above equation can be simplified to account only for R 697 : 𝑙𝑜𝑔(𝑘 𝐸𝑇 ) = 12.8 -0.6𝑅

As distances between heme rings are ~13.2 -15.2 Å, a crude estimation of the rates gives kET ~ 5×10 3 -8×10 4 s -1 . A typical steady-state turnover rate obtained for Cytbc1 or Cytb6f does not exceed 10 3 s -1 569,649 meaning that electron transfer between hemes bL/bp should not limit the catalytic turnover rate.

The experimental verification of whether intermonomer electron transfer is indeed as fast as theory predicts, required measuring the electron transfer rate between bL/bp hemes in the dimer, which in the native protein is generally difficult considering the structural symmetry of the complex, and the fact that electron transfer between the monomers leads to spectroscopically indistinguishable states. In fact, this has long been out of reach for direct experimental testing, due to the lack of a genetic tool to inactivate individual components of the dimer in an asymmetric manner. This is related to the fact that, in native systems, the genes coding for the subunits do not differentiate between monomers, thus any mutation is obligatorily introduced to both parts of the homodimer.

New strategies described in 2010 aimed at overcome these limitations. [START_REF] Świerczek | An Electronic Bus Bar Lies in the Core of Cytochrome Bc1[END_REF]329,[703][704][705][706][707] In these studies two distinct genetic approaches were used: a fusion duplicating gene or a co-expression of two variants of the gene. Both targeted the Cytb subunit of bacterial Cytbc1.

The first approach was based on extending the native gene encoding Cytb with a short linker followed by a second copy of the same gene [START_REF] Świerczek | An Electronic Bus Bar Lies in the Core of Cytochrome Bc1[END_REF][705][706][707] . As a result, each half of the fusion protein replaced one Cytb subunit of the dimer, providing a template for construction of derivatives in which the two halves could be mutated independently. Using such a template, different mutations inactivating key steps of electron transfer were introduced in various combinations to eliminate selected paths and to force electron transfer through other available paths within the dimer (see all forms in Fig. 37). This was used to test all possible electron transfer routes for electrons from the Qo to Qi site. [START_REF] Świerczek | An Electronic Bus Bar Lies in the Core of Cytochrome Bc1[END_REF]707 Because for a given combination of mutations, only one version of the complex with the fused Cytb is expressed in the cells, it was possible to monitor the kinetics of light-induced electron transfer in the native membranes. Analysis of the cross-inactivated complex (a variant WB-B N in Fig. 37), for which electrons derived from the Oo site must have crossed the dimer to reach the Qi site, revealed that electron transfer between hemes bL occurs on a millisecond time scale, as theory had predicted.

In addition, it was established that the complex remains enzymatically active even if the equivalent of only one monomer (W N B-B), or the equivalent of one monomer with a part of other monomer (WB-B, N B-B) are available. This revealed that any path connecting either or both Qo sites with either or both Qi sites is competent in supporting enzymatic turnover at high rates [START_REF] Świerczek | An Electronic Bus Bar Lies in the Core of Cytochrome Bc1[END_REF]704,705 . Based on these findings it was proposed that cofactor chains in the native dimer assemble into an H-shaped electron transfer system (two upper, two lower branches and a bridge) connecting the two Qo sites with the two Qi sites.

Interestingly, deactivation of only one branch (forms WB-B, N B-B) or both branches of the same monomer (W N B-B) decreases Vmax of the enzyme by approximately 50% when compared to the fusion protein without any disabling mutation (B-B) 569 . This suggests that the two monomers in the dimer contribute equally and independently of each other, thus any allosteric effect [START_REF] Mulkidjanian | Activated Q-Cycle as a Common Mechanism for Cytochrome Bc1 and Cytochrome b6f Complexes[END_REF][START_REF] Covian | Regulatory Interactions in the Dimeric Cytochrome Bc1 Complex: The Advantages of Being a Twin[END_REF][START_REF] Cooley | Across Membrane Communication between the Qo and Qi Active Sites of Cytochrome Bc1[END_REF]708 , if it exists, does not govern the catalytic turnover rate. Forcing electron transfer through the bL heme "bridge" decreases Vmax to ~70 s -1 , which is about 25 -40% of the average activity obtained for WB- B, W N B-B and N B-B forms, but is still enough to support multiple turnovers at high rates.

When using a fusion gene composed of two copies of the same gene, careful procedures must be employed in order to maintain high expression of the fusion protein during cell growth and prevent gene recombination. 706 Without rigorous growth and harvesting protocols there is a risk of losing the fusion or asymmetry of the mutations, which is especially critical if one wishes to test Cytbc1-related support of photosynthetic growth of bacterial cells. Indeed, just such recombination was observed, but incorrectly interpreted by Hong et al. 709 To overcome these limitations and gain the possibility to test functionality of the asymmetrically mutated complexes in vivo, the original system was modified by constructing a hybrid fusion system, in which the fusion protein was expressed from a gene linking two different genes encoding Cytb from closely related bacteria Rhodobacter sphaeroides and capsulatus 704,705 . Careful analysis of photosynthetic competence of the various complexes containing asymmetrically mutated hybrid fusion proteins (analogous to the ones described above) demonstrated that in agreement with kinetic results, the cross-inactivated form relying exclusively on inter-monomer electron transfer is fully functional in vivo, as is the form with just one branch inactivated. A second approach for obtaining asymmetrically mutated variants of Cytbc1 was based on coexpression of two plasmids, each carrying one copy of the gene encoding for Cytb with a sequence coding for a different tag attached (His-tag, Flag-tag or Strep-tag) 329,710,711 . The different mutations inactivating either the Qo or the Qi sites were placed on different plasmids and the two-step affinity chromatography was used to isolate the cross-inactivated form from the symmetric variants resulting from random assembly of the complexes in the cells 703,710 . These studies also demonstrated that intermonomer electron transfer through bL hemes occurs in milliseconds, thus in a catalytically-relevant timescale. In addition, some of the kinetic transients were interpreted as resulting from electron equilibration between the two Qi sites and the four hemes b in the dimer 703 . The same type of equilibration (see Fig. 35) was considered as rationale for the existence of fast-relaxing semiquinone (SQiF) in native Cytbc1 exposed to conditions favoring one electron reduction of heme bH by QH2 bound at the Qi site (described in section 8.3) 151 .

Although the existence of inter-monomer transfer has been confirmed by several studies, its possible physiological meaning and role in the catalysis remain unclear. [START_REF] Sarewicz | Electronic Connection between the Quinone and Cytochrome c Redox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling[END_REF]699 Electron transfer between the bL hemes could be just a simple coincidence of the conserved structure of the homodimer and as such does not play an important catalytic role, as all structural elements needed to perform catalytic cycle are present in each of the monomers [START_REF] Crofts | The Mechanism of Ubihydroquinone Oxidation at the Qo-Site of the Cytochrome Bc1 Complex[END_REF]206,709 .

On the other hand, it has been hypothesized that inter-monomer electron transfer to support efficient catalysis may offer some advantage for bioenergetic processes, in particular when unfavorable redox conditions and/or potential danger of superoxide generation exist. As pointed out by Sarewicz et al. [START_REF] Sarewicz | Electronic Connection between the Quinone and Cytochrome c Redox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling[END_REF]699 mitochondria usually posses several copies of genes coding for Cytb and the appearance of a mutation in one or more genes is expected to lead to expression of a fraction of the dimers having damage in only one part of the monomer (and thus possibly having a "dead end" for electrons). If this monomer was isolated functionally from undamaged monomer, it would inevitably catalyze only energy-wasting short-circuits or free-radical generating reactions. However, if the damaged monomer is linked functionally with an undamaged one, the existence of the heme bL-bL bridge can provide an efficient way to remove electrons from the low-potential chain in the damaged monomer (Fig. 38). With such a mechanism, the amount of ROS generated during the QH2 oxidation will be significantly lowered enabling electron flow through respiratory or photosynthetic chains. Also, as shown by Pintscher et al. 151 electron exchange between monomers may increase the rate of SQ -i reduction to QH2 by providing electron paths for dismutation of SQ -from two Qi sites of a dimer., which can be of advantage under shortage of QH2. Alternative to linear electron transfer (LET), which requires in series PSII and PSI activity, is the cyclic pathway (CET), which involves electron flow via PSI and the P700 reaction center complex via the plastoquinone pool through Cytb6f complex. It thus produces ATP without generating reducing power, and it is considered as a central element to readjust the ATP/NADPH balance for proper CO2 assimilation in plants and green algae 230 . Actors involved in CET and CET regulation have been recapitulated in recent reviews 374,[712][713][714][715] . Nonetheless, several aspects of this process remain enigmatic. While most of the CET electron carriers (the plastoquinone pool, Cytb6f, plastocyanin, PSI and ferredoxin and possibly NADPH) are shared with the LET pathway, electron transfer from Fd/NADPH to the quinone pool on the stromal side of the thylakoids may require an additional, CET specific, activity. This activity would be either by the NAD(P)H dehydrogenase (NDH) complex similar (but not identical) to Complex I of the mitochondrial respiratory chain 713,716 , or the FQR (ferredoxin quinone reductase) complex. FQR would correspond either to a PGRL1 and PGR5 complex 199 or to the Cytb6f itself either Cytb6f complex interacting with Fd, most likely via FNR (as discussed paragraph Erreur ! Source du renvoi introuvable.) and/or possibly PGRL1 and PGR5, although as considered above involvement of the latter components is supported only by very qualitative electron transfer data and stoichiometries are small.

Mutants lacking the NDH complex lack a clear growth phenotype under a variety of environmental conditions 716,717 , suggesting that the NDH activity, per se, is not essential for CET, since CET is an essential process for plant growth 235 . On the other hand, a mutant with enhanced CET activity, isolated in Arabidopsis, displays higher levels of the NDH complex 379 . Moreover, Arabidopsis photosynthetic embryos, which have a larger CET activity than mature leaves, also display an enhanced NDH activity 718 . While the FQR activity was initially identified with the Cytb6f 719 , this function has later been associated with a complex containing the PGR5, PGRL1 proteins 199 , i.e. previously identified as CET actors 713 . Similarly to the NDH, mutants lacking the PGR5:PGRL1 complex show WT like CET rates in Arabidopsis 386 and Chlamydomonas 387 , again pointing to a non-essential role of this complex in CET.

Overall, these results can be interpreted assuming that both the NDH and FQR pathways contribute to CET, each activity being compensated by the other when genetically disrupted.

Consistent with this conclusion, previous genetic studies have highlighted that severe growth phenotype in Arabidopsis can only be observed upon concomitant disruption of the NDH and PGR5:PGRL1 complexes 720 .

What is role for the Cytb6f (if any) in a context where the role of the NDH and PGRL1:PGR5 pathways in CET is still controversial? Interactions of the Cytb6f with PSI and/or FNR have been proposed as a mean to trigger CET 721,722 . This notion has received experimental support by the finding by Cramer and co-workers that Cytb6f co-purified with FNR in plants 180 . However, recent ITC experiments show that there is no significant interaction between FNR and Cytb6f. Moreover, interactions between subunit of PSI, Cytb6, FNR and PGR5 were reported in a yeast two-hybrid assay using Arabidopsis genes 236 . In the green alga Chlamydomonas reinhardtii, Minagawa and colleagues 197 were able to isolate a PSI:Cytb6f, which they considered as a specific component of CET. The existence/function of this complex is still debated. If real, why would a supercomplex be needed for CET? As mentioned above, most electron carriers are shared between the CET and LET pathways. Therefore, CET, which is a 'circular' process continually recycling electrons around PSI, would be easily overcompeted by LET every time that a cyclic electron is shunted towards the stromal sinks by LET. Two alternative models have been proposed to explain how LET does not overcompete CET 426,723,724 .

The first one considers that CET and LET are, to some extent, segregated ('segregated CET').

It is well-known that the photosynthetic complexes are unevenly distributed in the thylakoids: PSII is mainly concentrated in the appressed grana, PSI is mainly found in the non appressed membranes (stroma lamellae, grana margins), while the Cytb6f is roughly homogeneously distributed 725 . Moreover, the diffusion of PQ/PQH2 in the thylakoid membrane is highly restricted, probably due to the very high protein concentrations that characterize this membrane 272,726 . The 'segregated CET' model assumes that because the physical distance between the different domains is rather large and the PQ diffusion in the thylakoids is restricted, two electron carrier pools would exist with different functions. Photosynthetic complexes present in the grana and grana margins (PSII, Cytb6f and PSI) would be involved in LET, while those in the stroma lamellae (Cytb6f and PSI) would be the CET ones. Consistent with this hypothesis, recent affinity-mapping AFM studies has revealed nanodomains of co-localised PSII and Cytb6f complexes 727 . The domains (previously hypothesized by Lavergne and Joliot 272 ) would provide a structural rationale for the segregation of LET and CET and its regulation. Small changes in the distance between adjacent membranes in stacked grana regions might control the access of Cytb6f to the grana because of steric hindrance 5 . Because the width of the stromal gap is variable and light regulated, this model predicts that changes in the lateral distribution of the Cytb6f should affect CET and LET by modulating the amount of this complex present in the CET and LET compartments. Obviously, the presence of a supercomplex is an extreme case of the 'segregated CET' hypothesis, the CET domain becoming the PSI:Cytb6f supercomplex itself.

Alternative to the 'segregated CET' model, a 'dynamic' hypothesis to explain CET cyclic flow has been proposed 410,426,723,728 . According to this model, CET and LET would not be segregated but actually directly compete for electrons. Under most physiological conditions, this competition would favour LET, because of electron leak toward stromal sinks. However, under particular conditions (high light, CO2 limitation, low oxygen in microalgae) CET would become more efficient because the LET efficiency would be reduced by limitation at the PSI acceptor side. Competition would occur either at the PSI acceptor side (via competition for Fd, likely via the FNR-Cytb6f complex 723 or at the level of the PQ(H2) pool 374 . The model proposed by these authors is particularly interesting from a Cytb6f perspective, as it postulates that a function interaction between the two Cytb6f monomers at the PQ-reducing site would allow a 'direct' electron flow from the stromal to the luminal PQ, in a confined environment not in contact with the photoactive PQ(H2) pool in the thylakoids, similar to the semiquinone version of the Q cycle proposed earlier 729 . This hypothesis is therefore somehow in between the 'segregated CET' and the redox competition hypotheses previously described.

10.2

The "elusive" Cytb6f-PSI CET supercomplex.

As discussed above, the presence of a PSI-Cytb6f complexes represent the most extreme case of the 'segregated CET' hypothesis. In the green alga Chlamydomonas reinhardtii, where CET can be a prominent process 728,730,731 Iwai and colleagues used a sucrose density gradient to isolate a supercomplex composed of the PSI-LHCI supercomplex with LHCIIs, Cytb6f, FNR, and PGRL1 in a fraction heavier than that of the single complexes (PSI-LHCI, Cytb6f) alone. Its formation is enhanced by reducing conditions, which increases CET activity in this alga 221,732 . Spectroscopic analyses 732 indicate that PSI and Cytb6f are able to exchange electrons within the supercomplex, PSI being able to reduce Cytb6 in the presence of Fd and NADPH and to oxidise Cytf in the presence of plastocyanin 197 . Based on these results, the authors conclude that the PSI-Cytb6f supercomplex corresponds to the CET supercomplex, the existence of which had been anticipated by theoretical predictions 733 and functional studies (e.g. Joliot and Joliot 734 ).

Supercomplex formation in Chlamydomonas reinhardtii requires not only reducing conditions (anaerobiosis) but also Ca 2+ signaling in Chlamydomonas reinhardtii 197,735 . Regulation by anaerobiosis would be mediated by a protein, Anaerobic Response 1 (ANR1). ANR1, as well as the plastid localized Ca 2+ sensor (CAS) are found in the PSI-Cytb6f supercomplex, together with PGRL1

735 . The Cytb6f associated subunit PETO co-fractionates with other thylakoid proteins involved in CET (ANR1, PGRL1, FNR). The absence of PETO impairs the formation of the supercomplex in anaerobic conditions.

Since its discovery and biochemical characterization, the existence/function of the Chlamydomonas CET supercomplex has been debated. The biochemical significance of the green band of high molecular weight in sucrose density gradient that was used to purify this complex has been recently questioned, because various PSI complexes can migrate in these sucrose density regions 376 . On the other hand, single-particle analysis of the PSI-Cytb6f complex from Chlamydomonas 736 has provided a structural support for the existence of a true supercomplexes in this alga.

Until recently, no such supercomplex was found in plants 426 , and it was argued that no such supercomplex is required for CET to occur in plants 410,714 . However, the isolation a PSI-Cytb6f supercomplex has been reported starting from stroma lamellae of Arabidopsis 737 . However, even in this case the significance of this complex has been questioned 374 , because in this preparation Cytb6f complex bound to PSI was not only in its active (dimeric) conformation but also in the inactive (monomeric) one.

At variance with the respiratory chain, where several supercomplexes between different component of the electron flow chain have been characterized, no other photosynthetic supercomplex has been reported so far, besides the PSI-NDH supercomplex that was characterized in Arabidopsis and later in Arabidopsis, STN7 745 . It is as a ser/thr kinase located in the thylakoids. A phosphatase (TAP38/PPH1) has also been found, which dephosphorylates LHCII 746,747 during the state 2 to state 1 transition.

Several pieces of evidence suggest that the PQ(H2) pool activates the Stt7/STN7 kinase via Cytb6f.

Wollman and Lemaire 748 used Chamydomonas lines with mutations in the photosynthetic apparatus to demonstrate that state transitions were missing in mutants lacking the Cytb6f. Later, independent spectroscopic 749 and genetic evidenced 576 suggested that the Cytb6f mediates kinase activation upon PQH2 binding to its lumenal binding pocket, via a still largely unknown mechanism.

Several models have been proposed so far to account for the Cytb6f mediated activation of the sTT7/STN7 kinase 724,[750][751][752][753] . They are summarized in a multistep model proposed in Fig. 39. There, PQH2 binding to the cytochrome lumenal pocket would induce conformational changes (via movements of the ISP-HD) making the Cytb6f-kinase interaction possible. Formation of a Cytb6fkinase complex would activate this protein leading to the phosphorylation of the Cytb6f-associated subunit PetO 743,754,755 . This step would precede LHCII phosphorylaytion, implying the existence of an additional step, where the active kinase would be released from Cytb6f to interact with the PSII antenna. The existence of two separate steps for Cytb6f and LHCII phosphorylation is suggested by the finding that PQ analogue stigmatellin, which blocks Cytb6f activity likely by interfering with the movement of the ISP-HD [START_REF] Zhang | Electron Transfer by Domain Movement in Cytochrome Bc1[END_REF]554,755 prevents LHCII phosphorylation but not the phosphorylation of PetO.

This model also accounts for additional information concerning the induction of state transitions. The first one is how the signal generated by PQH2 binding in the lumenal part of the complex is transduced to the stromal site where the catalytic domain of STT7/STN7 is located.

Several hypotheses have been conceived to address this point: Vener and colleagues 749 proposed that the putative transmembrane helix of the kinase could directly sense PQH2 binding to the luminal site, leading to kinase activation. Consistent with this possibility, Shapiguzov and colleagues found that the N terminus moiety of the kinase (which is supposed to be on the lumenal side) interacts with the transmembrane part of the ISP, based on yeast two hybrid 751 . Alternatively, a chlorophyll-based hypothesis has been proposed by Zito and colleagues. These authors analyzed of a Cytb6f mutant of Chlamydomonas, in which the small subunit PetL was fused to subunit IV to "transform" the Cytb6f, which contains seven helices in its core complex subunits (Cytb6-and sub IV), into a Cytbc1-type of complex (which contains eight helices in its unique core complex subunit, Cytb). They found that while this mutation was not affecting electron flow, it was able to completely abolish state transitions 756 . They conclude therefore that the mutation was sterically hindering kinase docking to Cytb6f, i.e.

that the kinase docking site was located in the proximity of the C terminus of subunit IV (where the subunit PetL was fused in the mutant). There, the tetrapyrrole ring of Cytb6f bound chlorophyll is exposed to the lipid solvent. Conversely, the PQ phytol chain goes deeply into the complex structure ending up in the lumenal pocket (in Chlamydomonas 41 ) or in proximity of the F helix, which sandwiches the chlorophyll ring in the cyanobacterium Mastigocladus laminosus [START_REF] Kurisu | Structure of the Cytochrome b6f Complex of Oxygenic Photosynthesis: Tuning the Cavity[END_REF] . There, it could sense PQH2 binding to its luminal site via a conformational change 753 and transduce the signal via its chlorophyll ring. The possible involvement of the chlorophyll ring in kinase activation was later supported by the finding that mutants affecting the environment of the chlorophyll ring in the complex were altering the kinetics of state transitions in Chlamydomonas 695 . Hasan and colleagues further developed this concept, proposing that specific lipids located on the surface of Cytb6f close to the chlorophyll could provide an adaptable surface for interaction with Stt7/STN7 174 . More recently, this hypothesis has been refined by pinpointing several residues in the stromal loop linking helices F and G of Cytb6f subunit IV (where the chlorophyll ring is exposed) that are crucial for state transitions.

This has led to the idea of an interaction between Cytb6f and the Stt7 kinase mediated by the stromal side of the complex above the chlorophyll ring 696 .

The second information concerns inhibition of state transitions in high-light treated Chlamydomonas cells 757 and plants 758,759 . It was proposed that this inhibition would stem from redox control by reduced stromal thioredoxins targeting a disulfide bond in the kinase 758 . Analysis of the Stt7/STN7 protein sequences revealed indeed the presence of two conserved Cys residues close to the N-terminal end of this kinase, which are highly conserved and essential for kinase activity 274,744 . However, mutagenesis of the Cys pair of the Stt7/STN7 kinase showed no change in the redox state of these Cys during state transitions and high-light treatment. Based on these results, a different role for Cys in kinase activation by the Cytb6f was proposed 751 in which these residues would allow the formation of a dimer between two kinases bound to each monomer of the Cytb6f dimer via formation of intermolecular disulfide bridges. Consistent with this idea, while no STN7 dimer was detectable in vivo, it could be clearly detected in the Arabidopsis STN7-Cys mutants in which C1 or C2 was stabilized 760 . Such dimer would be rather unstable and revert rapidly to an intramolecular disulfide bridge in each monomer when the luminal site is empty, explaining why the kinase rapidly inactivates upon oxidation of the PQ(H2) pool.

The molecular description of the Stt7 kinase 753 and its interaction with Cytb6f are unknown or unclear. The Stt7 kinase has been cloned, expressed, and purified in a heterologous host. The kinase is active in vitro in the presence of reductant and has been purified as a tetramer, determined by analytical ultracentrifugation, electron microscopy, and electrospray ionization mass spectrometry, with a molecular weight of 332 kDa, consisting of an 83.41-kDa monomer. Far-UV circular dichroism spectra have shown Stt7 to be mostly α-helical and document a physical interaction with Cytb6f through increased thermal stability of Stt7 secondary structure. The activity of wild-type Stt7 and its Cys-Ser mutant at positions 68 and 73 in the presence of a reductant suggest that the enzyme does not require a disulfide bridge for its activity as suggested elsewhere. Kinase activation in vivo could result from direct interaction between Stt7 and Cytb6f or long-range reduction of Stt7 by superoxide, known to be generated in Cytb6f by quinol oxidation. Although kinase activation on the n side of the thylakoid membrane requires a transmembrane configuration of the kinase (Fig. 39), the only hydrophobic domain of the kinase, near the N-terminus, contains 3-4 prolines. There is no known precedent for such a transmembrane domain. The active kinase would be in a dimeric state, thanks to the interaction between conserved cysteines present on every monomer (black squares) 751 . It would interact with LHCII promoting its phosphorylation, and migration from PSII to PSI. Alternatively, a ternary binding complex might be formed between LHCII, Cytb6f and the kinase. Finally, monomerization of the kinase would inactivate it, closing the activation cycle. See also Singh et al. 753 , for a further discussion.

11 Comments on the nomenclature used for Cytbc1 and Cytb6f

In the literature there are number of differences in naming of similar structural and/or functional elements of Cytbc1 and Cytb6f. This section contains short comments on nomenclature to clarify the potentially confusing terms and, when needed, explains how they are used in this review.

Complexes:

The term "Cyt-bc" refers to both Cytbc1 and Cytb6f as enzymes belonging to one group traditionally named cytochromes bc. This is how it is used in the review. Based on phylogenic analysis, it has been proposed to rename this group as "Rieske/b complexes", as it would better reflect the key and evolutionary unchanged subunits. 761 However this name, in spite of being scientifically justified, did not gain popularity in the literature.

In mitochondrial electron transport chain, Cytbc1 is also named "mitochondrial complex III" or in short "complex III".

Figure 1 .

 1 Figure 1. Overview of electron transfer paths involving Cyt-bc complexes: Cytbc1 in mitochondrial respiratory chain (A) and in anoxygenic photosynthesis or respiration of purple bacteria (B); Cytb6f in oxygenic photosynthesis in chloroplasts (C). Q and PQ denote ubiquinone and plastoquinone pool, respectively. PC, Fd and FNR denote plastocyanin, ferredoxin and ferredoxin/NADP + oxidoreductase. LET depicts linear electron transfer in photosynthesis while CET shows cyclic electron transfer path which recycles the electrons from Fd pool back to the PQ(H2) pool. Scheme in A considers mitochondria of both plants and animals (the diiron alternative oxidase is not present in animal mitochondria except for rare cases, such as in Trypanosoma).

Figure 2 .

 2 Figure 2. Overview of structures and cofactor chains in dimeric Cyt-bc complexes. Top: Crystal structure representations of M. laminosus Cytb6f (left, PDB ID: 4H13), R. capsulatus Cytbc1 (middle, PDB ID: 1ZRT) and mitochondrial Cytbc1 (right, PDB ID: 1KYO). Bottom: Spatial arrangement of cofactors extracted from the structures above. The layout of cofactors in R. capsulatus and mitochondrial Cytbc1 is the same. The simplified cartoon models depict symbols for cofactors that are used throughout the figures.

Figure 3 .

 3 Figure 3. A simplified, static scheme, showing step-wise reactions taking place during the catalytic cycle of Cytbc1. (A) Quinone (Q) and Quinol (QH2) (yellow hexagon and white hexagon with 2 dots) bind to the Qi and Qo sites, respectively, while oxidized Cytc (oval with white rhombus) binds to the Cytc1 domain. (B) After binding, QH2 is oxidized, 2 protons are released (turquoise arrows) to the p side, one electron is transferred (red arrow) to Cytc through the high-potential chain, while the second electron goes (red arrow), through the low-potential chain, to Q bound at the Qi site. (C) Reduced Cytc (oval with red rhombus) and Q diffuse out of the respective sites (black arrows) while SQi (blue hexagon with dot) stays stably bound at the Qi site. (D) Second molecule of QH2 and oxidized Cytc bind to the Qo site and Cytc1, respectively.

Figure 4 :

 4 Figure 4: Two possible mechanisms of Q cycle in Cytb6f. (A) The "modified Q cycle" 39 which is similar to Cytbc1 -two electrons are delivered to the Qn site from Qp site, which requires oxidation of 2 PQH2 and reduction of 2 PC, per 1 reduced PQ at the Qn site. (B) Original Mitchell Q cycle 32 in which one electron delivered to the Qn site comes from the PQH2 oxidation at the Qp site and the second electron comes from electron donors at the n side (Fd/FNR). According to the original Mitchell mechanism 1P PQH2, 1 PC, 1 PQ and 1 n side donor are needed to complete the cycle. Color code and symbols as in Fig 3. Copper in PC is marked as blue dot. For simplicity proton paths are omitted and reactions in one monomer are shown while the second monomer is grayed out.

Figure 5 .

 5 Figure 5. Crystal structure and spatial arrangement of electron-transfer cofactors in Cytbc1 from Rhodobacter capsulatus (PDB ID: 1ZRT 48 ). A) Ribbon structure showing all catalytic subunits in the dimer with the overlaid cofactors. B) Redoxactive cofactors in the dimer with edge-to-edge (numbers) and iron-to-iron distances (numbers in brackets).

7. 4 )

 4 prompted scientists to look for structural factors responsible for controlled docking/release of the domain from the b-position during the catalysis. This seemed additionally justified by the specific effects of the Pf and Pm inhibitors on modulation of the position of ISP-HD and on interactions of ISP-HD with Cytb. None of the available structures succeeded in resolving the position of natural substrates, UQ/PQ or UQH2/PQH2, bound at the Qo/Qp site[START_REF] Berry | Unanswered Questions about the Structure of Cytochrome Bc1 Complexes[END_REF] which left room for interpretation of any observed structural effects in the context of possible effects of the presence of Q or QH2 at the catalytic site on the ISP-HD motion. Some additional clues have been drawn from MD simulations[START_REF] Postila | Key Role of Water in Proton Transfer at the Qo-Site of the Cytochrome Bc1 Complex Predicted by Atomistic Molecular Dynamics Simulations[END_REF][START_REF] Barragan | Identification of Ubiquinol Binding Motifs at the Qo-Site of the Cytochrome Bc1 Complex[END_REF][START_REF] Barragan | Mechanism of the Primary Charge Transfer Reaction in the Cytochrome Bc1 Complex[END_REF] .Structural changes in brief.Prior to discussions on the mechanisms of the ISP-HD motion, it is necessary to briefly describe key structural elements of ISP. The ISP subunit can be divided into 3 domains 98 : a) ISP-HD harbouring the redox-active [2Fe-2S] Rieske cluster (2Fe2S) b) the membranous anchor, consisting of a single hydrophobic helix (residues Rh 1 -38, Bt 1 -61), and c) the neck or hinge region (residues Rh 39 -48 or Bt 62 -74) containing highly conservative amino acid sequence[START_REF] Tian | Flexibility of the Neck Region of the Rieske Iron-Sulfur Protein Is Functionally Important in the Cytochrome Bc1 Complex[END_REF] connecting the anchor with the ISP-HD (see Fig.6). The two ISPs interwind the dimer in such a way that each of the ISP-HD, interacting with Cytc1 or Cytb of one monomer, has its anchor associated with Cytb of the second monomer (seeFig 5). The movement of ISP-HD is associated with conformational change within the flexible neck region (described below), rotation of the domain by approximately 57 to 65o 92,100 and displacement of the domain by approximately 2 nm.

Figure 6 .

 6 Figure 6. Structure of Cytb and two conformations of ISP in one monomer. (A) Structure based on PDB:1sqx with ISP-HD at the b-position (B) Structure based on PDB:1be3 with ISP-HD being at the c-position. The hydrophobic anchor of ISP (blue helix) is embedded in the lipid membrane (pale green square). In A and B the neck region connecting ISP-HD (green) with the anchor has two different conformations. Cytb is shown in gray.

Figure 7 .

 7 Figure 7. Several crystal structures of Cytbc1 monomers with different conformations of ISP-HD. The figure shows positions of 2Fe2S clusters (yellow and orange spheres) from the overlaid positions from bovine (PDB IDs: 1sqx, 1bgy, and 1be3), mouse (PDB ID 3cx5) and chicken (PDB ID: 1bcc) crystallographic structures of ISP. Blue, ochre and red ribbons represent ISP, Cytb and Cytc1, respectively. Hemes are shown as red sticks. Green sticks show the stigmatellin from 1sxq structure bound at the Qo site (yellow oval).

Figure 8 .

 8 Figure 8. Structural features and binding motifs of metallic cofactors of Cytbc1. A) Schematic representation of the transmembrane part of Cytb, showing helices involved in formation of the heme-bearing 4-helix bundle (A-D, pale orange rectangles). Hemes are shown as orange polyhedrons, positions of axial ligands of hemes are marked by black dots, R. capsulatus residue numbering is used. B) Structural model showing hemes b orientation within the transmembrane bundle and orientation of their axial ligands. Helices providing axial ligands are shown in pale orange cartoon, ligating histidines are colored orange. C) Structural model showing heme c1 binding site in the globular domain of Cytc1. Cysteines of the binding motif are colored light blue, axial ligands are colored pale green. D) Structural model showing the [2Fe-2S] cluster binding site in ISP. Coordinating residues are labeled in black, cysteines that form the disulfide bridge are colored and labeled in green. All representations are based on PDB 1ZRT entry.

Figure 9 .

 9 Figure 9. Spectral features of Cytbc1 redox cofactors. A) Optical spectra of α/β-band region of hemes. Spectrum of the fully oxidized enzyme is shown in black, spectrum after reduction with sodium ascorbate is shown in orange (showing prominent contribution from heme c1) and spectrum of the fully reduced enzyme (with dithionite) is shown in magenta. B) EPR spectra of gz transitions of hemes bL and bH in an air-oxidized sample (top), sample of the enzyme inhibited with antimycin A (middle) and a sample with generated SQi (bottom). Numbers indicate g values. C) EPR spectrum of a oneelectron reduced [2Fe-2S] cluster in isolated Cytbc1.

Figure 10 .

 10 Figure 10. The Dimeric cytochrome b6f complex. (A) Polypeptide subunits from the cyanobacterium Nostoc sp PCC 7120 (PDB ID 4OGQ) are shown in ribbon format-Cytb6 (yellow), subIV (light brown), Cytf (orange), ISP (dark green), PetL (red), PetM (light green), PetG (blue), PetN (black). (B) Prosthetic groups of Cytb6f. Transmembrane hemes bp, bn (red/blue), and cn (black/blue) are shown as sticks. On the p side, heme f (green/blue) is shown as sticks, the [2Fe-2S] cluster as spheres (brown/yellow), and the Chl-a (green/blue) and β-car (yellow) as sticks. (C) Heme-heme distances within the transmembrane domain of the complex (PDB ID 4OGQ). Distances shown as black dashes. Heme edge-edge, and center-center (Fe-Fe) distances are shown, respectively, outside and inside parentheses (selected atoms shown as spheres). The dielectric constant, εij, between each pair of hemes, εn1,p1, εn1,p2, εn2,p1, etc. 184 is shown. (D) Transmembrane heme ligation in Cytb6f. The central Fe atom of heme bp and bn is axially ligated, respectively, by residues His86/His187 and His100/His202 which bridge the B and D TMH of Cytb, Heme cn is covalently attached to the protein via Cys35.

oxide 186 .

 186 Electrochemically positive, negative sides of membrane labeled p, n. (Reprinted/adapted by permission from Springer Nature,Cytochrome Complexes: Evolution,Structures, Energy Transduction, and Signaling; Structure-Function of the Cytochrome b6f Lipoprotein Complex by William A. Cramer, S. Saif Hasan, 2016).

Figure 11 .

 11 Figure 11. High potential electron acceptor of Cyt-bc: Cytf of Cytb6f (A, B) and Cytc1 of Cytbc1 complex (C, D). Cytf

Figure 12 .

 12 Figure 12. Transmembrane helices and distribution of lipid in Cytb6f complex (Figure drawn by S. Saif Hasan). Reprinted/adapted by permission from Springer Nature,Cytochrome Complexes: Evolution,Structures, Energy Transduction, and Signaling; Structure-Function of the Cytochrome b6f Lipoprotein Complex by William A. Cramer, S.SaifHasan, 2016.

  followed 15 years after the original inference of the chemiosmotic concept of membrane energization 28 . The demonstration of oxidantinduced reduction of Cytb heme in the mitochondrial Cytbc1 and the requirement in this electron transfer reaction for the high potential (ca. +0.25 V) 2Fe2S was elucidated in studies by B. Trumpower 203 . An apparently anomalous reduction of the Cytb component was observed in a preparation of mitochondrial membrane protein 'complex III'. Support for the Q cycle model ofr proton translocation in the mitochondrial Cytbc1 was provided in studies by Wikstrom and Saraste 204 , and Rich 205 .

Figure 13 .

 13 Figure 13. Chemical structures of biologically important quinones with a different number of isoprenoid molecules comprising the hydrophobic side chains. From top to bottom there are: plastoquinone-9 (PQ-9), ubiquinone-10 (UQ-10) and menaquinone-7 (MK-7). The Em,7 values corresponds to the average redox midpoint potentials at pH 7 for the respective Q/QH2 couples.

(Em, 7 )

 7 is approximately +300 mV and decreases to +230 and +176 mV upon attachement of one or two methyl groups to the ring at position 2 and 2,3, respectively 246 . On the other hand, substituting the ring with chlorine at position 2 and 5 increases the Em to approximately +540 mV. Attachment of methoxy groups to the ring also increases the measured Em in relation to 1,4-bezoquinone. For example, the Em of 2,6-dimethoxy-1,4-benzoquinone is approx. +340 mV, which is higher by about 200 mV than the Em of 2,6-dimethylo-1,4-bezoquinone. The redox midpoint potentials of quinones belonging to the family of menaquinones are significantly lower than those of benzoquinone derivatives, for example the Em, 7 of 2-methy-1,4-naphtoquinone (menadione) is only -5 mV, which is much lower than +230 mV of methyl-1,4-benzoquinone. Due to such a low redox midpoint potentials of menaquinones, they are considered as "ancient devices" for electron transport in living organisms recruited at early stages of evolution by some eubacteria and/or archaebacteria, at a time, when the atmosphere on Earth was anaerobic160,241 .This general rule of changes in theEm values of quinones containing different groups substituted to the ring holds true for natural, biologically active quinones that are used by living organisms as substrates for Cytbc. It means, that the Em of Q/QH2 couples increases in the order: menaquinone/menaquinol (Em,7[MK/MKH2] = -60 mV), ubiquinone/ubiquinol (Em,7[UQ/UQH2] = 70 mV) and plastoquinone/plastoquinol (Em,7[PQ/PQH2] = 110 mV) (see fig. 13)

Figure 14 .

 14 Figure 14. Binding of Cytc to Cytbc1. A) Fragment of the structure of Cytbc1 co-crystallized with Cytc (PDB: 1KYO). B) Comparison of electrostatic potential of the surface of Cytbc1 accessible to Cytc in two different positions of ISP-HD. C) Schematic representation showing different molecular organization of Cytc near the binding domain of Cytbc1 at low and high ionic strength.

Figure 15 .

 15 Figure 15. Comparison of charge distribution on the n side surface of the Cytb6f with known Fd-binding sites. A) Cytb6f monomer from M. laminosa (taken from 2D2C), seen as looking down from the n side B) Cytb6f monomer from C. reinhardtii (taken from 1Q90), seen as looking down from the n side . C) Fd-dependent nitrite reductase (2AKJ), looking down on the Fd binding site, as defined by site directed mutagenesis studies. D) Fe:NADP(H) oxidoreductase (FNR, 1GAW), looking down on the Fd binding site, as defined by site directed mutagenesis studies and co-crystalization. Known Fd binding sites are highlighted with a black circle. The putative Fd binding site on Cytb6f suggested by Nawrocki et al (2019) 374 is indicated by a hashed line. Positively charged groups in blue, negatively charged groups in red.

Figure 16 .

 16 Figure 16. Structural features of antimycin and related compounds. A) Structural formulae of compounds belonging to the antimycin A family. The upper formula represents the general structural elements of antimycins A, the lower is a formula of antimycin A1b. B) Stick/ribbon representation of binding of antimycin A in the Qi site, as revealed by X-ray crystallography. Antimycin A (pale green sticks) forms a direct hydrogen bond to Bt Asp228 and additional, water-bridged bonds to Bt Lys227, Bt His201 and Bt Ser35 (gray sticks). Oxygen and nitrogen atoms are colored red and blue, respectively. Heme bH shown in brown. The model is based on PDB entry 1PPJ. C) Structural formulae of synthetic antimycin analogues. D) Structural formula of UK-2A. In A, C and D, pale-red, dotted frames indicate the toxophore moiety of each compound.

  A, upper middle), isolated from fungal strain Capnodium sp. 33985. The AS2077715 has been shown to exhibit selective and highly inhibitory properties towards Trichophyton mentagrophytes Cytbc1, while being a weak inhibitor of the mammalian counterpart. Therefore, it has been proposed as a potential drug for Trichophyton infections.453

  B, top and middle)462 or MJM170 (Fig.17 B, bottom)463 . The co-crystal structures of bovine enzyme with these three novel inhibitors indicate that they all bind close to heme bH, with the carbonyl group of the pyridone toxophore in a direct contact to the conserved Bt Ser35462,463 .

Figure 17 .

 17 Figure 17. Structural formulae of Qi site inhibitors: A) Naturally occurring 4-hydroxy-2-pyridone deriviatives. B) Synthetic 4-pyridone-based antimalarial drugs. C) Diuron. D) Sulfonamide-based commercial fungicides. Pale-red, dotted frames indicate the toxophore moiety of each compound.

  D left) and amisulbrom (Fig.17D right) are commercial, synthetic compounds which are imidazole and triazole sulfonamides, respectively. Both are fungicides with

Figure 18 .

 18 Figure 18. Qo site inhibitors that fix ISP-HD at b-position. A) Structural formulae of natural stigmatellin A and synthetic tridecyl-stigmatellin. Pale-red, dotted frames indicate the toxophore moiety of each compound. B) Stick/cartoon representation of binding of stigmatellin A in the Qo site, as revealed by X-ray crystallography. Stigmatellin A (yellow sticks) forms hydrogen bonds to Bt Glu271 of Cytb (gray sticks) and to Bt His161 of ISP (pale cyan sticks). Oxygen and nitrogen atoms are colored red and blue, respectively. Heme bL is shown in brown. Model is based on PDB entry 1PPJ. C) Structural formulae of atovaquone (left) and DBMIB (right). The toxophore of atovaquone is marked with a frame.

Figure 19 .

 19 Figure 19. Structural features of 6-hydroxy-4,7-benzothiazolediones. A) General structural formulae of nHDBT compounds. B) Possible tautomeric states of nHDBT compounds.

Figure 20 .

 20 Figure 20. Crocacins and famoxadone-like compounds. A) Structural formulae of natural crocacins A and D (top and middle, respectively) and synthetic derivative of crocacin D (bottom). B) Structural formulae of famoxadone (upper), JG144 (lower, left) and fenamidone (lower, right). Pale-red, dotted frames indicate the toxophore moiety of each compound.

Figure 21 .

 21 Figure 21. MOA-comprising myxobacterial toxins. A) Structural formulae of natural Qo site inhibitors with dithiazole moieties: myxothiazol A (top) comprising β-methoxyacrylamide toxophore and two other, comprising the βmethoxyacrylate toxophore, melithiazol A (middle) and cystothiazole A (bottom). B) Structural formula of cyrmenin B1. Pale-red, dotted frames indicate the toxophore moiety of each compound.

Figure 22 .

 22 Figure 22. Structural formulae of selected natural (A) and synthetic (B) strobilurins. Pale-red, dotted frames indicate the toxophore moiety of each compound. Numbers within frames indicate the type of toxophore (explained in text).

Figure 23 .

 23 Figure 23. Structural formulae of (A) macrolides isolated from sea sponges, with their methyl-carbamate toxophores marked with frames, and (B) pyrimorph. Pale-red, dotted frames indicate the toxophore moiety of each compound.

Figure 24 .

 24 Figure 24. Structural formulae of dual-mode inhibitors of Cytbc1. A) 1-hydroxyquinolin-4-ones. B) Tautomeric states of 1-hydroxyquinolin-4-one. C) Ametoctradin. D) Quinolones. E) Ascochlorin.

25 ,

 25 lower), have been shown to inhibit porcine Cytbc1 with efficiency comparable to commercially available inhibitors 546 .

Figure 25 .

 25 Figure 25. Structural formulae of karrikinolide (upper) and its 4-n-butylphenyl derivative (lower panel).

  Qp site. Typical inhibitors of this site are: DNP-INT, stigmatellin and DBMIB.DNP-INT(2,4-dinitrophenylether of 2-iodo-4-nitro-thymol) (Fig.26) belongs to the family of dinitrophenyl ethers 547 , i.e., compounds that compete with plastoquinol for its oxidising pocket without having redox properties. These compounds do not act as electron donors or redox mediators, unlike DBMIB (see below). DNP-INT inhibits photosynthetic electron flow in vitro, where its activity can be circumvented by addition of TMPD (N-tetramethyl-p-phenylenediamine), an artificial electron carrier able to mediate e-flow from PQH2 to the Cytf(Trebst 1980). DNP-INT is also active in vivo as demonstrated by the work of Delosme and colleagues in Chlorella sorokiniana548 . In this organism, DNP-INT lowers the rate of Cytb6 oxidation and of PC reduction under single turnover flash conditions. Furthermore, DNP-INT diminishes the amplitude of the slow phase of the Electrochromic Band Shift (ECS), a modification of the absorption spectrum of intra-membrane pigments in response to the transmembrane electrical field (ca. 3x10 5 V/cm) an expression of the quantummechanical Stark Effect, following the establishement of the proton motive force across the thylakoids membrane, 549 . The ECS slow phase (phase B) has been proposed to be a consequence of the transfer of an electron across the low potential chain of the cytochrome complex 550 . A study by Fitzpatrick and colleagues challenged the efficiency of DNT-INT as an inhibitor of photosynthetic electron flow. A possible explanation for this finding is that DNP-INT would be a poor inhibitor in continuous light, while efficiently blocking Cytb6f under single turnover illumination, as previously reported 551 .

Figure 26 .

 26 Figure 26. Chemical structure of DNP-INT inhibitor

  authors found that DNP-INT affects equally the low-and high-potential chain of Cytb6f decreasing the rates of both Cytf reduction and Cytb6 turnover. Conversely, DBMIB inhibits only the rate of Cytf reduction while reducing, at the same time, the amplitude of Cytb6 redox signals. The accessibility of DNP-INT to the Qp site was unaffected by preillumination, while that of DBMIB was greatly enhanced, even after a single turnover. Overall, the authors propose that oxidation of DBMIBH2 by a single turnover flash would trigger the same conformational changes of the ISP-HD that occurs during oxidation of PQH2 in unpoisoned Cytb6f. This would allow transferring one electron to the high-potential chain, and generating at the same time the inhibitory DBMIB species 551 . Slow reduction of DBMIB inside the Qp pocket (see Rich et al. 557 ) would regenerate DBMIBH2, which would diffuse out of the quinol binding pocket, unlashing the inhibition. A relatively recent study by Sarewicz/Bujnowicz et. al. revealed that the EPR spectrum of 2Fe2S in DBMIBsupplemented spinach Cytb6f is frequency-dependent which is a strong indication that DBMIB semiquinone at the Qp site is spin-spin coupled to the reduced 2Fe2S 482 .

  95 for [UQ]/[UQH2] and [Cytc 3+ ]/[Cytc 2+ ], respectively 569 . Taking +260 mV as the Em at the physiological pH of 7 (Em7) of Cytc 3+ /Cytc 2+ 570 , +70 mV as the Em7 of Q/QH2 488 and the number of electrons n = 2, one may calculate the energy released during the oxidation of 1 mol of QH2 and reduction of 2 mols of Cytc: ΔG = -nFΔEh = -66 kJ/mol. (eq. 15)

  KQH2-Qo = [Qo-QH2] / [Qo-empty] = ~ 10 (eq. 19) KISPox = [Qo-QH2-ISP-HDQo] / [Qo-ISP-HDremote] = ~10 (eq. 20) where: [Qo-QH2] and [Qo-empty] are the concentrations of QH2 at the Qo site and empty Qo site, respectively, at the time when ISP-HD containing oxidized 2Fe2S cluster is in position close to the Qo site. [Qo-QH2-ISP-HDQo] and [Qo-ISP-HDremote] denote relative concentrations of ISP-HD containing oxidized 2Fe2S at position close to the Qo site with bound QH2 and remote from the site, respectively.

Figure 27 .

 27 Figure 27. Comparison of structure of the Qo site from Bos taurus with bound myxothiazol (A) or stigmatellin (B). Prepared on basis of 1SQP 77 and 1PP9 123 , respectively. Amino acids of ISP are not shown in A, since myxothiazol induce c-position of ISP-HD. The imidazole ring of the 2Fe2S ligand -histidine 161, is shown in B as a structure forming an H bond to stigmatellin.

Figure 28 .

 28 Figure 28. Energy diagram of electron transfer through cofactor chains in Cytbc1. Horizontal black lines mark Em values of the respective oxidized/reduced couple and are labeled with an orange letter in bracket. Gray gradient denotes a split in potentials between Q/SQ -and SQ -/QH2 couples at the Qo site. The average Em value of membranous Q/QH2 pool is

Figure 29 .

 29 Figure 29. Scheme showing 4 types of short circuit reactions that are theoretically possible during the catalytic cycle of Cytbc1 and Cytb6f 259 . Red arrows show energy conserving ET while blue arrows shows energy-wasting short circuiting ET. Numbers denote the order of ET events. Short circuits start with QH2 (SC1 and SC2) or Q (SC3 and SC4) present at the Qo/Qn site. Gray rhombuses denote hemes and white-yellow circles show 2Fe2S. Scheme does not consider redox states of cofactors. Dashed arrows and 3 positions of 2Fe2S denote movement of the ISP-HD (necessary to complete SC1). See text for details.

  this context an interesting concept concerning the evolutionary adaptation of Cyt-bc complexes to the presence of oxygen was proposed recently by Bergdoll et al 160 . By comparing redox properties of organisms that use different quinones and live in aerobic and anaerobic conditions, they concluded that the Great Oxidation Event occurred about 2.5 billion years ago when the rise of atmospheric oxygen resulted in an up-shift of the ambient redox potential of hemes b and the 2Fe2S cluster. The global response of organisms, which included up-shift of the redox potential of the Q(H2) pool by 150 mV (menaquinone versus ubi/plastoquinone) and a commensurable increase in the entire set of redox potentials of the cofactors of Cytbc complexes, was aimed to avoid deleterious ROS generation.

Figure 30 .

 30 Figure 30. The "semireverse-Rieske off" model of ROS generation in the Qo site. (A) A simplified static scheme showing one of the possible sequences of reactions leading to ROS formation at the Qo site: oxidation of QH2 ("forward" box),

Figure 31 .

 31 Figure 31. Two forms of SQ detected by EPR for the Qo/Qp site of Cytbc: the spin coupled SQ-2Fe2S state (with prominent transition at g= 1.945) and the "free" SQo not coupled to 2Fe2S (with transition at g = 2.006). Experimental spectra of Cytbc1 (A,C,D) and Cytb6f (B) recorded at the X band. Spectra in A and B contain contributions from the spectrum of SQ-2Fe2S and the spectrum of 2Fe2S. C shows the spectrum of 2Fe2 alone, for comparison. The insert (D) presents the single-line spectrum of the SQo.

Figure 32 .

 32 Figure 32. A simplified energy diagram of Cytbc1 and b6f. The diagram shows the states of the catalytic reaction at the Qo/Qp site. 1) Conditions before the start of the bifurcation reaction -Qo/Qp is occupied by QH2,, 2Fe2S and heme bL/bp are oxidized. 2) Transition state -unstable semiquinone and the reduced 2Fe2S, oxidized heme bL/bp. 3) Conditions after full oxidation of QH2 -Qo/Qp is occupied by Q, cluster 2Fe2S and heme bL/bp are reduced. 4) Stage after electron transfer from heme bL/bp to heme bH/bn. This state has different energy in the Cytbc1 and Cytb6f due to the different potential of heme bH/bn in both enzymes and the potential differences between PQ and UQ. 5) Spin-coupled SQ-2Fe2S state with

Figure 33 .

 33 Figure 33. Structural details of the region around the Qi site. A) Overall view of the Q binding environment and elements involved in proton transfer to Q. B) Geometry of the Q binding at the Qi site. Labels indicate O1, O4, C1 and C4 atoms of the Q molecule and distances between Q and proton donors. Models based on PDB IDs: A206 and 1NTZ, A and B, respectively.

Figure 34 .

 34 Figure 34. A) Two populations of SQi detected by EPR. Upper: slow-relaxing SQi (SQiS) associated with the presence of reduced (and diamagnetic) heme bH near the Qi site. Green line illustrates the expected MP saturation profile of SQiS. Lower: fast-relaxing SQi (SQiF) associated with the presence of oxidized (and paramagnetic) heme bH near the Qi site. Blue line illustrates the expected saturation profile of SQiF. B) EPR-monitored redox titrations of SQiS and SQiF in a range of pH from 5 to 9. 163

Figure 35 .

 35 Figure 35. The cross-dimer electron equilibration leading to formation of SQiF (see details in the text)..

Figure. 36 .

 36 Figure.36. The charge polarization mechanisms of Q reduction at the Qi site. Boxes on the right represent the reaction steps A to G (see text for details). SQi bound at the Qi site (blue and green boxes refer to the Qi site occupied by SQiF and SQiS, respectively) is stabilized by equilibration between neutral and anionic form of the radical (upper and lower scheme

Figure 37 .

 37 Figure 37. Schematic representation of various forms of Cytbc1-like complexes containing Cytb fusion protein used to test possible combinations of electron transfer paths. A) Native-like system with all four branches active (B-B). B-E) Asymmetric forms containing one or more mutations inactivating either the Qo site (Cytb: Rh G158W; designated as WB) or heme bH (Cytb: Rh H212N; designated as B N ): WB-B -lower branch inactivated, N B-B -one upper branch inactivated; W N B-B -upper and lower branch on the same side inactivated; WB-B N -cross-inactivated enzyme.

Figure 38 .

 38 Figure 38. Putative advantage of dimers with possible ET through the hemes bL. A) Homodimeric Cytbc1 operates normally using two active monomers. B) A heterodimer in which one monomer has disabled heme bH and or Qi site (gray) is still still functional and able to oxidize QH2 at two Qo sites. C) A heterodimer containing two different mutations in each monomer, one with disabled Qo site (green) and second with heme bH or Qi site, still catalyze QH2 oxidation. D) and E) Homodimers containing mutations are inactive and/or contribute to elevated superoxide generation. Considering a sum of monomers from B to E, occurrence of intermonomer electron transfer gives: 3 active monomers, 3 inactive monomers

738.

  Formation of the PSI-NDH supercomplex requires the presence of specific forms of the PSI Light Harvesting Complex (LHCI, 739 ).

10 . 3

 103 Role of cytochrome b6f in state transitionsIn oxygenic photosynthesis, the two photosystems have distinct pigment containing antenna complexes, and consequently different spectral absorption features. PSI has an enriched light absorption capacity in the far-red region, while PSII has a slightly higher absorption capacity in the spectral regions where chlorophyll b absorbs. Under natural light conditions, where both the quality and quantity fluctuate with time 740 light absorption by these two complexes may be unbalanced, with negative consequences on the quantum yield of photosynthesis, which requires a commensurate light utilization by PSI and PSII for optimum photosynthetic yield.In plants and green algae, balanced light utilization is achieved via a regulatory mechanism called state transitions, which actively adjusts the absorption capacity of PSI and PSII, in response to chromatic changes in the light. Discovered byMurata and Sugahara (1969) andBonaventura and Myers (1969) 741,742 state transitions rely on a redox triggered phosphorylation of the PSII antenna complexes (LHCII), which dissociates from PSII binding to PSI under conditions where absorption of the former is enhanced (e.g. when the light available for photosynthesis enhances light absorption by chlorophyll b). This state is called state 2. Conversely, over-excitation of PSI (e.g. by far red light) leads to the detachment of LHCII form PSI, and its re-association to PSII, leading to state 1, 230 .Pharmacological and genetic experiments have pinpointed the PSII electron acceptors (the plastoquinone, PQ) pool as the sensor triggering LHCII phosphorylation 743 . This occurs via the activation of a specific kinase[START_REF] Rochaix | Protein Kinases and Phosphatases Involved in the Acclimation of the Photosynthetic Apparatus to a Changing Light Environment[END_REF] , mediated by the Cytb6f. The plastoquinone can act as the redox sensor because it is functionally 'located' between PSII and PSI. Thus, this redox active molecule 'senses' the relative imbalance in the light harvesting capacity of the two photosystems, becoming reduced when the PSII activity overcomes that of PSI, and oxidised when PSI is prevailing. The kinase responsible for LHCII phosphorylation has been first identified in Chlamydomonas, STT7744

Figure 39 .

 39 Figure39. Multistep model for the activation of the state transitions kinase by Cytb6f in photosynthetic eukaryotes. The model assumes that PQH2 (red) interaction with the Qn/Qp site leads to conformational changes within the complex, likely mediated by the movement of the ISP-HD 554 (yellow). These changes would to promote the association between the cytochrome and the kinase responsible for state transitions (Stt7 in algae, STN7 in plants). Cytb6f-kinase interaction would be mediated by residues surrounding the chlorophyll ring, as indicated by mutagenesis studies 756 . Upon binding, the kinase would become active and phosphorylate the Cytb6f-associated subunit PetO. The active kinase would then be released from the cytochrome complex, in a step possibly involving PQH2 oxidation, PQ release from the Qn site and conformational changes of the ISP, as suggested by the inhibitory effect of the PQ analogue stigmatellin on this step 755 .

Table 1 .

 1 . gz values measured for hemes in selected Cytbc1 variants.

	organism		gz value (concerns Cytbc1)
	(reference)	heme bL	heme bH	heme c1
	Rhodobacter capsulatus
	153	3.78	3.45	n.d.
	151	3.78	3.44	n.d.
			(3.47 /antimycin)
			(3.42 /SQi)
	152 , 150	3.78	3.44	3.16
	Rhodobacter sphaeroides
	153	3.78	3.49	3.36
	Saccharomyces cerevisiae
	141	3.76	3.60	3.49
	Bos taurus
	155	3.79	3.43	3.37

Table 2 .

 2 Em values of redox cofactors in selected Cytbc1 variants. Two values of Em for heme bH results from two-component fit to data obtained for Cytbc1 without antimycin.

		Em value [mV] (concerns Cytbc1)
	[2Fe2S]	heme c1	heme bL	heme bH *

*

table Erreur ! Source du renvoi introuvable..

	crystallization procedures, and does not include relatively weakly bound peptides which may be
	important for function but are lost during purification and crystallization. Molecular weights (MW)
	and pI values for the 8 core subunits of Cytb6f are summarized in Subunit MW (kDa)*	pI
	Cytf a	30.9	4.7
	Cytb6 a	24.7	8.5
	ISP a	19.3	5.3
	SubIV a	17.5	7.8
	Pet G a	3.9	8.1
	Pet L a	3.2	9.9
	Pet M a	3.5	4.3
	Pet N a	3.3	9.5
	FNR b	35.4	6.3
	PetO c	15.1	9.6
	PetP d	7.4	8.2
	a From cyanobacterium Nostoc sp. PCC 7120; b PetH; resolved on SDS-PAGE, spinach prep; MW,
	A. Thaliana; c PetO, C. reinhardtii ; d PetP, A. platens

Table 3 :

 3 Parameters of Subunits of Cytb6f

  287,288 . It is generally accepted that PC has evolved to replace a Cytc homologue -Cytc6, the primordial acceptor for electrons coming from the Cytf subunit of Cytb6f (see De la Rosa et al., and references therein 289 ). Following oxygenation of the atmosphere, oxidation of Fe 2+ to Fe 3+ in the ocean would have made it considerably less bioavailable, whilst oxidation of Cu to Cu 1+ would correspondingly have made it more soluble and therefore bioavailable.

  197,236 , and FNR is no longer recruited efficiently to the membrane in the classic pgr5 mutant of the alga C. reinhardtii 411 . Inhibitors of FNR indicate that the enzyme is essential to some CET pathways233,375,[412][413][414][415][416] , although FNR does not bind antimycin A in solution233 , and is relatively insensitive to this CET inhibitor 417 . Immuno-inhibition studies show that blocking Fd to NADP(H) electron transfer by FNR does not affect CET activity414,418 , indicating that any CET role for FNR does not involve the NADP

+ binding site. On the other hand, It has been reported that Cytb6f complexes purified from spinach and incorporated into liposomes are only capable of NADPH mediated quinone reduction if the purification method is mild enough to preserve FNR binding 419 . It should be noted that the severe washes employed to remove FNR in this paper may also have removed other proteins important for this function, such as Fd and/or PgrL1. Importantly, addition of NADPH to the purified FNR:Cytb6f complex only results in reduction of Cytb6 in the presence of Fd 180 .

  dibromothymoquinone) (Fig.18 C) is probably the most representative of a class of inhibitors based on halogenated or hydroxylated lipophilic benzoquinones 547 . DBMIB has been extensively used as a specific inhibitor of PQH2 oxidation. Early results by Haehnel indicate that it does not affect PQ reduction by PSII, while blocking its reoxidation by PSI 555 . The number of DBMIB molecules required for inhibition is one per 300 chlorophylls, i.e. one per electron-transport chain. This would argue in favour of a 1 to 1 binding stoichiometry with Cytb6f. However, studies of the consequences of DBMIB on the EPR spectrum of the 2Fe2S by Roberts and Kramer led to the conclusion that two molecules of DBMIB bind to each monomer of the spinach Cytb6f, both in the isolated form and in intact thylakoid membranes556 . Binding likely occurs in two sites: a high affinity one, where DBMIB binding would cause small shifts in the EPR spectrum of the 2Fe2S similar to those induced by stigmatellin, and a low-affinity site, where binding was induced by super-

stoichiometric amounts of the inhibitor. DBMIB binding is accompanied by the appearance of a new EPR signal (g = 1.94). The binding of two molecules of DBMIB to the Qp/Qo pocket would be consistent with the so-called "double-occupancy" models proposed in the case of Cytbc1 (see paragraph Erreur ! Source du renvoi introuvable.

Acknowledgments

AO acknowledges financial support from National Science Centre, Poland, (grant 2015/18/A/NZ1/00046) and from Foundation for Polish Science (programme TEAM, POIR.04.04.00-00-5B54/17-00) WAC acknowledges support for the research studies described in this Review, supported at different times by the U. S. National Institutes of Health/General Medical Sciences (GMS-038323) and the U.

List of abbreviations

𝛥𝜇 ~𝐻 +

The transmembrane electrochemical potential gradient 2Fe2S

Quinone binding catalytic sites: The quinol oxidation site is named the "Qo site" (in the case of Cytbc1, in particular bacterial Cytbc1) or "Qp site" (in the case of Cytb6f and sometimes eukariotic Cytbc1/complex III). The same rule applies for the quinone reduction site which is named either the "Qi site" (Cytbc1) or the "Qn site" (Cytb6f and Cytbc1/complex III). The subscripts "o " and "i " were originally used by Mitchell to indicate "out" and "in", respectively, as the direction of proton transfers catalyzed by the respective sites. The subscripts "p" and "n " refer to "positive" and "negative" side of the membrane. In brief : Qo = Qp while Qi = Qn.

In this review we use "Qo/Qp "and "Qi /Qn" when referring to the sites in both Cytbc1 and Cytb6f ; "Qo" and "Qi" -when referring specifically to Cytbc1; "Qp" and "Qn"-when referring specifically to Cytb6f

Cofactor chains: In Cytbc1 the "high-potential chain" is alternatively named the "c-chain" and the "low-potential chain" is the "b-chain".

Protein subunits: In Cytb6f, the assembly of two subunits: Cytb6 and Subunit IV corresponds to one larger subunit -Cytb of Cytbc1.

Cofactors:

The b hemes in Cytbc1 and Cytb6f are often named differently. Hemes b in Cytbc1 are usually named bH and bL (to denote higher and lower Em of the heme, respectively). Their counterparts in Cytb6f are named bn and bp (to denote proximity of the heme to negative and positive side of the membrane, respectively). In brief: bH = bn while bL= bp.

In this review we use heme bH/bn and bL/bp when referring to hemes in Cytbc1 and Cytb6f. Hemes bH and bL are used when referring specifically to hemes of Cytbc1. Hemes bn and bp are used when referring specifically to hemes of Cytb6f.

The atypical c-type heme present in Cytb6f is designated using different symbols such as cn and ci and also cx. All these symbols describe the same heme.

Other: When describing the motion of ISP-HD, the "Qo-position" or "b-position" of ISP-HD refers to the same position of ISP-HD (when it is bound at the Qo site/cytochrome b or the Qp site/cytochrome b6).

S. Dept. of Energy (DOE DE-SC0018238).

GF acknowledges support by the French national funding agency ANR ('Momix' Projet-ANR-17-CE05-0029); the excellence cluster LabEX GRAL (ANR-10-LABX-49-01 financed within the University Grenoble Alpes graduate school -Ecoles Universitaires de Recherche-CBH-EUR-GS ANR-17-EURE-0003) and the European Research Council (ERC AdG 'Chloro-mito', grant no.

833184)

Short biographical notes

TOC Figure