domain: sdv chim

text:
Christina Ferousi

Simon Lindhoud

Frauke Baymann
email: baymann@imm.cnrs.fr

Eric R Hester

Joachim Reimann
email: jokelreimann@gmail.com

Boran Kartal
email: bkartal@mpi-bremen.de

Discovery of a functional, contracted heme-binding motif within a multiheme cytochrome

Keywords: anaerobic ammonium oxidation (anammox), cytochrome c, heme proteins, nitrogen cycle, EPR spectroscopy, nitric oxide (NO), hydrazine synthase

Anaerobic ammonium-oxidizing (anammox) bacteria convert nitrite and ammonium via nitric oxide (NO) and hydrazine into dinitrogen gas by using a diverse array of proteins, including numerous c-type cytochromes. Many new catalytic and spectroscopic properties of ctype cytochromes have been unraveled by studies on the biochemical pathways underlying the anammox process. The unique anammox intermediate hydrazine is produced by a multiheme cytochrome c protein, hydrazine synthase, through the comproportionation of ammonium and NO and the input of three electrons. It is unclear how these electrons are delivered to hydrazine synthase. Here, we report the discovery of a functional tetraheme c-type cytochrome from the anammox bacterium Kuenenia stuttgartiensis with a naturally occurring contracted Cys-Lys-Cys-His (CKCH) heme-binding motif, which is encoded in the hydrazine synthase gene cluster. The purified tetraheme protein (named here KsTH) exchanged electrons with hydrazine synthase. Complementary spectroscopic techniques revealed that this protein harbors four low-spin hexacoordinated hemes with His/Lys (heme 1), His/Cys (heme 2), and two His/His ligations (hemes 3 and 4). A genomic database search revealed that c-type cytochromes with a contracted CXCH heme-binding motif are present throughout the bacterial and archaeal domains in the tree of life, suggesting that this heme recognition site may be employed by many different groups of microorganisms.

Introduction

Anaerobic

ammonium-oxidizing (anammox) bacteria use nitrite as their terminal electron acceptor and produce dinitrogen gas (N2). These microorganisms first reduce nitrite to nitric oxide (NO) and then use hydrazine synthase (HZS), an enzyme unique to anammox bacteria, to combine NO with ammonium to form hydrazine. HZS is a heterotrimeric protein complex that harbors two spatially separated active site hemes and catalyzes the formation of hydrazine [START_REF] Kartal | Molecular mechanism of anaerobic ammonium oxidation[END_REF][START_REF] Dietl | The inner workings of the hydrazine synthase multiprotein complex[END_REF]. In HZS, the reductive conversion of nitric oxide (NO) yields hydroxylamine (NH2OH), which is then combined with ammonia through a comproportionation reaction [START_REF] Dietl | The inner workings of the hydrazine synthase multiprotein complex[END_REF].

The produced hydrazine is subsequently oxidized to N2 by hydrazine dehydrogenase (HDH) [START_REF] Maalcke | Characterization of Anammox Hydrazine Dehydrogenase, a Key N2-producing Enzyme in the Global Nitrogen Cycle[END_REF][START_REF] Akram | A 192-heme electron transfer network in the hydrazine dehydrogenase complex[END_REF] releasing four low-potential electrons that are used for carbon fixation and the preceding catabolic reactions such as hydrazine synthesis [START_REF] Kartal | How to make a living from anaerobic ammonium oxidation[END_REF][START_REF] Hu | Nitric oxide-dependent anaerobic ammonium oxidation[END_REF].

The entire HZS gene cluster (kuste2854-61)

is conserved in all sequenced anammox genomes, and two of the gene products (kuste2855-56 or KSMBR1_3597-3578 in the anammox bacterium Kuenenia stuttgartiensis) form a membraneassociated protein complex [START_REF] De Almeida | Membrane-bound electron transport systems of an anammox bacterium: A complexome analysis[END_REF][START_REF] Frank | Resolving the complete genome of Kuenenia stuttgartiensis from a membrane bioreactor enrichment using Single-Molecule Real-Time sequencing[END_REF]. This complex is suggested to form the membrane-bound, quinol-interacting electron transfer module (ETM) of HZS, and either interact directly with HZS, or work in tandem with a soluble electron carrier [START_REF] Kartal | How to make a living from anaerobic ammonium oxidation[END_REF]. The latter is the product of another gene from the HZS cluster (kuste2854 or KSMBR1_3596), hereafter referred to as KsTH ('Ks' standing for Kuenenia stuttgartiensis and 'TH' for Tetraheme). KsTH is a 236-amino-acid cytochrome c protein that contains three canonical heme c binding sites and belongs to the c554 protein family [START_REF] Sonnhammer | Pfam: a comprehensive database of protein domain families based on seed alignments[END_REF]. Cytochrome c554 is a crystallographically resolved tetraheme c-type cytochrome that is suggested to accept electrons from hydroxylamine oxidation catalyzed by hydroxylamine oxidoreductase (HAO) [START_REF] Hooper | Enzymology of the oxidation of ammonia to nitrite by bacteria[END_REF][START_REF] Arp | The Impact of Genome Analyses on Our Understanding of Ammonia-Oxidizing Bacteria[END_REF]. C554 harbors three hexa-coordinated hemes (i.e., heme 1, 3, and 4 in order of appearance in the sequence) and one pentacoordinated high-spin heme center (heme 2) [START_REF] Yamanaka | Cytochrome c-552 and cytochrome c-554 derived from Nitrosomonas europaea. Purification, properties, and their function in hydroxylamine oxidation[END_REF]. Heme 2 of c554 has also been implicated in NO binding and catalysis, albeit the latter remains inconclusive [START_REF] Arciero | Resolution of the four hemes of cytochrome c554 from Nitrosomonas europaea by redox potentiometry and optical spectroscopy[END_REF][START_REF] Iverson | High-resolution structures of the oxidized and reduced states of cytochrome c554 from Nitrosomonas europaea[END_REF][START_REF] Upadhyay | NO reductase activity of the tetraheme cytochrome c554 of Nitrosomonas europaea[END_REF][START_REF] Mcgarry | Upon further analysis, neither cytochrome c554 from Nitrosomonas europaea nor its F156A variant display NO reductase activity, though both proteins bind nitric oxide reversibly[END_REF].

Many reactions in the anaerobic ammonium oxidation pathway involve the activity of ctype cytochromes [START_REF] Kartal | Anammox Biochemistry: a Tale of Heme c Proteins[END_REF]. Indeed, anammox bacteria encode more than 60 cytochrome c proteins in their genomes, and their discovery enriched our knowledge on metabolic reactions that occur in nature [START_REF] Kartal | Molecular mechanism of anaerobic ammonium oxidation[END_REF][START_REF] Dietl | The inner workings of the hydrazine synthase multiprotein complex[END_REF][START_REF] Akram | A 192-heme electron transfer network in the hydrazine dehydrogenase complex[END_REF][START_REF] Maalcke | Structural basis of biological NO generation by octaheme oxidoreductases[END_REF]. Investigation of the biochemical by guest on [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] http://www.jbc.org/ Downloaded from reactions that underlie the anammox metabolism through the characterization of key enzymes has led to the discovery of novel properties of c-type cytochromes and the reactions they can catalyze (1-4, 18, 19).

Cytochrome c proteins are characterized by one or more c-type heme cofactors that are covalently bound to the protein backbone.

The two vinyl groups of a heme b moiety form thioether bonds with the sulfhydryls of two cysteine residues that are commonly arranged in the highly conserved amino acid sequence CXXCH, the canonical hemebinding motif [START_REF] Kranz | Cytochrome c biogenesis: mechanisms for covalent modifications and trafficking of heme and for heme-iron redox control[END_REF]. Although the functional implications of covalent heme attachment are not fully understood, it has been proposed that it enhances protein stability and allows for more solvent-exposed heme centers [START_REF] Kleingardner | Biological significance and applications of heme c proteins and peptides[END_REF]. The histidine residue of the CXXCH motif typically serves as the proximal ligand to the heme iron, whereas the distal ligand is often a histidine or a methionine residue at a variable distance from the motif in the amino acid sequence [START_REF] Liu | Metalloproteins Containing Cytochrome, Iron-Sulfur, or Copper Redox Centers[END_REF]. Even though the canonical CXXCH is by far the most common heme-binding motif, several cytochromes with more than two residues separating the cysteines have been described.

Results and Discussion

KsTH harbors four c-type heme cofactors

The gene product of kuste2854 was purified 2).

To probe the covalent attachments formed between the heme moieties and the protein backbone, the reduced alkaline pyridine hemochromogen spectrum of KsTH was recorded. Bis-pyridine heme adducts that are linked to the protein via two thioether bonds result in an absorbance maximum of the alpha band at 550 nm, whereas the presence of only a single thioether bond results in a red-shifted maximum at 553 nm [START_REF] Ishida | Design and synthesis of de novo cytochromes c[END_REF]. In perfect agreement, the reduced alkaline bis-pyridine adduct of KsTH displayed the typical absorbance maximum at 550 nm (Figure 3), which was indicative of two thioether bonds linking the heme to the protein backbone [START_REF] Ishida | Design and synthesis of de novo cytochromes c[END_REF]. Taken together, these results further corroborated that the CKCH motif served as the additional hemebinding site in KsTH.

All four hemes are low-spin and hexacoordinated

Visible and electron paramagnetic resonance (EPR) spectroscopy were used to investigate the spin state of the KsTH hemes as well as the nature of their ligation.

In optical spectroscopy, oxidized high-spin hemes with a single axial ligand exhibit a Soret maximum around 390 nm and a charge transfer absorption band around 600 nm, whereas in the reduced state a broad signal around 430 nm is generally observed [START_REF] Arciero | Resolution of the four hemes of cytochrome c554 from Nitrosomonas europaea by redox potentiometry and optical spectroscopy[END_REF][START_REF] Moore | Cytochromes c. Evolutionary, Structural and Physicochemical Aspects[END_REF]. Neither of these two features were observed in the KsTH spectra (Figure 4). In EPR spectroscopy high-spin hemes give rise to a strong signal centered at g=6 [START_REF] Moore | Cytochromes c. Evolutionary, Structural and Physicochemical Aspects[END_REF][START_REF] Palmer | The electron paramagnetic resonance of metalloproteins[END_REF], which was absent from EPR spectra of KsTH recorded under different conditions (Figure 5). Interactions between a high spin heme and a neighboring co-factor can result in spectral features different from the g=6 Furthermore, the EPR spectrum of KsTH was very similar to the spectrum of c554 at pH 12, where all hemes of c554 have been shown to be low-spin [START_REF] Andersson | Tetraheme cytochrome c554 from Nitrosomonas europaea. Heme-heme interactions and ligand binding[END_REF].

Heme axial ligation is inferred based on spectroscopy and sequence homology

The UV/Vis electronic absorption spectrum of the as-isolated (fully oxidized) KsTH displayed a broad Soret band with a maximum at 409 nm while, upon reduction, the Soret band maximum shifted to 418 nm with a pronounced increase in amplitude and decrease in half width. Absence of a shoulder at 437 nm and 564 nm (Figure 4A) ruled out the possibility of a hydroxide ligand for any of the hemes of KsTH [START_REF] Zhong | Ligation and Reactivity of Methionine-Oxidized Cytochrome c[END_REF] and absence of a charge transfer band at 695 nm most likely excluded methionine as a heme axial ligand [START_REF] Moore | Cytochromes c. Evolutionary, Structural and Physicochemical Aspects[END_REF]. Upon reduction, the Soret bands of His/Lys, His/His, and His/Cys ligated hemes shift from 408 nm to 417 nm [START_REF] Ubbink | Characterization of mutant Met100Lys of cytochrome c550 from Thiobacillus versutus with lysine-histidine heme ligation[END_REF], 407 nm to 420 nm [START_REF] Andersson | Tetraheme cytochrome c554 from Nitrosomonas europaea. Heme-heme interactions and ligand binding[END_REF], and 418 nm to 416 nm [START_REF] Zhong | Redoxdependent stability, protonation, and reactivity of cysteine-bound heme proteins[END_REF], respectively.

Although thiolate heme ligation in the ferric form usually exhibits red-shifted Soret His/His ligation [START_REF] Moore | Cytochromes c. Evolutionary, Structural and Physicochemical Aspects[END_REF]. The other signal features g values at 2.54, 2.29, and 1.84, which were compatible with a His/Cys ligated oxidized heme [START_REF] Zhong | Redoxdependent stability, protonation, and reactivity of cysteine-bound heme proteins[END_REF][START_REF] Motomura | Crystal structure and redox properties of a novel cyanobacterial heme-protein with a His/Cys heme axial ligation and a per-arnt-sim (PAS)-like domain[END_REF]36).

In order to retrieve more information about KsTH based on its primary structure, homology searches were performed against non-redundant protein sequences using

KsTH hemes have low redox midpoint potentials

To assess the redox properties of the heme cofactors of KsTH electrochemical redox titration monitored by optical spectroscopy was performed (Figure 6). KsTH titrated in two waves, one centered around -200 mV (vs. SHE) accounting for 66-85% of the total signal amplitude and the remaining 15-33% around -400 mV (vs. SHE).

EPR spectra recorded on samples prepared at a few selected potentials between the fully oxidized TH and -400 mV (Figure 5B)

showed characteristic individual EPR signatures for two of the hemes and interactions signatures at higher potentials when all four hemes were oxidized.

The three hemes of the high-potential wave Additionally, in the alkaline conformer of mitochondrial cytochrome c the native methionine distal ligand of the heme iron is replaced by a lysine [START_REF] Amacher | Lysine-Ligated State: Loop Refolding and Functional Implications of a Conformational Switch[END_REF].

Heme environments are flexible: external ligands can bind to the hemes

To further examine the heme ligand environment, potential external ligands were added to KsTH. Upon addition of CO to the reduced protein, 75% of the α band changed its shape, indicating that CO became a ligand to three out of four hemes (Figure 4B). The Soret band became narrower but, unexpectedly, did not increase in amplitude. Addition of a ~9-fold excess of NO/heme (600 µM NO for 65 µM heme) to the oxidized protein resulted in an optical spectrum of NO-bound heme (Figure 4C) and in loss of all EPR features (Figure 5C). Upon addition of an equimolar amount of NO, the EPR signal amplitude diminished by a factor of two (Figure 5C).

Contracted heme-binding motifs are present in a variety of genomes

The discovery of a naturally occurring contracted heme-binding motif in TH prompted us to investigate whether contracted binding sites also occurred in proteins other than KsTH and its orthologs.

We found 107 multiheme proteins that also contained the CXCH motif, which were widely spread across different phyla and present in both Archaea and Bacteria (Figure 8; Supplementary

Experimental Procedures

All chemicals used were purchased from Sigma-Aldrich, unless stated otherwise.

High-performance liquid chromatography (HPLC)-grade chemicals were purchased from Baker, USA. All purification steps took place in ambient air and at 4°C.

Cell-free extract preparation

Cells from a 10-liter laboratory scale enriched (~95% pure) K. stuttgartiensis

continuous

Protein Sequence Analyses

Protein sequence homology searches were performed against selected entries using the BlastP program at the NCBI website. Nterminal signal cleavage sites were predicted with SignalP 4.1 [START_REF] Petersen | SignalP 4.0: discriminating signal peptides from transmembrane regions[END_REF]. Multiple sequence alignments were performed using Muscle [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF] as implemented in the EMBL web server and then manually curated.

Mass spectrometry

Samples

Other analytical methods

Protein concentrations were measured with the Bio-Rad protein assay, based on the method of Bradford (50), using bovine serum albumin as standard. The pyridine hemochrome assay was performed as described previously [START_REF] Berry | Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra[END_REF].

Bioinformatic analyses

The NCBI non-redundant (nr) database and all publicly available genomes from the NCBI were surveyed for the contracted heme-binding motif. Sequences that contained a CXCH motif, while also containing a CXXCH motif were retrieved.

Sequences were excluded if the CXCH motif was CCCH, CXCCH, or CCXCH.

  directly from K. stuttgartiensis biomass by a three-step purification protocol. The purified protein migrated as a monomeric species on both native and SDS-denaturing PAGE. Peptide mass fingerprinting using MALDI-TOF mass spectrometry verified the identity of the protein as kuste2854. Tandem mass spectrometry analysis of the intact, albeit denatured full-length protein established a molecular mass of 26095.3 Da for the monoisotopic species (Figure 1A). The theoretical value of the apoprotein without the N-terminal targeting sequence (aa: 1-29) was 23634.79 Da. The +2460.4844 Da offset in mass could only be explained by the covalent binding of four heme groups to the protein backbone, resulting in a total theoretical mass of 26095.46 Da for the holoprotein, which was an exact match to the mass measured for the denatured full-length protein. Collisioninduced dissociation tandem mass spectrometry (CID MS/MS) detected only one dominant fragment ion of 617.18 [M+H] 1+ Da, and its spectrum matched the simulated isotope envelope of Feprotoporphyrin IX (heme b) (Figure 1B). Consequently, the observed mass discrepancy was most likely due to the covalent attachment of four heme groups instead of the three that were predicted from the presence of three canonical CXXCH heme c binding motifs in the KsTH

  bands around 418 nm[START_REF] Amacher | Lysine-Ligated State: Loop Refolding and Functional Implications of a Conformational Switch[END_REF], superposition of the optical contribution from a His/Cys ligated heme to spectra of His/His and/or His/Lys ligated hemes could account for the broad Soret band of oxidized KsTH and the pronounced narrowing and rise in the Soret band upon reduction. EPR spectroscopic studies on purified KsTH agreed with the above conclusions. The EPR spectrum of oxidized KsTH at 15 K had broad spectral features in the g=2.67 to g=1.74 range that relaxed slowly. The shape of these signals slightly narrowed at higher temperature, above 70 K (Figure5A). The broad shape and temperature-dependent behavior were both characteristic of a spin-spin interacting system of low-spin hemes. This was consistent with the EPR spectra of another tetraheme c-type cytochrome, cytochrome c554, where pairwise interaction between hemes 1 and 3 and hemes 2 and 4 induced interaction signals in EPR[START_REF] Upadhyay | Spectroscopic characterization and assignment of reduction potentials in the tetraheme cytochrome c554 from Nitrosomonas europaea[END_REF]. Partial reduction of the sample by small amounts of dithionite induced disappearance of the interaction signal and the appearance of two heme signals (Figure5B). One of these had a gz-value of 3.28, characteristic of highly anisotropic low-spin hemes with His/Lys or

  BlastP. While there was no significant hit to any characterized homolog, a conserved domain belonging to the c554 protein family was identified[START_REF] Sonnhammer | Pfam: a comprehensive database of protein domain families based on seed alignments[END_REF]. This domain was found close to the C-terminus and accounted for approximately one third of the total KsTH sequence length (i.e., amino acids 41-110).Multiple sequence alignments of KsTH orthologs with cytochrome c554 (Figure2) combined with structural insights from the tertiary structure of c554 allowed us to assign the above-mentioned identified heme ligations to specific hemes. For two of the hemes, the ligands that were detected in KsTH using EPR spectroscopy, could be identified in the amino acid sequence of the protein by sequence alignment between anammox TH and c554. They were conserved between all TH sequences from all known anammox species and situated within the stretch that was homologous to c554. The distal histidine ligand to heme 4 was conserved among c554 and TH (His30), and in TH there was a fully conserved lysine residue (Lys97) instead of the distal histidine ligand of heme 1 of c554. The His ligand to heme 3 of c554 was outside the protein sequence which was homologous to TH. However, in addition to the above discussed His ligands, there was only one further histidine fully conserved in TH sequences which was likely the sixth ligand to heme 3. As discussed above, all hemes of KsTH had two axial ligands, while heme 2 of c554 had only one. Based on the sequence alignment of KsTH with the structure of c554 we predicted a stretch of 6 amino acids (aa: 147-152) at the distal side of heme 2 of KsTH. This loop was fully conserved in all sequenced anammox genomes and contained one cysteine and three lysine residues that could potentially serve as iron ligands. EPR and UV-VIS spectroscopy points towards the presence of a Cys-ligated heme and we therefore propose Cys150 as the sixth ligand for heme 2 in KsTH. Taken together, our results showed that KsTH harbored four hexa-coordinated hemes with His/Lys (heme 1), His/Cys (heme 2), and two His/His ligations (heme 3 and heme 4).

  (-200 mV) exhibited slightly different redox midpoint potentials. Reduction of the highest potential heme induced loss of its interaction with the neighboring heme and, therefore, appearance of a signal at g=3.28 (pair of hemes 1 and 3). Further decrease of the potential first brought up the signal of the Cys ligated heme (heme 2) due to reduction of its interacting partner (heme 4), and then induced disappearance of the g=3.28. The low-potential wave (-400 mV) could be attributed to the Cys ligated heme, which could only be partially reduced by dithionite. Such low redox midpoint potentials have been reported earlier for His/Cys-ligated hemes[START_REF] Zhong | Redoxdependent stability, protonation, and reactivity of cysteine-bound heme proteins[END_REF][START_REF] Motomura | Crystal structure and redox properties of a novel cyanobacterial heme-protein with a His/Cys heme axial ligation and a per-arnt-sim (PAS)-like domain[END_REF].In optical spectroscopy the difference spectra of the hemes contributing to the transition at -200 mV could not be resolved. The spectrum showed an α band maximum at 552 nm and a Soret band at 418 nm. The spectrum of the low-potential component exhibited a blue-shifted α band at 549 nm accompanied by a split Soret band with a maximum at 418 nm and a shoulder at 425 nm (Figure 6B). Splitting of variable intensity was observed, depending on the sample preparation and the redox direction. A possible explanation for the variability of these spectroscopic signals could involve either conformational changes around the heme pocket and/or ligand exchange of the respective heme iron that might have occurred upon electrochemical reduction. An interesting case of redox-dependent ligand change has been reported for cytochrome c'' from Methylophilus methylotrophus in which the detachment of the distal histidine ligand upon reduction leads to spin-state transition (37).

  The reaction of NO with reduced TH was rapid as judged from the instant color change of the sample upon addition of NOsaturated buffer, even though extreme conditions such as low or high pH or incubation with high concentration of NO or CO for hours are usually necessary to achieve NO and CO binding to reduced and oxidized cytochrome c, respectively[START_REF] Butt | Absorption Spectra and Some Other Properties of Cytochrome c and of Its Compounds with Ligands[END_REF][START_REF] Hoshino | Photochemistry of nitric oxide adducts of water-soluble iron(III) porphyrin and ferrihemoproteins studied by nanosecond laser photolysis[END_REF]. Binding of NO to an oxidized heme can pull an electron from the heme iron resulting in loss of its paramagnetic properties. Our results suggested that NO became a ligand to all hemes of oxidized KsTH. However, exposure of pre-reduced KsTH to NO resulted in loss of the α band, corresponding to approximately two thirds of the hemes and concomitant appearance of a typical signal for NO-bound heme at 540/560 nm (Figure4D). A heme-NO signal appeared in EPR accounting for less than one heme. The lines of the hyperfine split signal were separated by 17 Gauss indicating that NO was bound as a fifth ligand to a reduced heme (Figure4C).Otherwise, the EPR spectra were nearly featureless, indicating that the NO-bound hemes were oxidized, except for the abovementioned NO-heme signal around g=2 which could be a degradation product. NO, therefore, partially oxidizes KsTH with an unknown reaction product. Whether this reaction is of physiological importance remains to be determined. Additions of either imidazole or azide to the reduced protein did not result in observable spectroscopic changes.KsTH and hydrazine synthase exhibit redox interactionTH is conserved within the hydrazine synthase gene cluster across all sequenced anammox genomes and was hypothesized to provide three electrons for the first halfreaction of hydrazine synthesis, i.e. NO reduction to hydroxylamine[START_REF] Kartal | How to make a living from anaerobic ammonium oxidation[END_REF].Therefore, electron transfer from KsTH to HZS was assessed. In a double-compartment cuvette, reduced KsTH and oxidized HZS gave rise to a Soret band at 415 nm with a shoulder at 425 nm, and an α band at 552 nm (Figure 7A), in agreement with the simulated combined spectrum. Mixing of the two enzymes resulted in loss of the shoulder at 425 nm and in the appearance of Soret band maxima at 409 and 417 nm. The 417 nm absorbance decreased over a time period of ten minutes accompanied by a shift of 409 nm band to 407 nm. EPR spectra on a 1:1 mixture of reduced KsTH and oxidized HZS (Figure 7B) resulted in loss of lines in the g=3 region due to reduction of the corresponding heme(s) of HZS. The g=2.54 signal of the Cys ligated heme 2 and the g=3.28 signal of either heme 1 or 3 of KsTH appeared. Disappearance of the shoulder at 425 nm together with the rise of the g=2.54 signal revealed oxidation of the low-potential heme of KsTH. Subsequent shift of the Soret band maximum from 409 to 407 nm indicated further oxidation of TH. Indeed, oxidized TH exhibited a blue-shifted Soret band maximum at 407 nm compared to HZS at 409 nm. This observation was corroborated by the g=3.28 signal of oxidized heme 1 or 3 that arose in EPR spectra. Therefore, about two electrons were transferred from KsTH to HZS, resulting in the rapid oxidation of heme 2 and the subsequent slower oxidation of either heme 1 or 3.

  figure legends. Several scans were

2 -

 2 Da peptide tolerance, and at most one trypsin mis-cleavage. Samples for ultrahigh resolution quadrupole time-of-flight (UHR-qTOF) mass spectrometry were prepared as follows. Purified protein sample buffer was exchanged for 0.1% formic acid 20% methanol using 3 kDa molecular weight cut-off filters (Amicon Ultracel) and subsequently analyzed by direct infusion electrospray ionization tandem mass spectrometry (ESI-MS/MS) using an ultrahigh resolution quadrupole time-offlight mass spectrometer (maXis 5G, Bruker Daltonics). The instrument was operated in positive ionization mode with the following parameters: capillary voltage: 4500 V, offset: 500 V, nebulizer gas pressure: 0.4 bar, N2 drying gas: 4L . min -1 at 200°C, in-source CID energy: 20 eV, mass range 300-3,700 m/z, 1 Hz acquisition rate. The z=31 + precursor ion (m/z 843.3) was isolated and subsequently fragmented using 25 eV collision induced dissociation (CID) energy. Acquired data was processed in Data Analysis 4.2 software (Bruker Daltonics). Spectral peak detection and charge deconvolution for MS and MS/MS spectra was performed by the SNAP2 algorithm. In addition, a high-resolution charge-deconvoluted MS spectrum was generated using the maximum entropy charge deconvolution algorithm (MaxEnt) which was compared to a simulated isotope pattern of KsTH (AA 30-236) plus four heme groups. The processed MS/MS spectrum was exported to BioTools 3.2 and compared against in silico b-and yfragment ion masses of KsTH (AA 30-236) plus four heme groups (0.05 Da mass tolerance).

SignalP 4 . 1 (

 41 [START_REF] Petersen | SignalP 4.0: discriminating signal peptides from transmembrane regions[END_REF] was used to predict if the sequence contained a signal peptide.Positive hits that also contained a signal peptide were manually curated to exclude (i) sequences of which either the annotation excluded that they were c-type cytochromes, (ii) sequences in which the contracted motif falls within the signal sequence, (iii) sequences in which the contracted motif overlapped with the canonical motif (i.e., either CXCHXCH or CXXCHCH). The lineage of the organism in which the protein originated was obtained using the Entrez efetch protocol via the Entrez python package. In order to investigate the phylogenetic relationship of taxa containing the contracted hemebinding motif, their genomes were downloaded from the NCBI database for phylogenetic analysis. The UBCG pipeline was used to extract conserved phylogenetic marker genes and build multiple alignments (52). A final concatenated alignment of the marker genes was used to infer the phylogenetic relationship of the taxa and visualized as an unrooted tree using the Interactive Tree of Life software (53).

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 12345 Figure 1: Mass spectrometry analysis of intact KsTH

Figure 6 :

 6 Figure 6: Optical redox titration

Table 1

 1

	),

by guest on November 19, 2019 http://www.jbc.org/ Downloaded from

K, microwave power 1 mW, modulation amplitude 10 Gauss. C) Spectra recorded on KsTH by guest on November 19, 2019 http://www.jbc.org/ Downloaded from

difference in absorbance between 552-565 nm (solid black line), resulting in midpoint potentials of -190±20 mV and -400±20 mV, respectively. Open and closed circles represent data recorded in the oxidative or reducing direction, respectively. B) Difference spectra corresponding to the redox transition at -400 mV (black line) and -190 mV (grey line). by guest on November 19, 2019 http://www.jbc.org/ Downloaded from

Acknowledgements

We thank Wolfgang Nitschke for insightful discussions. Guylaine Nuijten is acknowledged for maintenance of the K. stuttgartiensis culture. We thank MSM Jetten (MSMJ) for discussions and support. We acknowledge Hans Wessels for carrying out the MS/MS analysis. C.F. is supported by a Spinoza Prize awarded to M.S.M.J. by the Netherlands Organization for Scientific [NWO 62001581, 2012], S.L. by [NWO 824.15.011, 2015, to B.K.], B.K. by the European Research Council [ERC 640422, 2014]. J.R. by [ERC 339880, 2014, to M.S.M.J.]. This research was support by a van Gogh travel grant from EP-Nuffic and Campus France awarded to M.S.M.J. [BA/GP.16/23] and F.B. EPR work was supported by TGE RPE FR3443.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Author contributions

CF, SL, FB, JR and BK designed the experiments. CF, SL, FB performed protein purification, identification, and spectroscopic analyses. ERH performed the database search and phylogenetic tree construction. All authors interpreted the results and contributed to the writing of the manuscript.