Bcs1p can rescue a large and productive cytochrome bc(1) complex assembly intermediate in the inner membrane of yeast mitochondria.

The yeast cytochrome bc(1) complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc(1) complex, some ancillary proteins, such as the chaperone Bcs1p, are actively involved. The deletion of the nuclear gene encoding this chaperone caused the arrest of the bc(1) assembly and the formation of a functionally inactive bc(1) core structure of about 500-kDa. This immature bc(1) core structure could represent, on the one hand, a true assembly intermediate or, on the other hand, a degradation product and/or an incorrect product of assembly. The experiments here reported show that the gradual expression of Bcs1p in the yeast strain lacking this protein was progressively able to rescue the bc(1) core structure leading to the formation of the functional homodimeric bc(1) complex. Following Bcs1p expression, the mature bc(1) complex was also progressively converted into two supercomplexes with the cytochrome c oxidase complex. The capability of restoring the bc(1) complex and the supercomplexes was also possessed by the mutated yeast R81C Bcsp1. Notably, in the human ortholog BCS1L, the corresponding point mutation (R45C) was instead the cause of a severe bc(1) complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc(1) core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc(1) complex and gives new insights into the molecular mechanisms involved in the last steps of bc(1) assembly.

Direct demonstration of half-of-the-sites reactivity in the dimeric cytochrome bc1 complex: enzyme with one inactive monomer is fully active but unable to activate the second ubiquinol oxidation site in response to ligand binding at the ubiquinone reduction site.

We previously proposed that the dimeric cytochrome bc(1) complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc(1) monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289-22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc(1) complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10-16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c(1) by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.

Probing binding determinants in center P of the cytochrome bc(1) complex using novel hydroxy-naphthoquinones.

Atovaquone is a substituted 2-hydroxy-naphthoquinone used therapeutically against Plasmodium falciparum (malaria) and Pneumocystis pathogens. It acts by inhibiting the cytochrome bc(1) complex via interactions with the Rieske iron-sulfur protein and cytochrome b in the ubiquinol oxidation pocket. As the targeted pathogens have developed resistance to this drug there is an urgent need for new alternatives. To better understand the determinants of inhibitor binding in the ubiquinol oxidation pocket of the bc(1) complex we synthesized a series of hydroxy-naphthoquinones bearing a methyl group on the benzene ring that is predicted to interact with the nuclear encoded Rieske iron-sulfur protein. We have also attempted to overcome the metabolic instability of a potent cytochrome bc(1) complex inhibitor, a 2-hydroxy-naphthoquinone with a branched side chain, by fluorinating the terminal methyl group. We have tested these new 2-hydroxy-naphthoquinones against yeast and bovine cytochrome bc(1) complexes to model the interaction with pathogen and human enzymes and determine parameters that affect efficacy of binding of these inhibitors. We identified a hydroxy-naphthoquinone with a trifluoromethyl function that has potential for development as an anti-fungal and anti-parasitic therapeutic.

Biogenesis of the yeast cytochrome bc1 complex.

The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.

The dimeric structure of the cytochrome bc(1) complex prevents center P inhibition by reverse reactions at center N.

Energy transduction in the cytochrome bc(1) complex is achieved by catalyzing opposite oxido-reduction reactions at two different quinone binding sites. We have determined the pre-steady state kinetics of cytochrome b and c(1) reduction at varying quinol/quinone ratios in the isolated yeast bc(1) complex to investigate the mechanisms that minimize inhibition of quinol oxidation at center P by reduction of the b(H) heme through center N. The faster rate of initial cytochrome b reduction as well as its lower sensitivity to quinone concentrations with respect to cytochrome c(1) reduction indicated that the b(H) hemes equilibrated with the quinone pool through center N before significant catalysis at center P occurred. The extent of this initial cytochrome b reduction corresponded to a level of b(H) heme reduction of 33%-55% depending on the quinol/quinone ratio. The extent of initial cytochrome c(1) reduction remained constant as long as the fast electron equilibration through center N reduced no more than 50% of the b(H) hemes. Using kinetic modeling, the resilience of center P catalysis to inhibition caused by partial pre-reduction of the b(H) hemes was explained using kinetics in terms of the dimeric structure of the bc(1) complex which allows electrons to equilibrate between monomers.

Regulatory interactions in the dimeric cytochrome bc(1) complex: the advantages of being a twin.

The dimeric cytochrome bc(1) complex catalyzes the oxidation-reduction of quinol and quinone at sites located in opposite sides of the membrane in which it resides. We review the kinetics of electron transfer and inhibitor binding that reveal functional interactions between the quinol oxidation site at center P and quinone reduction site at center N in opposite monomers in conjunction with electron equilibration between the cytochrome b subunits of the dimer. A model for the mechanism of the bc(1) complex has emerged from these studies in which binding of ligands that mimic semiquinone at center N regulates half-of-the-sites reactivity at center P and binding of ligands that mimic catalytically competent binding of ubiquinol at center P regulates half-of-the-sites reactivity at center N. An additional feature of this model is that inhibition of quinol oxidation at the quinone reduction site is avoided by allowing catalysis in only one monomer at a time, which maximizes the number of redox acceptor centers available in cytochrome b for electrons coming from quinol oxidation reactions at center P and minimizes the leakage of electrons that would result in the generation of damaging oxygen radicals.

Mutations in cytochrome b that affect kinetics of the electron transfer reactions at center N in the yeast cytochrome bc1 complex.

We have examined the pre-steady-state kinetics and thermodynamic properties of the b hemes in variants of the yeast cytochrome bc1 complex that have mutations in the quinone reductase site (center N). Trp-30 is a highly conserved residue, forming a hydrogen bond with the propionate on the high potential b heme (bH heme). The substitution by a cysteine (W30C) lowers the redox potential of the heme and an apparent consequence is a lower rate of electron transfer between quinol and heme at center N. Leu-198 is also in close proximity to the b(H) heme and a L198F mutation alters the spectral properties of the heme but has only minor effects on its redox properties or the electron transfer kinetics at center N. Substitution of Met-221 by glutamine or glutamate results in the loss of a hydrophobic interaction that stabilizes the quinone ligands. Ser-20 and Gln-22 form a hydrogen-bonding network that includes His-202, one of the carbonyl groups of the ubiquinone ring, and an active-site water. A S20T mutation has long-range structural effects on center P and thermodynamic effects on both b hemes. The other mutations (M221E, M221Q, Q22E and Q22T) do not affect the ubiquinol oxidation kinetics at center P, but do modify the electron transfer reactions at center N to various extents. The pre-steady reduction kinetics suggest that these mutations alter the binding of quinone ligands at center N, possibly by widening the binding pocket and thus increasing the distance between the substrate and the bH heme. These results show that one can distinguish between the contribution of structural and thermodynamic factors to center N function.

Differential efficacy of inhibition of mitochondrial and bacterial cytochrome bc1 complexes by center N inhibitors antimycin, ilicicolin H and funiculosin.

We have compared the efficacy of inhibition of the cytochrome bc1 complexes from yeast and bovine heart mitochondria and Paracoccus denitrificans by antimycin, ilicicolin H, and funiculosin, three inhibitors that act at the quinone reduction site at center N of the enzyme. Although the three inhibitors have some structural features in common, they differ significantly in their patterns of inhibition. Also, while the overall folding pattern of cytochrome b around center N is similar in the enzymes from the three species, amino acid sequence differences create sufficient structural differences so that there are striking differences in the inhibitors binding to the three enzymes. Antimycin is the most tightly bound of the three inhibitors, and binds stoichiometrically to the isolated enzymes from all three species under the cytochrome c reductase assay conditions. Ilicicolin H also binds stoichiometrically to the yeast enzyme, but binds approximately 2 orders of magnitude less tightly to the bovine enzyme and is essentially non-inhibitory to the Paracoccus enzyme. Funiculosin on the other hand inhibits the yeast and bovine enzymes similarly, with IC50 approximately 10 nM, while the IC50 for the Paracoccus enzyme is more than 10-fold higher. Similar differences in inhibitor efficacy were noted in bc1 complexes from yeast mutants with single amino acid substitutions at the center N site, although the binding affinity of quinone and quinol substrates were not perturbed to a degree that impaired catalytic function in the variant enzymes. These results reveal a high degree of specificity in the determinants of ligand-binding at center N, accompanied by sufficient structural plasticity for substrate binding as to not compromise center N function. The results also demonstrate that, in principle, it should be possible to design novel inhibitors targeted toward center N of the bc1 complex with appropriate species selectivity to allow their use as drugs against pathogenic fungi and parasites.

Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins.

We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc(1) complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8(m)). Subsequently replacing ARG8(m) with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit(-)). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.

Parameters determining the relative efficacy of hydroxy-naphthoquinone inhibitors of the cytochrome bc1 complex.

Hydroxy-naphthoquinones are competitive inhibitors of the cytochrome bc(1) complex that bind to the ubiquinol oxidation site between cytochrome b and the iron-sulfur protein and presumably mimic a transition state in the ubiquinol oxidation reaction catalyzed by the enzyme. The parameters that affect efficacy of binding of these inhibitors to the bc(1) complex are not well understood. Atovaquone, a hydroxy-naphthoquinone, has been used therapeutically to treat Pneumocystis carinii and Plasmodium infections. As the pathogens have developed resistance to this drug, it is important to understand the molecular basis of the drug resistance and to develop new drugs that can circumvent the drug resistance. We previously developed the yeast and bovine bc(1) complexes as surrogates to model the interaction of atovaquone with the bc(1) complexes of the target pathogens and human host. As a first step to identify new cytochrome bc(1) complex inhibitors with therapeutic potential and to better understand the determinants of inhibitor binding, we have screened a library of 2-hydroxy-naphthoquinones with aromatic, cyclic, and non-cyclic alkyl side-chain substitutions at carbon-3 on the hydroxy-quinone ring. We found a group of compounds with alkyl side-chains that effectively inhibit the yeast bc(1) complex. Molecular modeling of these into the crystal structure of the yeast cytochrome bc(1) complex provides structural and quantitative explanations for their binding efficacy to the target enzyme. In addition we also identified a 2-hydroxy-naphthoquinone with a branched side-chain that has potential for development as an anti-fungal and anti-parasitic therapeutic.

A comparison of stigmatellin conformations, free and bound to the photosynthetic reaction center and the cytochrome bc1 complex.

We describe in detail the conformations of the inhibitor stigmatellin in its free form and bound to the ubiquinone-reducing (Q(B)) site of the reaction center and to the ubiquinol-oxidizing (Q(o)) site of the cytochrome bc(1) complex. We present here the first structures of a stereochemically correct stigmatellin in complexes with a bacterial reaction center and the yeast cytochrome bc1 complex. The conformations of the inhibitor bound to the two enzymes are not the same. We focus on the orientations of the stigmatellin side-chain relative to the chromone head group, and on the interaction of the stigmatellin side-chain with these membrane protein complexes. The different conformations of stigmatellin found illustrate the structural variability of the Q sites, which are affected by the same inhibitor. The free rotation about the chi1 dihedral angle is an essential factor for allowing stigmatellin to bind in both the reaction center and the cytochrome bc1 pocket.

Modeling the molecular basis of atovaquone resistance in parasites and pathogenic fungi.

Atovaquone is a substituted hydroxynaphthoquinone that is used therapeutically for treating Plasmodium falciparum malaria, Pneumocystis jirovecii pneumonia and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting parasite and fungal respiration by binding to the cytochrome bc1 complex. The recent, growing failure of atovaquone treatment and increased mortality of patients with malaria or Pneumocystis pneumonia has been linked to the appearance of mutations in the cytochrome b gene. To better understand the molecular basis of drug resistance, we have developed the yeast and bovine bc1 complexes as surrogates to model the molecular interaction of atovaquone with human and resistant pathogen enzymes.

Identification and characterization of cytochrome bc(1) subcomplexes in mitochondria from yeast with single and double deletions of genes encoding cytochrome bc(1) subunits.

We have examined the status of the cytochrome bc(1) complex in mitochondrial membranes from yeast mutants in which genes for one or more of the cytochrome bc(1) complex subunits were deleted. When membranes from wild-type yeast were resolved by native gel electrophoresis and analyzed by immunodecoration, the cytochrome bc(1) complex was detected as a mixed population of enzymes, consisting of cytochrome bc(1) dimers, and ternary complexes of cytochrome bc(1) dimers associated with one and two copies of the cytochrome c oxidase complex. When membranes from the deletion mutants were resolved and analyzed, the cytochrome bc(1) dimer was not associated with the cytochrome c oxidase complex in many of the mutant membranes, and membranes from some of the mutants contained a common set of cytochrome bc(1) subcomplexes. When these subcomplexes were fractionated by SDS/PAGE and analyzed with subunit-specific antibodies, it was possible to recognize a subcomplex consisting of cytochrome b, subunit 7 and subunit 8 that is apparently associated with cytochrome c oxidase early in the assembly process, prior to acquisition of the remaining cytochrome bc(1) subunits. It was also possible to identify a subcomplex consisting of subunit 9 and the Rieske protein, and two subcomplexes containing cytochrome c(1) associated with core protein 1 and core protein 2, respectively. The analysis of all the cytochrome bc(1) subcomplexes with monospecific antibodies directed against Bcs1p revealed that this chaperone protein is involved in a late stage of cytochrome bc(1) complex assembly.

A concerted, alternating sites mechanism of ubiquinol oxidation by the dimeric cytochrome bc(1) complex.

A refinement of the protonmotive Q cycle mechanism is proposed in which oxidation of ubiquinol is a concerted reaction and occurs by an alternating, half-of-the-sites mechanism. A concerted mechanism of ubiquinol oxidation is inferred from the finding that there is reciprocal control between the high potential and low potential redox components involved in ubiquinol oxidation. The potential of the Rieske iron-sulfur protein controls the rate of reduction of the b cytochromes, and the potential of the b cytochromes controls the rate of reduction of the Rieske protein and cytochrome c(1). A concerted mechanism of ubiquinol oxidation reconciles the findings that the ubiquinol-cytochrome c reductase kinetics of the bc(1) complex include both a pH dependence and a dependence on Rieske iron-sulfur protein midpoint potential.An alternating, half-of-the-sites mechanism for ubiquinol oxidation is inferred from the finding that some inhibitory analogs of ubiquinol that block ubiquinol oxidation by binding to the ubiquinol oxidation site in the bc(1) complex inhibit the yeast enzyme with a stoichiometry of 0.5 per bc(1) complex. One molecule of inhibitor is sufficient to fully inhibit the dimeric enzyme, and the binding is anti-cooperative, in that a second molecule of inhibitor binds with much lower affinity to a dimer in which an inhibitor molecule is already bound. An alternating, half-of-the-sites mechanism implies that, at least under some conditions, only half of the sites in the dimeric enzyme are reactive at any one time. This provides a raison d'être for the dimeric structure of the enzyme, in that bc(1) activity may be regulated and capable of switching between a half-of-the-sites active and a fully active enzyme.

RPL29 codes for a non-essential protein of the 60S ribosomal subunit in Saccharomyces cerevisiae and exhibits synthetic lethality with mutations in genes for proteins required for subunit coupling.

RPL29 (YFR032c-a) is a non-essential gene that codes for a 60S ribosomal subunit protein in Saccharomyces cerevisiae. Deletion of RPL29 leads to a moderate accumulation of half-mer polysomes with little or no change in the amounts of free 60S subunits. In vitro translation and the growth rate are also delayed in the Deltarpl29 strain. Such a phenotype is characteristic of mutants defective in 60S to 40S subunit joining. The Deltarpl29 strain exhibits synthetic lethality with mutations in RPL10, the gene encoding an essential 60S ribosomal subunit protein that is required for 60S to 40S subunit joining. The Deltarpl29 strain also exhibits synthetic lethality with RSA1, a gene encoding a nucleoplasmic protein required for the loading of Rpl10p onto the 60S subunit. Over-expression of RPL10 suppresses the half-mer phenotype of the Deltarpl29 strain, but does not correct the growth defect of the deletion strain. We conclude that absence of Rpl29p impairs proper assembly of proteins onto the 60S subunit and that this retards subunit joining and additionally retards protein synthesis subsequent to subunit joining.

Protonmotive pathways and mechanisms in the cytochrome bc1 complex.

The cytochrome bc(1) complex catalyzes electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which electron transfer is linked to proton translocation across the inner mitochondrial membrane. In the Q cycle mechanism proton translocation is the net result of topographically segregated reduction of quinone and reoxidation of quinol on opposite sides of the membrane, with protons being carried across the membrane as hydrogens on the quinol. The linkage of proton chemistry to electron transfer during quinol oxidation and quinone reduction requires pathways for moving protons to and from the aqueous phase and the hydrophobic environment in which the quinol and quinone redox reactions occur. Crystal structures of the mitochondrial cytochrome bc(1) complexes in various conformations allow insight into possible proton conduction pathways. In this review we discuss pathways for proton conduction linked to ubiquinone redox reactions with particular reference to recently determined structures of the yeast bc(1) complex.

Design of anti-parasitic and anti-fungal hydroxy-naphthoquinones that are less susceptible to drug resistance.

Atovaquone is a hydroxy-naphthoquinone that is used to treat parasitic and fungal infections including Plasmodium falciparum (malaria), Pneumocystis jivorecii (pneumonia) and Toxoplasma gondii (toxoplasmosis). It blocks mitochondrial oxidation of ubiquinol in these organisms by binding to the ubiquinol oxidation site of the cytochrome bc(1) complex. Failure of atovaquone treatment has been linked to the appearance of mutations in the mitochondrially encoded gene for cytochrome b. In order to determine the optimal parameters required for inhibition of respiration in parasites and pathogenic fungi and overcome drug resistance, we have synthesized and tested the inhibitory activity of novel hydroxy-naphthoquinones against blood stage P. falciparum and liver stage P. berghei and against cytochrome bc(1) complexes isolated from yeast strains bearing mutations in cytochrome b associated with resistance in Plasmodium, Pneumocystis, and Toxoplasma. One of the new inhibitors is highly effective against an atovaquone resistant Plasmodium and illustrates the type of modification to the hydroxy-naphthoquinone ring of atovaquone that might mitigate drug resistance.

Ilicicolin Inhibition and Binding at Center N of the Dimeric Cytochrome bc1 Complex Reveal Electron Transfer and Regulatory Interactions between Monomers.

We have determined the kinetics of ilicicolin binding and dissociation at center N of the yeast bc(1) complex and its effect on the reduction of cytochrome b with center P blocked. The addition of ilicicolin to the oxidized complex resulted in a non-linear inhibition of the extent of cytochrome b reduction by quinol together with a shift of the reduced b(H) heme spectrum, indicating electron transfer between monomers. The possibility of a fast exchange of ilicicolin between center N sites was excluded in two ways. First, kinetic modeling showed that fast movement of an inhibitor between monomers would result in a linear inhibition of the extent of cytochrome b reduction through center N. Second, we determined a very slow dissociation rate for ilicicolin (k = 1.2 x 10(-3) s(-1)) as calculated from its displacement by antimycin. Ilicicolin binding to the reduced bc(1) complex occurred in a single phase (k(on) = 1.5-1.7 x 10(5) m(-1) s(-1)) except in the presence of stigmatellin, where a second slower binding phase comprising approximately 50% of the spectral change was observed. This second kinetic event was weakly dependent on ilicicolin concentration, which suggests that binding of ilicicolin to one center N in the dimer transmits a slow (k = 2-3 s(-1)) conformational change that allows binding of the inhibitor in the other monomer. These results, together with the evidence for intermonomeric electron transfer, provide further support for a dimeric model of regulatory interactions between center P and center N sites in the bc(1) complex.

The rate-limiting step in the cytochrome bc1 complex (Ubiquinol-Cytochrome c Oxidoreductase) is not changed by inhibition of cytochrome b-dependent deprotonation: implications for the mechanism of ubiquinol oxidation at center P of the bc1 complex.

Quinol oxidation at center P of the cytochrome bc(1) complex involves bifurcated electron transfer to the Rieske iron-sulfur protein and cytochrome b. It is unknown whether both electrons are transferred from the same domain close to the Rieske protein, or if an unstable semiquinone anion intermediate diffuses rapidly to the vicinity of the b(L) heme. We have determined the pre-steady state rate and activation energy (E(a)) for quinol oxidation in purified yeast bc(1) complexes harboring either a Y185F mutation in the Rieske protein, which decreases the redox potential of the FeS cluster, or a E272Q cytochrome b mutation, which eliminates the proton acceptor in cytochrome b. The rate of the bifurcated reaction in the E272Q mutant (<10% of the wild type) was even lower than that of the Y185F enzyme ( approximately 20% of the wild type). However, the E272Q enzyme showed the same E(a) (61 kJ mol(-1)) with respect to the wild type (62 kJ mol(-1)), in contrast with the Y185F mutation, which increased E(a) to 73 kJ mol(-1). The rate and E(a) of the slow reaction of quinol with oxygen that are observed after cytochrome b is reduced were unaffected by the E272Q substitution, whereas the Y185F mutation modified only its rate. The Y185F/E272Q double mutation resulted in a synergistic decrease in the rate of quinol oxidation (0.7% of the wild type). These results are inconsistent with a sequential "movable semiquinone" mechanism but are consistent with a model in which both electrons are transferred simultaneously from the same domain in center P.