Paulina Wakula

Monique Beullens
email: monique.beullens@med.kuleuven.be

Aleyde Van Eynde

Hugo Ceulemans

Willy Stalmans

Mathieu Bollen

The translation initiation factor eIF2 is an interactor of protein phosphatase-1

Keywords: protein (de)phosphorylation, eIF2, protein phosphatase-1

It is reasonably well understood how the initiation of translation is controlled by reversible phosphorylation of the translation initiation factors eIF2 , eIF2B and eIF4E. Other initiation factors, including eIF2 are also established phosphoproteins but the physiological impact of their phosphorylation is not known.

Using a sequence homology search we found that the central region of the eukaryotic translation initiation factor eIF2 contains a putative PP1-binding RVxF motif. The predicted eIF2 -PP1 interaction was confirmed by PP1 binding and coimmunoprecipitation assays on cell lysates as well as with the purified components.

Site-directed mutagenesis showed that eIF2 contains, in addition to an RVxF-motif, at least one other PP1-binding site in its C-terminal half. eIF2 functioned as an inhibitor for the dephosphorylation of glycogen phosphorylase and eIF2 (Ser51) by PP1, but did not affect the dephosphorylation of eIF2B (Ser464) by this phosphatase. Strikingly, eIF2 emerged as an activator of its own dephosphorylation (Ser2, Ser67, Ser218) by associated PP1, since the substrate quality of eIF2 was decreased by the mere mutation of its RVxF-motif. These data make eIF2 an attractive in vivo candidate substrate for associated PP1. The overexpression of wild-type eIF2 or eIF2 with a mutated RVxF-motif did not differentially affect the rate of translation, indicating that the binding of PP1 is not rate-limiting for translation under basal circumstances.

INTRODUCTION

The pre-initiation complex of translation contains eIF1, GTP-bound eukaryoticinitiation-factor (eIF) 2, Met-tRNA i Met and the 40S ribosomal subunit, and is converted to the 43S preinitiation complex by association with mRNA and eIF5. A conformational change in this 43S preinitiation complex activates the hydrolysis of GTP. Subsequently, the recognition of the AUG start codon triggers the release of the resulting inorganic phosphate from eIF2 and the dissociation of eIF1 from the complex. Next, the release of eIF2-GDP from the complex enables the recruitment of the 60S ribosomal subunit [START_REF] Preiss | Starting the protein synthesis machine: eukaryotic translation initiation[END_REF][START_REF] Algire | P i release from eIF2, not GTP hydrolysis is the step controlled by start-site selection during eukaryotic translation initiation[END_REF]. The recycling of the eIF2-GTPase complex for a next round of translation depends on the eIF2B-mediated exchange of eIF2associated GDP by GTP. eIF2 consists of , and subunits eIF2 with its intrinsic GTPase activity functions as the catalytic subunit and interacts with both GTP and Met-tRNAi Met . eIF2 is a regulatory subunit and its phosphorylation on Ser51 by one of at least four stress-induced protein kinases turns it into an inhibitor of the GTP exchange factor eIF2B and thus initation of translation [START_REF] Holcik | Translational control in stress and apoptosis[END_REF]. Finally, eIF2 interacts with mRNA, eIF1, eIF5 and eIF2B [START_REF] Kapp | The molecular mechanics of eukaryotic translation[END_REF]. The recruitment of eIF1 prevents premature GTP hydrolysis by eIF2 [START_REF] Unbehaun | Release of initiation factors from 48S complexes during ribosomal subunit joining and the link between establishment of codon-anticodon base-pairing and hydrolysis of eIF2-bound GTP[END_REF]. The interaction between eIF2 and eIF2 is required for the correct usage of the AUG start codon [START_REF] Hashimoto | Translation initiation at non-AUG codons mediated by weakened association of eukaryotic initiation factor (eIF) 2 subunits[END_REF]. eIF2B is the catalytic subunit of the GTP exchange factor eIF2B and its recruitment by eIF2 requires phosphorylation by protein kinase CK2 [START_REF] Wang | Eukaryotic initiation factor 2B: Identification of multiple phosporylation sites in the epsilon-subunit and their functions in vivo[END_REF] Phosphorylation of eIF2B by protein kinase CK1 enhances its catalytic activity while phosphorylation by DYRK and the insulin-regulated protein kinase GSK3 has an inhibitory effect on the catalytic activity of eIF2B [START_REF] Hashimoto | Translation initiation at non-AUG codons mediated by weakened association of eukaryotic initiation factor (eIF) 2 subunits[END_REF]. eIF5 acts as a GTPase-activating protein for the catalytic centre of eIF2 [START_REF] Paulin | Eukaryotic translation initiation factor 5 (eIF5) acts as a classical GTP-ase activator protein[END_REF][START_REF] Das | Eukaryotic translation initiation factor 5 functions as a GTP-ase activating protein[END_REF] and is known to be phosphorylated by protein kinase CK2.

However, the role of this phosphorylation is unknown. eIF2 is also a phosphoprotein in vivo. Phosphorylation of eIF2 by protein kinase A increases the guanine-nucleotide exchange activity of associated eIF2B [START_REF] Kimball | Identification of interprotein interactions between the subunits of eukaryotic initiation factors eIF2 and eIF2B[END_REF]. eIF2 is also phosphorylated by the DNA-dependent protein kinase as well as by protein kinases C and CK2, but the significance of phosphorylation by these kinases is not understood [START_REF] Ting | DNAdependent protein kinase interacts with antigen receptor response element binding proteins NF90 and NF45[END_REF][START_REF] Welsh | Identification of novel phosphorylation sites in the -subunit of translation initiation factor eIF-2[END_REF].

Protein phosphatase-1 (PP1) is a major protein Ser/Thr phosphatase that regulates numerous cellular processes by its ability to dephosphorylate key regulatory proteins. The catalytic subunit of PP1 is present in all eukaryotic cells and forms dozens of different PP1 holoenzymes by association with a host of regulatory polypeptides. These protein interactors function as targeting subunits, substratespecifiers, inhibitors and/or substrates for associated PP1 [START_REF] Ceulemans | Functional diversity of protein phosphatase-1, a cellular economizer and reset button[END_REF]. The large majority of PP1 interactors contain a degenerate, so-called RVxF-motif that actually conforms

to the consensus sequence [RK]-x 0-1 -[VI]-{P}-[FW]
, where x denotes any residue and {P} any residue except Pro [START_REF] Bollen | Combinatorial control of protein phosphatase-1[END_REF][START_REF] Wakula | Degeneracy and function of the ubiquitous RVXF motif that mediates binding to protein phosphatase-1[END_REF]. This motif binds with high affinity to a hydrophobic channel that is remote from the catalytic site of PP1. The binding of the RVxF-motif per se has no major effects on the conformation or activity of PP1 [START_REF] Egloff | Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1[END_REF].

However, the RVxF-mediated anchoring of PP1 promotes the occupation of secondary, lower-affinity binding sites and this often does affect the activity and/or substrate specificity of PP1 [START_REF] Bollen | Combinatorial control of protein phosphatase-1[END_REF].

Various approaches have been used to identify novel protein interactors of PP1. Initially, interactors of PP1 were mainly identified by the purification and characterisation of newly purified PP1 holoenzymes. Later, the identification of new PP1 interactors was mainly driven by yeast two-hybrid screens. Other PP1 interacting proteins were identified by affinity chromatography on an affinity matrix of microcystin, a cyclic heptapeptide that potently inhibits PP1 [START_REF] Ceulemans | Functional diversity of protein phosphatase-1, a cellular economizer and reset button[END_REF]. More recently, mass spectrometry and tools of bioinformatics have been used to extend our knowledge of the PP1 interactome [START_REF] Ceulemans | Regulator-driven functional diversification of protein phosphatase-1 in eukaryotic evolution[END_REF][START_REF] Meiselbach | Structural analysis of the protein phosphatase 1 docking motif: molecular description of binding specificities identifies interacting proteins[END_REF]. Using a combination of sequence alignment and biochemical data we show here that the eukaryotic initiation factor eIF2 is a direct interactor of PP1 in vitro and in vivo. We also report that eIF2 has at least two PP1-interaction sites and functions as a substrate-specifier of PP1.

EXPERIMENTAL

Reagents

The catalytic subunit of PP1 and glycogen phosphorylase b were purified from rabbit skeletal muscle [START_REF] Deguzman | Preparation of low-molecular-weight forms of rabbit muscle protein phosphatase[END_REF][START_REF] Fischer | The isolation and crystallization of rabbit skeletal muscle phosphorylase b[END_REF]. Protein kinase CK2 was isolated from pig spleen [START_REF] Agostinis | Phosphorylation of the phosphatase modulator subunit (inhibitor-2) by casein kinase-1[END_REF]. The catalytic subunit of beef protein kinase A and recombinant rat DYRK1A were obtained from Sigma-Aldrich and Upstate, respectively. A synthetic peptide comprising the C-terminus of eIF2 (C 321 VTGKRAQLRAKA 332 ) and coupled to keyhole limpet hemocyanin, was used to generate a rabbit polyclonal antibody. The anti-eIF2 antibodies were affinity-purified on the peptide coupled to bovine serum albumin and linked to CNBr-activated Sepharose 4B (GE Healthcare). Monoclonal anti-PP1 antibodies were purified on protein-A Sepharose CL-4B (GE Healthcare).

The hybridoma clone producing these antibodies was a gift from Dr. J.

Vandenheede (University of Leuven). Monoclonal anti-FLAG antibodies were obtained from Stratagene. Polyclonal anti-EGFP antibodies were obtained from Santa Cruz. Polyclonal antibodies specific for eIF2 phosphorylated at Ser51 were delivered by Biosource International. Swine anti-rabbit and rabbit anti-mouse antibodies were purchased from DakoCytomation.

Plasmids

The sequence encoding rabbit PP1 was introduced between the XhoI and BamHI sites of pEGFP-C1 (Clonetech), yielding an expression vector for EGFP-PP1 . The sequence encoding human eIF2 was subcloned in the XhoI-BamHI sites of pEGFP-C1 and the EGFP sequence in this vector was replaced by an adaptor of 1 x FLAG-sequence, generating an expression vector for FLAG-eIF2 .

Preparation of recombinant proteins

The polyhistidine-tagged polypeptides and GST-fusions were produced in Escherichia coli BL21(DE3)pLysS cells by transformation with the pET plasmid and the pGEX plasmid, respectively. The His-tagged proteins were purified on Ni 2+ -Sepharose™6 Fast Flow (GE Healthcare). The GST-fusion proteins were purified on glutathione-agarose (Sigma-Aldrich).

Pull-down experiments with Ni 2+ -Sepharose

Equimolar amounts of BSA, polyhistidine-tagged eIF2 wt and eIF2 mutants were pre-incubated for 1.5 hour at 10°C with Ni 2+ -Sepharose™6 Fast Flow (GE Healthcare) that had been pre-blocked with TBS plus BSA (1 mg/ml) and 0.1% Nonidet P-40. After washing of the beads, the suspension was incubated for 45 minutes at 10°C with 0.2 pmol PP1. Subsequently, the beads were sedimented (1 min at 1000 x g), washed 3 times, first with TBS buffer plus 0.25 M LiCl, then with TBS buffer that was supplemented with 0.1% Nonidet P-40, and finally with 50 mM imidazole at pH 7.4, 0.5 mM dithiothreitol and 5 mM -mercaptoethanol. The washed beads were used for the assay of trypsin-revealed phosphorylase phosphatase activity.

Cell culture

HEK293T cells (human embryonic kidney cells) were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum on 10-cm plates or in sterile 6well plates (Greiner). One day after seeding, the cells were transfected with plasmid DNA, using the FuGene TM 6 Transfection reagent (Roche), according to the protocol of the manufacturer, or using polyethylenimine (PEI) obtained from Sigma-Aldrich.

Cell transfection experiments with PEI were performed in DMEM with 5% fetal calf serum. One day after transfection, the medium was replaced with fresh DMEM supplemented with 10% fetal calf serum.

Cells were blocked in mitosis by the addition of 500 ng/ml Nocodazole (Sigma-Aldrich) for 17 hours. Cells were blocked in the G1/G2-phases by addition of 1 µM

Trichostatin A (BioMol) for 17 hours. To induce ER stress, cells were incubated for 1 hour with 100 µM thapsigargin (Alexis Biochemicals). UV-C irradiation at 254 nm of cells was carried out using a UV Stratalinker 1800 (Stratagene).

48 hours after transfection, the cells were lysed in PBS buffer, supplemented with 0.5 mM phenylmethanesulfonyl fluoride, 0.5 mM benzamidine, and 5 µM leupeptin.

Cell lysates were sonicated (3 x 30 seconds) and centrifuged at 20,000 x g for 10 min. The supernatants were used for immunoprecipitation experiments. Within the same experiment, the total protein concentration in the various conditions was equalized.

Immunological procedures

For immunoprecipitation, cell lysates were incubated with antibodies against eIF2 , EGFP or the FLAG-peptide for 3 hours at 4°C. Subsequently, protein-A-TSK-Sepharose (Affiland) was added, and the mixture was rotated during 30 min at 4°C.

Following centrifugation (1 min at 1000 x g), the pellet was washed twice with TBS buffer supplemented with 0.1% Nonidet P-40, and once with 50 mM imidazole at pH 7.4, 0.5 mM dithiothreitol and 5 mM -mercaptoethanol. The pellets were either boiled in SDS-sample buffer for immunoblotting or assayed for trypsin-revealed phosphorylase phosphatase activity. For immunoblotting, the retained antibodies were visualized by enhanced chemiluminescence (Western Lightning, Perkin Elmer).

For overlay assays, proteins were directly spotted on a Polyvinylidene difluoride (PVDF) membrane, and the membrane was blocked in 20 mM Tris-HCl at pH 7.5, 0.5 M NaCl, 3% milk powder for 3 hours at 4°C. Subsequently, the blots were incubated overnight in TBS buffer with 1 mg/ml BSA and digoxygenin-labelled PP1. After extensive washes with TBS, the retained PP1 was detected with antidigoxygenin antibodies (Roche Diagnostics), according to the manufacturer's protocol. For far-western overlays, the blocked blots were incubated in TBS with 1 mg/ml BSA and PP1 for 5 hours at room temperature. After washing twice with TBS buffer and once with TBS buffer with 5% milk powder and 0.2% Triton X-100, PP1

was visualized by immunoblotting.

Assays

Protein phosphatase activities were assayed at 30°C with phosphorylase a as a substrate, by the measurement of released acid-stable radioactivity [START_REF] Beullens | The isolation of novel inhibitory polypeptides of protein phosphatase 1 from bovine thymus nuclei[END_REF]. To measure the total protein phosphatase activity of PP1 that co-sedimented with eIF2 , the samples were pretreated with trypsin (0.1 mg/ml) for 5 minutes at 30°C to release the free, fully active catalytic subunit. Subsequently, trypsin inhibitor (1mg/ml) was added and the phosphorylase phosphatase activity was measured.

Other, recombinant substrates of PP1 were phosphorylated by incubation for 30-60 minutes at 30°C in 20 mM Tris-HCl at pH 7.5 plus 0.1 mM ATP (with or without 0.1 µCi/µl [ -32 P]ATP), 2mM magnesium acetate and the indicated protein kinase.

Phosphorylation reactions were terminated by the addition of 6.6 mM EDTA.

Subsequently, the mixtures were incubated with PP1 with or without eIF2 . At the indicated times aliquots were boiled in SDS-sample buffer and the extent of phosphorylation was visualized by autoradiography after SDS-PAGE or by immunoblotting with eIF2 phosphoserine51-specific antibodies.

Protein synthesis measurements

Equal numbers of HEK293T cells were split into 6-well dishes and then transfected with plasmids encoding Flag-eIF2 or Flag-eIF2 -V131A/F133A. After 48 hours, the cells were labelled for 2 hours at 37°C with 50 µCi of [ 35 S]methionine (GE Healthcare)/ml in methionine-free medium (Invitrogen), supplemented with 10% fetal bovine serum. Cells were washed three times in PBS, and disrupted with lysis buffer. After sonication, the lysates were centrifuged (10,000 x g, 10 min). An aliquot of 5 µl of the supernatants was diluted in 2 ml scintillation liquid Lumasafe Plus (Lumac LSC) and analyzed by scintillation counting to determine the incorporation of [ 35 S]methionine per mg of proteins.

RESULTS

Identification of eIF2 as a novel interactor of PP1

The sequence-profile alignment method MaxHom (http://www.cbi.pku.edu.cn/mirror/predictprotein/) [START_REF] Sander | Database of homology-derived structures and the structural meaning of sequence alignment[END_REF] suggested structural similarities between the established PP1 interactor NIPP1 (nuclear inhibitor of protein phosphatase-1) and the translation initiation factor eIF2 (Fig. 1A). The similarities included residues 129-133 of eIF2 , KNVKF, which forms an instance of the consensus PP1-binding 'RVxF'-motif, i.e. [RK]-x 0-1 -[VI]-{P}-[FW], as defined by Wakula et al. [START_REF] Wakula | Degeneracy and function of the ubiquitous RVXF motif that mediates binding to protein phosphatase-1[END_REF]. Moreover, as in NIPP1 and various other established PP1interactors [START_REF] Wakula | Degeneracy and function of the ubiquitous RVXF motif that mediates binding to protein phosphatase-1[END_REF], the RVxF-instance of eIF2 is flanked N-terminally by a stretch of basic residues and C-terminally by an acidic stretch. This instance is conserved in eIF2 from mammals, birds, amphibia and some fish, but not in non-chordate eIF2 (Fig. 1B). Clearly, the acquisition of the RVxF-instance postdates the primary function of eIF2 in translation, which qualifies the protein as a 'secondary' interactor of PP1, i.e. a polypeptide that acquired its PP1-binding site later in evolution [START_REF] Ceulemans | Regulator-driven functional diversification of protein phosphatase-1 in eukaryotic evolution[END_REF].

To experimentally explore the predicted interaction between PP1 and eIF2 , we generated bacterially expressed His-eIF2 , affinity-purified on Ni 2+ -Sepharose.

His-eIF2 bound to digoxigenin-labelled PP1 in overlay assays (Fig. 2A). NIPP1 served as a positive control in this experiment while NIPP1-(1-329)RATA and BSA were used as negative controls. His-eIF2 also inhibited PP1 with an IC 50 of around Copyright 2006 Biochemical Society 50 nM, using glycogen phosphorylase a as substrate (Fig. 2B). In contrast, eIF2

did not affect the phosphorylase phosphatase activity of the structurally related protein phosphatase-2A, indicating that it is a specific inhibitor of PP1. Importantly, the activity of eIF2 -inhibited PP1 could be fully recovered by the destruction of eIF2 with trypsin (not shown). Thus, unlike the PP1 interactor Inhibitor-2, eIF2

does not convert PP1 into an inactive, trypsin-sensitive conformation [START_REF] Bollen | The structure, role, and regulation of type 1 protein phosphatases[END_REF].

Subsequently, we have examined the association between eIF2 and PP1 in cell lysates. For this purpose, we performed immunoprecipitations with anti-FLAG or anti-EGFP antibodies, following the expression of FLAG-eIF2 and EGFP-PP1 in HEK293T cells. These experiments revealed a reciprocal co-immunoprecipitation of FLAG-eIF2 and EGFP-PP1 (Fig. 3A). In addition, we found that the immunoprecipitation of endogenous eIF2 resulted in the co-precipitation of endogenous PP1, as revealed by immunoblotting (Fig. 3B) and phosphorylase phosphatase assays (Fig. 3C). The latter activity was completely inhibited by the addition of NIPP1, showing that it was derived from PP1.

We have also explored whether the eIF2 -PP1 interaction is affected by treatments that are known to influence the rate of translation by affecting the phosphorylation of eIF2 , such as the addition of thapsigargin, a treatment with UV-C irradiation, or the synchronization of cells in the G 0 -phase (serum starvation), Mphase (nocodazole treatment) or G1/G2-phases (addition of trichostatin A).

However, none of these treatments measurably affected the association between eIF2 and PP1, but we could confirm that these treatments indeed affected the phosphorylation level of eIF2 (not illustrated).

Mapping of the eIF2 -PP1 interaction sites

To examine whether residues 129-133 of eIF2 , KNVKF, constitute a functional RVxF-instance, we mutated Val131 and Phe133 into an alanine. The resulting peptide His-eIF2 -V131A/F133A, further referred to as eIF2 -KNAKA, bound to Ni 2+ -Sepharose, was used as an affinity matrix for the binding of purified PP1.

Compared to the wild-type protein, eIF2 -KNAKA displayed a severely decreased ability to bind (Fig. 4A) and inhibit PP1 (Fig. 4B). The importance of the RVxFinstance for the inhibition of PP1 was further examined by using a synthetic RVxF-containing decapeptide, namely NIPP1-(197-206), as a competitor for the binding to the RVxF-binding site on PP1. The addition of 50 µM of NIPP1-(197-206), but not the corresponding RATA-peptide, abrogated the inhibition of PP1 by eIF2 (Fig. 4C), indicating that the RVxF-instance of eIF2 is indeed required for its ability to inhibit the phosphorylase phosphatase activity of PP1.

Although eIF2 -KNAKA was clearly deficient in the inhibition of PP1 (Fig. 4B), it could still retain about 25% of the amount of PP1 that interacted with wild-type eIF2 (Fig. 4A). Consistent with the presence of a second PP1-interaction site, we found that (1) RVxF-containing peptides could only partially disrupt the binding between PP1 and eIF2 wt, (2) PP1 still bound to the eIF2 -KNAKA in overlay assays, and (3) eIF2 -KNAKA, when expressed in HEK293T cells, also coimmunoprecipitated with EGFP-PP1 from cell lysates in a reciprocal manner (not illustrated). To gain more insight into the region of eIF2 that harbours a second PP1 binding site, we generated C-terminal deletions of eIF2 -KNAKA and examined the ability of these eIF2 fragments to bind PP1 (Fig. 5). Carboxyterminal truncation mutants of eIF2 -KNAKA clearly bound less PP1 and eIF2 -(1-145)-KNAKA barely retained any PP1 (Fig. 5) and did not bind to PP1 in overlay assays (Fig. 2A), pointing to the existence of a second, C-terminal binding site for PP1. Consistent with this view, we found that eIF2 -(187-341) bound about as much PP1 as did eIF2 -KNAKA (Fig. 5). Thus, binding of eIF2 to PP1 is mediated by the RVxF-motif and at least one additional binding site in the C-terminal half of eIF2 .

Functional analysis of the eIF2 -PP1 interaction

Mammalian eIF2 interacts with eIF5, eIF2B and eIF2 , and the latter binds in turn to eIF2 . Since the initiation factors eIF2 , eIF2B and eIF5 are established phosphoproteins, we examined whether these phosphoproteins are substrates for PP1 in the absence or presence of eIF2 . Ser51 of eIF2 , phosphorylated in vitro with protein kinase MELK [START_REF] Beullens | Substrate specificity and activity regulation of protein kinase MELK[END_REF] was readily dephosphorylated by PP1. The addition of eIF2 wt, but not of eIF2 KNAKA, inhibited the activity of PP1 towards eIF2 (Fig. 6A). Thus, eIF2 is an inhibitor of the dephosphorylation by PP1 of both glycogen phosphorylase a (Fig. 2B) and eIF2 (Fig. 6A). eIF2B is phosphorylated by protein kinases CK2 (Ser712, Ser713), DYRK1 (Ser539) and CK1 (Ser464).

However, only CK1-phosphorylated eIF2B was dephosphorylated by purified PP1 (Fig. 6B and not shown) and, surprisingly, this dephosphorylation was only very mildly affected by the addition of eIF2 or eIF2 -KNAKA (Fig. 6B). eIF5 phosphorylated by protein kinase CK2 (Ser387, Ser388) was not dephosphorylated by PP1, whether or not eIF2 was added (not shown). Finally, we have examined whether eIF2 , phosphorylated by protein kinase CK2 (Ser2, Ser67) or protein kinase A (Ser218) [START_REF] Welsh | Identification of novel phosphorylation sites in the -subunit of translation initiation factor eIF-2[END_REF], is itself a substrate for associated PP1. The phosphorylation of eIF2 by either kinase was reversed by PP1 but, surprisingly, the dephosphorylation of eIF2 -KNAKA was clearly less efficient (Fig. 6C). These results imply that the binding of PP1 to the RVxF-instance promotes the dephosphorylation of eIF2 by associated PP1.

Since the in-vivo function of the phosphorylation of eIF2 by protein kinases A and CK2 is not known, we have examined whether the recruitment of PP1 by eIF2 is a determinant of the rate of translation. For this purpose, wild-type eIF2 or eIF2 -KNAKA were transiently expressed in HEK293T cells and the rate of translation was derived from the incorporation of [ 35 S]methionine into proteins. The expression of eIF2 or the KNAKA-mutant inhibited the rate of translation by 42 ± 7 % and 53 ± 4 % (n= 10), respectively. Thus, the severely decreased ability of the KNAKA-mutant to interact with PP1 did not differentially affect the rate of translation.

Consistent with this observation, we found that the addition of equimolar concentrations of eIF2 -wt or eIF2 -KNAKA to reticulocyte lysates decreased the rate of translation of a reporter transcript, encoding the histone methyltransferase EZH2, to a similar extent (supplementary Fig. S1). It is worthy of note that EZH2 is not an interactor of PP1 and has no effect on translation. Collectively, these data suggest that the eIF2 -PP1 interaction is not rate-limiting for protein synthesis.

DISCUSSION

A bioinformatics approach has previously already led to the identification of new members of established families of PP1 interactors [START_REF] Ceulemans | Regulator-driven functional diversification of protein phosphatase-1 in eukaryotic evolution[END_REF]. Also, Meiselbach et al. [START_REF] Meiselbach | Structural analysis of the protein phosphatase 1 docking motif: molecular description of binding specificities identifies interacting proteins[END_REF] recently showed that a more stringent definition of the RVxF-motif enables a reasonably specific identification of functional RVxF-instances. Here, we report the sequence-alignment assisted identification of eIF2 as an interactor of PP1. The sequence similarity between NIPP1 and eIF2 was not expected since these proteins are functionally unrelated. Indeed, NIPP1 has been implicated in transcription and pre-mRNA splicing but not in translation [START_REF] Jin | The protein phosphatase-1 (PP1) regulator, nuclear inhibitor of PP1 (NIPP1), interacts with the polycomb group protein, embryonic development (EED), and functions as a transcriptional repressor[END_REF]28]. The structural similarities between NIPP1 and eIF2 not only comprised the RVxF-motif but also the flanking sequences of this motif (Fig. 1A). The RVxF-instance of eIF2 is absent from non-chordates and from some fish species (Fig. 1B), indicating that PP1 binding, via its RVxF-instance, is a property that eIF2 acquired during metazoan evolution. We have demonstrated by various independent biochemical approaches that the predicted RVxF-instance of eIF2 is functional (Fig. 4). Moreover, the region of eIF2 that harbours the RVxF-motif is predicted by the secondary structure prediction programme SCRATCH (http://www.igb.uci.edu/tools/scratch) to form an exposed loop, which is also true for RVxF-motifs in other established PP1 interactors [START_REF] Ceulemans | Regulator-driven functional diversification of protein phosphatase-1 in eukaryotic evolution[END_REF]. In addition the RVxF-instance of eIF2 corresponds to the more stringent definition of RVxF-motifs described by Meiselbach et al. [START_REF] Meiselbach | Structural analysis of the protein phosphatase 1 docking motif: molecular description of binding specificities identifies interacting proteins[END_REF].

Two PP1 interactors, namely GADD34 and CreP, were already known to target PP1 to the eIF2 complex and to promote thereby the dephosphorylation of Ser51 of eIF2 [START_REF] Novoa | Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha[END_REF][START_REF] Connor | Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1[END_REF][START_REF] Jousse | Inhibition of a constitutive translation initiation factor 2alpha phosphatase, CreP, promotes survival of stressed cells[END_REF]. Here, we have shown that another subunit of eIF2, namely the -subunit, directly recruits PP1 (Figs. 2 and3) and is itself a likely substrate for dephosphorylation by associated PP1 (Fig. 6C andD). It remains to be examined whether eIF2 is also an in vivo substrate of associated PP1 and whether this somehow affects translation. Our preliminary data show that the binding of PP1 to eIF2 is not rate-limiting for translation under basal circumstances. However, it can not be excluded that eIF2 -associated PP1 only exerts a translational control for a selected group of transcripts or under specific conditions. Consistent with the role of PP1 as a cellular 'reset' button [START_REF] Ceulemans | Functional diversity of protein phosphatase-1, a cellular economizer and reset button[END_REF], it can also be envisaged that the dephosphorylation of eIF2 by associated PP1 is implicated in the recycling of eIF2 for a next round of translation. Also, since the addition of leptomycin-B, an inhibitor of nuclear export, causes the nuclear accumulation of eIF2 [START_REF] Bohnsack | Exp5 exports eEF1A via tRNA from nuclei and synergizes other transport pathways to confine translation to the cytoplasm[END_REF], PP1 may play a regulatory role in the poorly studied nuclear functions of eIF2 . Intriguingly, the binding of PP1 to eIF2 promoted the dephosphorylation of eIF2 but inhibited the dephosphorylation of glycogen phosphorylase and eIF2 , and did not affect the dephosphorylation of eIF2B (Fig. 6). Thus, like many PP1-interactors [START_REF] Ceulemans | Functional diversity of protein phosphatase-1, a cellular economizer and reset button[END_REF], eIF2 Copyright 2006 Biochemical Society appears to have a substrate-specifying function. Interestingly, eIF2 not only binds to PP1 (this work) but also interacts directly with protein kinase CK2 [START_REF] Llorens | Eukaryotic translation initiation factor eIF2beta binds to protein kinase CK2: effects on CK2alpha activity[END_REF]. Thus, eIF2 may function as a scaffold for the assembly of a signalling complex that includes CK2 and PP1, and that somehow controls the initiation of translation.

In conclusion, we have shown here that PP1 is a physiological interactor of eIF2 . We have identified a functional RVxF-motif and a second PP1-binding site in the C-terminal half of eIF2 and have provided evidence that eIF2 functions as a substrate-specifier for associated PP1. Strikingly, eIF2 itself emerges as the most likely substrate for associated PP1 and its dephosphorylation depends on a functional RVxF-motif. The figure shows the His-tagged eIF2 mutants/fragments that were used for PP1 binding assays. Equimolar concentrations of the fusions, linked to Ni 2+ -Sepharose, were incubated with an equal amount of purified PP1. The right column shows the amount of retained PP1, as derived from the trypsin-revealed phosphorylase phosphatase activity and expressed as a percentage of binding to wild-type eIF2 .

The results are means ± S.E.M. for 4-8 phosphatase assays.

  to scale) of non-conserved fragments in eIF2 . Identical residues are boxed in black. Residues conserved whithin the same group [positively charged (H,K,R); negatively charged (D,E); small residues (G,A,S,T); bulky hydrophobic residues (I,L,V,M,F,Y)] are boxed in grey. (B) The table shows the sequence of RVxF-motifs with respect to a conserved poly-lysine tract. The residues of the RVxF-motif are in bold. The N-terminal basic residues and the C-terminal acidic residues are underlined.

Figure 2 eIF2

 2 Figure 2 eIF2 is a specific inhibitor of PP1 (A) An overlay with digoxigenin-labelled catalytic subunit of PP1 shows binding to eIF2 wt but not to the mutant eIF2 -(1-145)-KNAKA (see also Fig. 5). NIPP1 was included as a positive control and NIPP1-(1-329)-RATA, a NIPP1-mutant that does not bind to PP1 (34), and BSA were used as negative controls. 380 pmol of each protein was used. (B) The effect of the indicated concentrations of eIF2 on the phosphorylase phosphatase activity of PP1 and PP2A. The results represent means ± S.E.M. of three assays.

  Figure 3 Co-immunoprecipitation experiments confirm the eIF2 -PP1 interaction (A) Lysates from HEK293T cells co-expressing EGFP-PP1 and FLAG-eIF2 were used for immunoprecipitations with protein-A-TSK and anti-EGFP antibodies, anti-FLAG antibodies or without antibodies (control). The presence of EGFP-PP1 and FLAG-eIF2 was detected by western analysis with anti-EGFP and anti-FLAG antibodies. (B and C) Endogenous eIF2 from HEK293T cell lysates was immunoprecipitated and the precipitates were screened for the presence of eIF2 and PP1 by immunoblotting (B) and for trypsin-revealed phosphorylase phosphatase activities in the absence and presence of 1 µM NIPP1 (C).

  Figure 4 eIF2 contains a functional RVxF-motif (A) Equimolar amounts of recombinant His-eIF2 -wt and His-eIF2 -KNAKA, coupled to Ni 2+ -Sepharose, were incubated with the catalytic subunit of PP1, purified from rabbit skeletal muscle. The retained phosphorylase phosphatase activities were quantified after the release of PP1 from the beads by pre-incubation

Figure 5

 5 Figure 5Mapping of a second PP1 interaction site in eIF2

Figure 6

 6 Figure 6In vitro dephosphorylation of eIF2 , eIF2B and eIF2 by PP1The panels show time courses of dephosphorylation of the indicated substrates by 88 nM PP1 in the presence of buffer, 1 µM eIF2 -wt or eIF2 -KNAKA. Prior to the addition of PP1, phosphorylation reactions were terminated by the addition of EDTA. (A) Dephosphorylation of MELK-phosphorylated Ser51 in eIF2 , as visualized by anti-phospho-Ser51 antibodies. (B) Dephosphorylation of eIF2B phosphorylated by protein kinase CK1, as visualized by autoradiography. (C and D). An autoradiogram of the dephosphorylation of eIF2 and eIF2 -KNAKA, phosphorylated by protein kinase CK2 (C) or protein kinase A (D).

Biochemical Journal Immediate Publication. Published on 20 Sep 2006 as manuscript BJ20060758

Biochemical Journal Immediate Publication. Published on 20 Sep 2006 as manuscript BJ20060758

Biochemical Journal Immediate Publication. Published on 20 Sep 2006 as manuscript BJ20060758 Copyright 2006 Biochemical Society

ACKNOWLEGDEMENTS

This work was financially supported by the Fund for Scientific Research-Flanders (Grant G.0290.05), a Flemish Concerted Research Action and the Prime Minister's office (IAP/V-05). Nicole Sente provided expert technical assistence. We thank Drs. U. Maitra (Albert Einstein College of Medicine), T. Matsui (Kanazawa Medical University), C. Proud (University of British Columbia), P. Roach (Indiana University) and J. Vandenheede (University of Leuven) for the generous gifts of expression vectors. H.C. is a post-doctoral fellow of the Fund for Scientific Research-Flanders.

Abbreviations

BSA, bovine serum albumin; eIF, eukaryotic initiation factor; PBS, phosphate buffered saline; PP1, protein phosphatase-1; TBS, Tris buffered saline

LEGENDS TO THE FIGURES

Similarities between eIF2 and the central domain of NIPP1


Steven Sherwood

Judy Hirst

Investigation of the mechanism of proton translocation by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria: does the enzyme operate by a Q-cycle mechanism ?

Keywords: Electron transport chain, Complex I, NADH:ubiquinone oxidoreductase, Q cycle mechanism, Reductant induced oxidation, Respiratory chain

NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the membrane bound electron transport chain in mitochondria. It conserves energy, from the reduction of ubiquinone by NADH, as a proton motive force across the inner membrane, but the mechanism of energy transduction is not known. The structure of the hydrophilic arm of thermophilic complex I supports the idea that proton translocation is driven at (or close to) the point of quinone reduction, rather than at the point of NADH oxidation, with a chain of iron-sulphur clusters transferring electrons between the two active sites. Here we describe experiments to determine whether complex I, isolated from bovine heart mitochondria, operates via a Q-cycle mechanism analogous to that observed in the cytochrome bc 1 complex. No evidence for the 'reductantinduced oxidation' of ubiquinol could be detected -therefore, no support for a Q-cycle mechanism was obtained. Unexpectedly, in the presence of NADH, complex I inhibited by either rotenone or piericidin A was found to catalyse the exchange of redox states between different quinone and quinol species, providing a possible route for future investigations into the mechanism of energy transduction.

INTRODUCTION

NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the membrane bound electron transport chain in mitochondria [START_REF] Schultz | Structures and proton-pumping strategies of mitochondrial respiratory enzymes[END_REF][START_REF] Saraste | Oxidative phosphorylation at the fin de siècle[END_REF]. It oxidizes NADH, produced predominantly by the tricarboxylic acid cycle in the mitochondrial matrix, and reduces ubiquinone in the inner mitochondrial membrane. The ubiquinol is reoxidised by the cytochrome bc 1 complex and the electrons are used ultimately to reduce oxygen. In addition, complex I conserves energy as a proton motive force across the inner mitochondrial membrane, supporting ATP synthesis.

Complex I from bovine heart mitochondria has a molecular mass of approximately 1 MDa and comprises 46 different subunits [START_REF] Hirst | The nuclear encoded subunits of complex I from bovine heart mitochondria[END_REF][START_REF] Walker | The NADH-ubiquinone oxidoreductase (complex I) of respiratory chains[END_REF]. It has an L-shaped structure, with one arm in the membrane plane and the other extending into the mitochondrial matrix (Scheme 1A) [START_REF] Hofhaus | Electron-microscopic analysis of the peripheral and membrane parts of mitochondrial NADH dehydrogenase (complex I)[END_REF]. The hydrophilic arm contains the active site for NADH oxidation (containing a flavin mononucleotide (FMN)), and eight iron-sulphur clusters, two [2Fe-2S] clusters and six [4Fe-4S] clusters [START_REF] Sazanov | Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus[END_REF][START_REF] Ohnishi | Iron-sulphur clusters / semiquinones in complex I[END_REF].

In the bovine enzyme the hydrophilic arm contains at least 14 subunits, and seven of them are conserved throughout all known complexes I [START_REF] Hirst | The nuclear encoded subunits of complex I from bovine heart mitochondria[END_REF][START_REF] Walker | The NADH-ubiquinone oxidoreductase (complex I) of respiratory chains[END_REF]. Recently, a structural model of the hydrophilic arm of complex I from Thermus thermophilus, containing homologues to these seven subunits, has been reported, confirming that the clusters form a chain between the FMN and the ubiquinone binding site [START_REF] Sazanov | Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus[END_REF].

Many varied reaction mechanisms have been proposed for proton translocation by complex I (see, for recent reviews, [START_REF] Brandt | Proton pumping by NADH:ubiquinone oxidoreductase. A redox driven conformational change mechanism[END_REF][START_REF] Hirst | Energy transduction by respiratory complex I -an evaluation of current knowledge[END_REF][10]). However, the structure of the hydrophilic arm [START_REF] Sazanov | Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus[END_REF] supports the notion that proton translocation is driven at (or close to) the point of quinone reduction. The iron-sulphur clusters are likely to act predominantly as innocent electron carriers, although the final cluster in the chain, cluster N2, is close to the putative quinone binding site and may play a more intimate role in catalysis. Consequently, realistic mechanistic proposals for energy transduction by complex I fall into three categories -directly coupled proton translocation (as observed in cytochrome c oxidase [11]), indirectly coupled proton translocation (proton transfer is driven by (or drives) a reaction at a remote site, as in ATP synthesis [12,13]), and Qcycle mechanisms (as in the cytochrome bc 1 complex [14,15]). Here, we aim to determine whether complex I operates by a Q-cycle-type mechanism.

Various Q-cycle mechanisms have been proposed previously for catalysis by complex I [16,17]. We focus on the elegant but simple proposal of Dutton and coworkers [17], that the Q-cycle mechanism in complex I is the 'mirror-image' of that established for the cytochrome bc 1 complex. Because a direct analogy of the mechanism of the bc 1 complex alone can account for only two of the four protons which are pumped by complex I for every NADH oxidized, an additional 'proton-pumping' component was incorporated into Dutton's mechanism [17]. The strategy adopted here does not address this component. A number of experimental observations are consistent with a Q-cycle in complex I, including evidence for more than one Q-binding site [18], and observations of semiquinone intermediates, at least one of which responds to the proton motive force [19]. However, no bound cofactor has yet been identified in the membrane domain of complex I to mediate electron transfer between two Q-sites located on opposite sides of the membrane, and evidence has been presented also which challenges proposals for more than one Q-binding site [20].

Here, we describe experiments designed to test whether a Q-cycle mechanism can be detected in complex I from bovine mitochondria. The principle of our experiment is to ask whether 'reductant-induced oxidation' of ubiquinol occurs during catalysis. Thus, our experiment is inspired by the transient increase in reduction of the b-haems in the cytochrome bc 1 complex, observed by Chance upon a pulse of oxygen to slowly respiring mitochondria [21], which led to development of the modified Q-cycle mechanism [14]. In the bc 1 complex the status of the individual cofactors can be defined spectroscopically, but the poor spectroscopic signals of the cofactors in complex I (flavin, iron-sulphur clusters and quinones) preclude this option.

Consequently, our experiments are aimed at detecting the oxidation of ubiquinol, induced by the reduction of complex I by NADH.

EXPERIMENTAL

Preparation of complex I from bovine mitochondria

Mitochondria were isolated from bovine hearts and used to prepare mitochondrial membranes [22,23]. Complex I was purified as described previously [24], at 4 °C, with minor modifications. 30 ml of membranes (protein ∼12 mg ml -1 ) were solubilised with 1 % n-dodecyl-β-Dmaltopyranoside (DDM, Anatrace) for 30 min. in the presence of 2 mM dithiothreitol (DTT, Melford Laboratories) and 0.005 % phenylmethylsulphonyl fluoride (PMSF, Sigma), and -20 °C, and HPLC analysis demonstrated less than 1 % reoxidation after a week. However, after an hour exposure to air, 17 % reoxidation was observed. Consequently, all experiments involving quinols were carried out in the anaerobic glovebox. HPLC experiments carried out at varying flow rates confirmed that quinol reoxidation during the HPLC run is negligible.

Reverse phase HPLC separation and quantification of Q and QH 2 species HPLC analysis was carried out using an Agilent 1100 series HPLC system equipped with a manual injector (Rheodyne injection valve), column thermostat (30 ºC) and multiple wavelength detector, and controlled by an Agilent ChemStation. DQ, DQH 2 , Q 2 and Q 2 H 2 were eluted from a Nucleosil C18 column (5 μm, 25 cm x 3.2 mm, Hichrom) in a mobile phase of 50 mM sodium perchlorate in 53:25:22:0.1 v/v/v/v ethanol / water / methanol / 70 % perchloric acid, with detection at 278 and 290 nm (adapted from a published protocol [29]). All solvents were HPLC grade (Sigma) and water was from a MilliQ purification system. The four Q species were separated at 0.4 ml min -1 during a run time of 40 minutes, and elute at ∼8.2 min. (Q 2 H 2 ), ∼12.0 min. (Q 2 ), ∼14.5 min. (DQH 2 ) and ∼23.0 min. (DQ). The extinction coefficients are discussed below and reported in Table 1. Standard solutions were prepared in the HPLC mobile phase at concentrations between 0 and 100 μM.

Preparation of samples for HPLC analysis

Complex I (24 μl of 10 -20 mg ml -1 ) was placed in the bottom of a glass vial and mixed with either 30 μl of 1 % bovine heart polar phospholipids (Avanti Polar Lipids) or 45 μl of 1 % asolectin (solubilised in 2 or 1 % CHAPS respectively). ∼2850 μl (calculated for a final volume of 3 ml) of 10 mM HEPES buffer (pD 7.5 in D 2 O, 30 °C) and 100 μM NADH (30 μl from a 10 mM stock solution) were added, and the solution was mixed and incubated for two minutes whilst stirring slowly. The reaction was initiated by addition of the appropriate Q species to a final concentration of approximately 100 μM (60 μl of a 50 x concentrated stock solution in acidified ethanol). Aliquots of 100 μl were removed at set time points, and added to 300 μl of quenching solution (53:22 v/v ethanol/methanol), allowing analysis by HPLC without further manipulation. HPLC analysis confirmed that this procedure quenched all reactions efficiently. The samples were sealed anaerobically, frozen, and stored in liquid nitrogen. For analysis, they were removed one at a time from the liquid nitrogen, thawed, filtered using a 0.2 μm filter (Sartorius Minisart RC 4), and loaded into the injection syringe (all procedures in the glove box), then injected quickly onto the HPLC column. The NADH concentration of each aliquot was determined by UV-visible spectroscopy (340 nm, ε = 6.37 cm -1 mM -1 in this solvent mixture).

Data modelling calculations

Data sets from HPLC analysis were fit by minimizing the least squares error value (LSQ) between calculated curves and experimental data sets. The initial concentration of each Q species was allowed to vary within a small range to account for experimental error, and rate constants were defined by calculating curves for a wide range of possible values, then refining the best fit values. Analytical solutions describing concentration as a function of time were used when possible. Otherwise, digital step calculations were used. Digital simulations use rate equations to calculate small changes in concentration which occur upon small steps in time -when decreasing the step size does not alter the results the simulation has converged to the analogue solution.

RESULTS AND DISCUSSION

Experimental strategy

Scheme 1A depicts complex I in the inner-mitochondrial membrane, catalysing the oxidation of NADH coupled to the reduction of ubiquinone and to proton translocation. The mechanism of proton translocation is not defined. Scheme 1B illustrates one possible mechanism: complex I catalysing by a modified Q-cycle mechanism analogous to that employed by the cytochrome bc 1 complex [17]. Quinone (Q) binds at the matrix side of the membrane and is reduced by two electrons, one from the iron-sulphur clusters (derived from NADH) and the other from a quinol (QH 2 ) bound on the outside membrane face. This step is the reverse of the bifurcation reaction in the bc 1 complex [14,15] and it leads to a bound semiquinone on the outside face -which is oxidized completely upon reduction of a second quinone. Thus, oxidation of one NADH is coupled to the reduction of two quinones and to the oxidation of one quinol, with spatial separation of the redox processes providing net proton translocation. The experimental strategy applied here is to provide complex I with different (distinguishable) species of Q and QH 2 , and to ask whether any QH 2 is oxidized during catalysis (see Figure 1). Directly or indirectly (conformationally) coupled mechanisms of proton translocation should not lead to 'reductant induced oxidation' (Figure 1A), whereas a Q-cycle mechanism should result in the oxidation of one QH 2 for every NADH oxidized (Figure 1B). Key requirements for the success of our strategy are: i) the availability of two quinone species with similar physical and catalytic properties; ii) a method of accurately quantifying the four quinone/ol species present; iii) the absence of significant alternative routes for the oxidation of QH 2 . Decylubiquinone (DQ) and ubiquinone-2 (Q 2 ) both contain 10-carbon sidechains and have similar physical properties [30,31]. The reactions of both of them with complex I have been studied [31][32][33][34][35][36], and the general picture is that they are both reduced by complex I in an inhibitor-sensitive manner, and their reduction is coupled to proton translocation. However, differences in their catalytic properties have been observed, notably Q 2 supports a lower rate of turnover than DQ [31,33,35], and Q 2 shows a greater tendency than DQ to react at the 'inhibitor-insensitive site' on complex I, commensurate with its higher hydrophilicity [33]. Here we have taken these differences into account by, first, quantifying the behaviour of both quinones under the conditions used here (Scheme 2A), and second, by carrying out all experiments in duplicate (interchanging the identities of each of the quinone / quinol species) to balance out any variations. Importantly, both oxidation states of DQ and Q 2 are readily distinguished and quantified by HPLC analysis (see below). The main obstacle to our strategy was found to be the 'self-exchange reaction' between Q and QH 2 (Scheme 2B) [28], which exchanges the redox states of the two species and provides an alternative route for QH 2 oxidation. This reaction precluded an end-point or 'pulse' approach (taking catalytic turnover to completion with varying amounts of NADH), and required catalysis to be analysed during an experimental timecourse. However, the exchange reaction could be quantified accurately according to a simple bimolecular reaction (Scheme 2B) thus, providing that its rate is not prohibitively fast, it can be readily factored into simulations of experimental data.

Quantification of DQ, Q 2 , DQH 2 and Q 2 H 2 by HPLC

The reverse phase HPLC method was capable of separating and quantifying all four species in a single experiment (see Figure 2). For each species, peak area was directly proportional to the concentration loaded onto the column, and determinations of the concentration of an unknown sample were reproducible (multiple injections gave values which differed by less than 1 %), with a limit of quantification of ∼1 μM.

Accurate values of extinction coefficients in the HPLC mobile phase and in ethanol were determined as follows. The UV-visible spectra of stock solutions of DQ and Q 2 were recorded over a range of dilutions. Then, complex I was used to catalyse the reaction between NADH (0 to 100 μM) and different estimated concentrations of DQ or Q 2 (0 -100 μM) under standard assay conditions. The reaction was incubated until no further NADH oxidation was observed (∼ 10 min.), and the total amount of NADH oxidised was calculated. HPLC analysis confirmed that when [NADH] > [Q] only negligible quinone remained (< 2 μM). The amount of DQ or Q 2 present in the stock solution was calculated and the extinction coefficient determined. Each determination was carried out at least three times, and the values found agree well with published values [28,31,33,37] (see Table 1). Corresponding values for the quinols were calculated by an analogous method: upon reaction with NADH the decrease in quinone concentration is equal to the increase in quinol concentration, so that comparison of the HPLC peak areas from sets of experiments from the same stock solution defined the ratio of the extinction coefficients (see Table 1). Comparison with previously reported values shows good agreement for DQH 2 [28,38] but significant discrepancy with the single reported value for Q 2 H 2 [39]. However, this published value is not consistent with reported values from other quinols. All data reported below have been calculated using the extinction coefficients determined as described above.

Development of a protocol for preparing samples for HPLC analysis

The quinone-quinol self-exchange reaction (Scheme 2B) is slow relative to enzyme turnover, but fast enough to perturb the reaction mixture significantly after a 10 min. incubation. Because it occurs in water, but not significantly in ethanol or methanol, a range of conditions were investigated in an attempt to minimize its rate. A mixture of Q 2 and DQH 2 was incubated for 10 min. at 30 °C in various test solutions, then evaluated by HPLC. Exchange is slower at lower pH (6.0 < 7.5 < 9.0, consistent with results described previously for plastoquinol-1 and ubiquinone-1

[28]), slower in D 2 O than in H 2 O (at equivalent pL), and accelerated by phospholipids. Different types and amounts of detergent, ethylene glycol, the ionic strength [28], and the order in which the different reagents (buffer, phospholipid and quinones) were added exerted no significant effect. However, the relative rates (exchange versus catalysis) are most important here, and catalysis is retarded significantly in the absence of phospholipids [24], and at pH 6.0 or 9.0 relative to 7.5 -whereas exchanging H 2 O for D 2 O had little effect. Therefore, the chosen conditions were 0.1 mg ml -1 of bovine heart phospholipids or 0.15 mg ml -1 asolectin (decreased below the optimal 'catalytic' level to minimise the exchange reaction), in D 2 O, at pD = 7.5 and 30 °C. The enzyme was incubated for 2 min. in the presence of 100 μM NADH before the reaction was initiated with the Q / QH 2 mixture. Finally, quenching the reaction with a 3-fold excess of ethanol / methanol (53:22 v / v) rapidly stopped both catalysis and exchange and allowed storage and anaerobic freeze / thawing without alteration in composition. In addition, the quenched mixture could be injected directly onto the HPLC column, and NADH in the solution was stable, allowing its analysis by UV-visible spectroscopy.

The reduction of DQ and Q 2 by NADH and validation of the experimental approach

In catalytic activity assays variation in the initial rate of NADH oxidation as a function of the concentrations of DQ and Q 2 defines apparent values for K M and k cat (in applying the Michaelis-Menten equation no assumption about the actual mechanism is intended). Values of K M and k cat derived here are reported in Table 2, and compared with published values. First, the K M values are significantly higher than values derived using submitochondrial particles (SMPs) [31,40], but not necessarily higher than the values of Hano and coworkers derived using isolated complex I [35]. K M is known to depend strongly on the hydrophobic phase volume [31], as the concentration of quinone in the hydrophobic phase is much higher than the average concentration in the cuvette. Previously, Fato and coworkers calculated 'true' K M values of 120 -138 mM for DQ and 11.5 -36 mM for Q 2 [31], the K M for endogenous Q 10 is in the mM range in membrane lipids [41], and values recorded here varied according to phospholipid concentration. Thus, the smaller values determined using SMPs probably correspond to small hydrophobic phase volumes. Second, k cat values of 3.1 (DQ) and 1.8 (Q 2 ) μmol min -1 mg -1 were typical under the conditions employed in generating samples for HPLC analysis, though, as expected, they are slightly lower than corresponding values recorded previously [24] because of the decreased phospholipid concentration in the assay buffer. As expected, k cat is smaller for Q 2 than for DQ, and although the absolute rates differ between preparations, good agreement in k cat DQ/k cat Q 2 is found throughout Table 2. All catalytic rates described here are >90 % sensitive to inhibition by rotenone or piericidin A (the sensitivity of DQ is higher than that of Q 2 , as described previously [33]).

In significant quinol concentrations the characteristics of product inhibition should also be defined. Product inhibition in complex I is poorly understood. Ohshima and coworkers suggested that quinols are competitive inhibitors, and showed that 40 μM Q 2 H 2 was sufficient to inhibit Q 1 reduction in SMPs completely [40]. However, for other combinations of Q and QH 2 only 40 % inhibition could be attained, incompatible with a simple competitive inhibition model.

Later, Nakashima and coworkers suggested that Q 1 H 2 is a competitive inhibitor of Q 1 reduction, but no kinetic parameters were reported [42]. In fact, it would be surprising if quinol inhibition did conform to any simple pattern. For example, the relative binding affinities of Q and QH 2 are likely to depend on the oxidation states of the enzyme's redox cofactors and so to depend on the relative rates of NADH oxidation and Q reduction -and complex I may adopt multiple conformations during the proton pumping cycle also. In addition, appropriate experiments are technically difficult (for example, quinols are sensitive to O 2 ) and difficult to interpret (for example, because of differences in the solubility and partition coefficients of the reactant and product species). In the experiments described here we found that a naive competitive inhibition model (Scheme 2A) was able to reproduce measured data satisfactorily, though it is unlikely to reflect the actual mechanism. Thus, sets of ten assay traces, covering a range of initial DQ and DQH 2 (or Q 2 and Q 2 H 2 ) concentrations, were recorded and modelled according to Scheme 2A, using the corresponding equation in Table 3 and the K M values in Table 2. Then, effective values for K I(DQH2) and K I(Q2H2) were estimated by optimising the fit between the model and the data (Table 2).

Figure 3 shows how the reduction of a single quinone species (DQ or Q 2 ), by complex I and NADH, could be monitored accurately using HPLC and modelled using Scheme 2A and the values reported in Table 2. In Scheme 2A the rate is controlled completely by quinone reduction, not NADH oxidation, because NADH:ferricyanide or hexaammine ruthenium oxidoreduction by complex I is significantly faster than NADH:ubiquinone oxidoreduction [26,43,44], showing that NADH oxidation is not rate limiting. Figure 3 validates Scheme 2A and our experimental approach because: i) the total amount of quinone species (Q + QH 2 ) calculated using the extinction coefficients reported in Table 1 remains

Quantitative characterization of the exchange reaction

Figure 4 shows data from HPLC analysis of the exchange reaction. In all cases the total amounts of each species {DQ + DQH 2 } and {Q 2 + Q 2 H 2 }, and of each oxidation state {DQ + Q 2 } and {DQH 2 + Q 2 H 2 } remain constant throughout. Scheme 2B shows the reaction mechanism used to model the data, a bimolecular reaction between quinol and quinone, where k 1 refers to both the forward and backward reactions [28]. Figure 4 shows clearly that the equation derived from Scheme 2B (see Table 3) describes the experimental data accurately. Only slight improvements (typically the LSQ value decreases by ~5 %) resulted from letting the forward and backward rate constants differ. The measured exchange rate constants are summarized in Table 4, demonstrating that the rate constant does not depend on the combination of quinones used (rows 1 and 2 vs. 3 and 4), or on the presence of NADH, complex I, or inhibitor alone (rows 1 to 4), but that it is larger in the presence of bovine phospholipids than asolectin phospholipids (rows 1 and 3 vs. 2 and 4).

Reactions to quantify the self-exchange reaction were also carried out in the presence of all the components of the assay (buffer, phospholipids, complex I, NADH, Q and QH 2 ) but with a complex I inhibitor (rotenone or piericidin A) present (see Figure 5). Complex I is thought to reduce quinone at two different sites: the 'inhibitor sensitive site' is associated with hydrophobic quinone substrates and proton translocation, while the 'inhibitor insensitive site' accepts more hydrophilic substrates and is decoupled from the proton-motive force [33,[45][46][47]. The two substrates used here, DQ and Q 2 , display a small component of inhibitor insensitive activity, so that the inhibited enzyme exhibits a slow rate of ubiquinone reduction. Quinone substrates with higher hydrophobicity (to increase the inhibitor sensitivity) are precluded by their insolubility in aqueous buffers [31][32][33][34]. Figure 5 shows three typical data sets, and the data fits obtained using Scheme 2B in conjunction with Scheme 2C to describe the inhibitor insensitive reduction (see Table 3). The agreement between the data and the model is excellent, and Table 4 includes the rate constants derived for the exchange reaction. Unexpectedly, the exchange reaction is accelerated significantly in the presence of inhibited complex I (all the substrates are present and turnover is actively inhibited). Because the inhibitor insensitive reaction is not associated with

Catalysis of NADH:ubiquinone oxidoreduction in the presence of ubiquinol

Figure 6 shows two data sets from the complex I catalysed reduction of either DQ or Q 2 , in the presence of Q 2 H 2 or DQH 2 respectively. In each case the total amount of each Q-species remains constant throughout the assay, and the decrease in the total quinone concentration is equal to the increase in the total quinol concentration, and to the decrease in the concentration of NADH.

Therefore, both data sets are internally consistent. On first sight it is obvious that Figure 6 resembles Figure 1B, rather than Figure 1A, indicating the operation of a Q-cycle mechanism.

However, as described below, the transient quinol oxidation may be explained quantitatively by the combination of the model of Figure 1A with the known rate of the self-exchange reaction.

Consequently, our experiments do not provide any evidence for the operation of a Q-cycle mechanism in complex I.

It is immediately apparent, from the relative rates of NADH oxidation and quinone reduction, that Q 2 H 2 slows the reduction of DQ severely, and that DQH 2 slows the reduction of Q 2 also. The relative effects of Q 2 H 2 and DQH 2 in this 'cross-over' reaction were confirmed in solution assays, but are only qualitatively consistent with the values of K M , k cat and K I reported in Table 2 (calculated according to Scheme 2D and the corresponding equations in Table 3). It is likely that Scheme 2D is not a sufficient (or correct) description of the mechanism.

Consequently, instead of attempting to apply Scheme 2D to the data shown in Figure 6, a pragmatic approach (avoiding any definition of the characteristics of product inhibition) was adopted as follows. The NADH concentration, at a given time, defines the total amount of quinone which has been reduced. Thus, changes in the individual quinone and quinol concentrations can be estimated from the amount of NADH oxidised, provided that the 'preference' of the enzyme to react with either DQ or Q 2 is known. First, calculations using Scheme 2D and the K M and k cat values given in Table 2 suggest that complex I should display a slight preference for DQ. For example, in the presence of 50 μM DQ and 50 μM Q 2 , DQ reduction is expected to account for 59 % (not 50 %) of the overall reduction rate. 50 μM DQ and 50 μM Q 2 is the point (in a total concentration of 100 μM Q) at which complex I is predicted to deviate most from a 'chance encounter' model, in which the enzyme reacts simply according to the concentration of the different species which are present (for example, in 70 μM DQ and 30 μM Q 2 DQ reduction would account for 70 % of the overall rate). Subsequently, experiments employing HPLC to analyse a range of DQ and Q 2 concentrations suggested that the chance encounter model actually provides an accurate picture, with very little deviation found in any case (the ratio of (DQ reduced / total reduction) to (DQ concentration / total concentration) varied only from 0.95 to 1.05). Therefore, the simple chance encounter model was applied to model the data presented in Figure 6. Future investigations, using a wider range of quinones, may help to define whether the chance encounter model reflects the catalytic mechanism, or whether its accuracy, in this case, results simply from the similarity in the apparent K M and k cat values (Table 2).

Figure 7 shows that excellent agreement with the experimental data is obtained by using the chance encounter model in combination with measured changes in NADH concentration.

The fits in Figure 7 rely on only a single variable, the exchange rate constant (k 1 in Table 3), and derived values from different data sets lie between 40 and 75 mol -1 dm 3 s -1 . Thus, the data reported in Figure 7 can be explained without using a Q-cycle mechanism, and by using an exchange rate constant equivalent to values measured in the presence of inhibited complex I (see Table 4). Clearly, this is evidence against the operation of a Q-cycle mechanism. However, the exchange rate constants required to fit the data are those from the inhibited enzyme (complex I + NADH + inhibitor), and they are significantly larger than equivalent values from complex I alone, NADH alone, or complex I + inhibitor in the absence of NADH. Two features are common to the inhibited complex I and to complex I during turnover. i). The flavin and the ironsulphur clusters are reduced in each case (NADH oxidation is much faster than Q reduction during catalysis). ii). The slow, inhibitor insensitive reaction occurs in both cases. If the reduced, inhibited enzyme facilitates the self-exchange reaction then it may reflect the existence of a alternative conformation, or a state in which the environment of one or more redox-active centres is altered. However, it is perhaps more likely that the inhibitor insensitive reaction increases exchange via the formation of semiquinone intermediates. Interestingly, redox equilibration between duroquinol and Q 2 has been reported to be catalysed by the cytochrome bc 1 complex also [48]. The reaction proceeds by reduction of cofactors in the enzyme by duroquinol, followed by the reduction of Q 2 to Q 2 H 2 . However, it is unlikely that complex I catalyses the reaction by this mechanism as the oxidised enzyme does not increase the exchange rate, and because Q 2 H 2 and DQH 2 have much higher potentials than any of the known complex I cofactors.

As expected, no evidence for any reduced FeS clusters was observed by EPR when DQH 2 or Q 2 H 2 were used to attempt to reduce complex I, irrespective of whether an inhibitor was present or not.

Finally, note that the experimental data can also be fit reasonably well by using an analogous approach, but with a mechanism that comprises the reaction of two quinones and one quinol, again using a chance-encounter model. However, the fits are compromised because data sets acquired using DQ and Q 2 H 2 can be fit well only if the rate of exchange is zero (with the best fits using slightly negative values) and because those acquired using Q 2 and DQH 2 show systematic errors with LSQ values several times higher than provided by the simple model.

Consequently, although our data do not support a Q-cycle mechanism in complex I, we must accept that the increased rate of the exchange reaction observed during turnover and with the inhibited enzyme (observations which are worthy of future investigation) compromises the certainty of our conclusion.

Implications for the mechanism of complex I

The experiments described here provide no support for the operation of a Q-cycle mechanism in complex I, and all data can be explained accurately by the 'simple' reduction of Q to QH 2 . However, our data do not exclude a Q-cycle mechanism unambiguously. First, the increased rate of Q / QH 2 exchange, catalysed by the inhibited enzyme and proposed to occur also during turnover, remains unexplained and is worthy of future consideration. We believe that it does not indicate a Q-cycle mechanism because 'inhibitor insensitive' catalysis is not coupled to the proton motive force. Second, we cannot exclude the possibility that Q, generated from QH 2 , is retained in the vicinity of the 'reducing' active site, promoting its immediate re-reduction and so precluding its detection. Most simply, Q could be released from the enzyme at a point which is close to the entry to the Q-binding site (at present no information on the spatial arrangement of the Q-binding sites in complex I exists). An additional possibility is that Q is retained in the enzyme and 'switched' internally to the second active site, requiring controlled directional uptake and release of protons from a single large site to alternate sides of the membrane. A semiquinone 'switch' mechanism has been proposed previously, conforming to this suggestion [49]. Finally, if complex I does not catalyse by a Q-cycle type mechanism then it is likely to translocate protons either by direct coupling at cluster N2 or at the site of quinone reduction, or to use a conformational change mechanism (see, for recent reviews, [START_REF] Brandt | Proton pumping by NADH:ubiquinone oxidoreductase. A redox driven conformational change mechanism[END_REF][START_REF] Hirst | Energy transduction by respiratory complex I -an evaluation of current knowledge[END_REF][10]). Clearly, determination of the mechanism of energy transduction by this complicated enzyme will require new structural information and creative approaches to functional studies of catalysis.

TABLES

ε in ethanol (mM -1 cm -1 ) ε in mobile phase (mM

( ) ( ) x ] Q [ K x ] QH [ 1 K x ] Q [ ] CI [ k dt dx 0 I 0 2 M 0 cat - + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ + + - = ( ) 0 t k ] Q [ x ] Q [ ln ] Q [ a b ax cat 0 0 0 = + - + + 1 K K a I M - = 0 I 0 M ] Q [ K ] P [ 1 K b + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ + = B. Exchange reaction - - - = ) x ] H * Q )([ x ] Q ([ k dt dx 0 2 0 1 ) x *] Q )([ x ] QH ([ k 0 0 2 1 + + ( ) Q ) 1 e ( ] H * Q [ ] Q [ *] Q [ ] QH [ x Q t k 0 2 0 0 0 2 1 Σ - - = Σ -
ΣQ is the sum of the concentrations of all the Q and QH 2 species. A). DQ and Q 2 H 2 incubated in the presence of complex I and bovine heart phospholipids, modelled using k 1 = 15 mol -1 dm 3 s -1 . B). DQ and Q 2 H 2 incubated in the presence of NADH and asolectin, modelled using k 1 = 7 mol -1 dm 3 s -1 . C). DQH 2 and Q 2 incubated in the presence of complex I and asolectin, modelled using k 1 = 8 mol -1 dm 3 s -1 .

C. Inhibitor insensitive reaction

) x ] Q ([ k dt dx 0 2 - = ( ) t k 0 2 e 1 ] Q [ x - - = D. Catalysis - two Qs with product inhibition ⎪ ⎭ ⎪ ⎬ ⎫ ⎪ ⎩ ⎪ ⎨ ⎧ + + + + = ⎟ ⎠ ⎞ - 2 2 2 2 Q M 2 DQ M H Q I 2 2 DQH I 2 DQ M catDQ 2 Q K ] Q [ K ] DQ [ K ] H Q [ K ] DQH [ 1 ] DQ [ K k dt ] DQ [ d
Figure 5. The self-exchange reaction of ubiquinone and ubiquinol in the presence of inhibited complex I. Different species of ubiquinone and ubiquinol were incubated together in the assay buffer at 30 °C, in the presence of complex I, phospholipids, NADH, and either rotenone or piericidin A, and their self-exchange reaction monitored over time by HPLC analysis ( DQ, Q 2 , DQH 2 , Q 2 H 2 ). A). DQ and Q 2 H 2 in the presence of complex I inhibited by piericidin A (bovine phospholipids) modelled using k 1 = 52 mol -1 dm 3 s -1 (inhibitor-insensitive catalysis k 2 = 17 mol -1 dm 3 s -1 ). B). DQH 2 and Q 2 incubated in the presence of complex I inhibited by rotenone (asolectin) modelled using k 1 = 65 mol -1 dm 3 s -1 (inhibitor insensitive catalysis k 2 = 44 mol -1 dm 3 s -1 ). C). DQH 2 and Q 2 incubated in the presence of complex I inhibited by piericidin A (asolectin) modelled using k 1 = 77 mol -1 dm 3 s -1 (inhibitor insensitive catalysis k 2 = 33 mol -1 dm 3 s -1 ).

  constant throughout; ii) the decrease in NADH concentration matches the decrease in the quinone concentration and the increase in the quinol 11 Biochemical Journal Immediate Publication. Published on 8 Aug 2006 as manuscript BJ20060766 Copyright 2006 Biochemical Society concentration; iii) agreement between the experimentally recorded data and the model is excellent, and the errors in each data point are small.

  proton translocation[33] the increased exchange rate is unlikely to be associated with energy transduction. Possible explanations are discussed below.

Figure 1 .

 1 Figure 1. Experimental strategy aimed at detecting a Q-cycle in complex I. A). Complex I catalyses the simple reduction of quinone (Q) to quinol (QH 2 ), so that a different species of quinol (Q*H 2 ) is unaffected and remains reduced throughout. B). Complex I catalyses a Q-cycle reaction (two quinones reduced and one quinol oxidised for every NADH oxidised), so that at the start of the reaction the oxidation of Q*H 2 to Q* is observed. As the reaction progresses both Q* and Q are reduced to their corresponding quinols completely. Thus, detection of Q* is indicative of 'reductant-induced oxidation' and indicative of a Q-cycle mechanism. (+, concentration of NADH).

Figure 2 .

 2 Figure 2. HPLC analysis of a mixture of the four quinone species. 20 μl of a mixture containing DQ (2.2 μM), DQH 2 (4.1 μM), Q 2 (3.2 μM) and Q 2 H 2 (5.8 μM) was loaded onto a Nucleosil C18 column and eluted using a mobile phase of 50 mM sodium perchlorate in 53:25:22:0.1 v/v/v/v ethanol / water / methanol / 70 % perchloric acid (0.4 ml min -1 ), and monitored at 290 nm (reported in milli-absorption units (mAU)). The four species elute at approximately 8.2 min. (Q 2 H 2 ), 12.0 min. (Q 2 ), 14.5 min. (DQH 2 ) and 23.0 min. (DQ).

Figure 3 .

 3 Figure 3. The reduction of DQ and Q 2 by NADH, catalysed by complex I. The reaction was initiated by the addition of approximately 100 μM DQ or Q 2 to assay buffer containing NADH, complex I and asolectin phospholipids, and monitored by HPLC analysis ( DQ, Q 2 , DQH 2 , Q 2 H 2 ) and UV-visible spectroscopy (+ NADH). A). Conversion of DQ to DQH 2 modelled using K M = 24 μM , K I = 26 μM and k cat = 3.0 mol -1 dm 3 s -1 . B). Conversion of Q 2 to Q 2 H 2 modelled using K M = 20.4 μM , K I = 15.7 μM and k cat = 1.2 mol -1 dm 3 s -1 .

Figure 4 .

 4 Figure 4. The self-exchange reaction of ubiquinone and ubiquinol. Different species of ubiquinone and ubiquinol were incubated together in the assay buffer at 30 °C, and their selfexchange reaction monitored over time by HPLC analysis ( DQ, Q 2 , DQH 2 , Q 2 H 2 ).

Figure 6 .

 6 Figure 6. The reduction of a quinone by complex I, in the presence of a different species of quinol. The quinone and quinol (DQ and Q 2 H 2 (A) or Q 2 and DQH 2 (B)) were added to an assay buffer at 30 °C, containing complex I, phospholipids (asolectin) and NADH, and the reaction was

Table 1 . Extinction coefficients for the two quinone and quinol species.

 1 Extinctioncoefficients for DQ and Q 2 were determined in ethanol and in the HPLC mobile phase, and the values determined in ethanol are consistent with values reported previously (enclosed in {}).Extinction coefficients for DQH 2 and Q 2 H 2 were determined in the HPLC mobile phase only.The value for DQH 2 matches previously reported values in ethanol but the value for Q 2 H 2 does not. The error values reported are 95 % confidence intervals.

	-1 cm -1 )

Table 2 . Values for K M , k cat and K I for DQ, Q 2 , DQH 2 and Q 2 H 2 determined in this work and compared to values reported previously.

 2 Error values are 95 % confidence intervals.

	Rate equation	Integrated rate equation
	A.
	Catalysis -
	single Q
	with
	product
	inhibition

Table 3 . Rate equations describing the four reactions shown in Scheme 2. x

 3 is the amount of the starting material which has reacted to form products after time t and [A] 0 is the concentration of species A at time zero. In D) only the rate equation for the decrease in DQ at constant Q 2 concentration is given; an analogous equation is readily derived for Q 2 .

	Quinone species	Phospholipid	Control condition	Rate constant (mol -1 dm 3 s -1 )
	DQ + Q 2 H 2	bovine	-NADH or -CI ± inhibitor	14.4 ± 4.6
	DQ + Q 2 H 2	asolectin	-NADH or -CI ± inhibitor	8.9 ± 2.6
	Q 2 + DQH 2	bovine	-NADH or -CI ± inhibitor	18.3 ± 7.3
	Q 2 + DQH 2	asolectin	-NADH or -CI ± inhibitor	9.1 ± 3.0
	DQ + Q 2 H 2 or Q 2 + DQH 2	bovine	+ NADH + CI + rotenone or piericidin A	68.0 ± 35.4
	DQ + Q 2 H 2 or Q 2 + DQH 2	asolectin	+ NADH + CI + rotenone or piericidin A	76.5 ± 15.7
	DQ + Q 2 H 2 or Q 2 + DQH 2	bovine or asolectin	+ NADH + CI + rotenone	74.8 ± 23.2
	DQ + Q 2 H 2 or Q 2 + DQH 2	bovine or asolectin	+ NADH + CI + piericidin A	72.3 ± 19.6

Table 4 . Rate constants for the self-exchange reaction measured under different control conditions.

 4 In data rows 1 to 4 there was no significant difference in the measured rate constants if the reaction was carried out in the absence of NADH or complex I (CI), or if an inhibitor was added (rotenone 2.3 μM or piericidin A 0.25 μM). The rate constants for the reactions between DQ and Q 2 H 2 and DQH 2 and Q 2 do not differ significantly, but the exchange reaction is faster in bovine phospholipids than in asolectin. In data rows 5 to 8 all the components of the assay are present and complex I is inhibited by either rotenone (2.3 or 11.5 μM) or piericidin A (0.25 or 1.25 μM). The rate constant does not depend on which inhibitor is present, on which combination of quinones is investigated, or on whether the reaction is carried out in bovine phospholipids or asolectin. All values are reported ± their 95 % confidence interval. Reaction schemes corresponding to the equations presented in Table3. A).Reduction of a single quinone species (reaction rate determined by the reduction of Q, not the oxidation of NADH), inhibited competitively by quinol. B). The quinone:quinol self-exchange reaction. C). The inhibitor insensitive reduction of Q by complex I. D). The reaction scheme in A) expanded to describe the reduction of both DQ and Q 2 , inhibited by both DQH 2 and Q 2 H 2 .

	LEGENDS
	Scheme 1. A). Schematic representation of complex I in the inner-mitochondrial membrane
	catalysing NADH oxidation, quinone reduction and proton translocation. B). Proposed Q-cycle
	mechanism of catalysis (reduction of two quinones and oxidation of one quinol for every NADH
	oxidised). Top: Q is reduced by one electron from the FeS chain (derived from NADH) and by
	one electron from quinol to form QH 2 and a semiquinone (right). Bottom: a second Q is reduced
	to form a second QH 2 and the SQ is oxidised to Q (left). Q: quinone; SQ: semiquinone; QH 2 :
	quinol.
	Scheme 2.

Biochemical Journal Immediate Publication. Published on 8 Aug 2006 as manuscript BJ20060766 Copyright 2006 Biochemical Society

Biochemical Journal Immediate Publication. Published on 8 Aug 2006 as manuscript BJ20060766

Value reported per mg of total protein.

Biochemical Journal Immediate Publication. Published on 8 Aug 2006 as manuscript BJ20060766 Copyright 2006 Biochemical Society Scheme 1.

Biochemical Journal Immediate Publication. Published on 8 Aug 2006 as manuscript BJ20060766 Copyright 2006 Biochemical Society Scheme 2.

ACKNOWLEDGEMENTS

This work was supported by The Medical Research Council.

 6). The modelled values were calculated using the change in NADH concentration (smoothed data derived from that shown in Figure 6) and the 'chance encounter' model (see text) with self-exchange rate constants of 44 mol -1 dm 3 s -1 (A) and 75 mol -1 dm 3 s -1 (B).