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 (b_H 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 b_H heme. These results show that one can distinguish between the contribution of structural and thermodynamic factors to center N function.     

The dimeric structure of the cytochrome bc1 complex prevents center P inhibition by reverse reactions at center N

Energy transduction in the cytochrome bc₁ 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 c₁ reduction at varying quinol/quinone ratios in the isolated yeast bc₁ 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 c₁ 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 c₁ 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 bc₁ complex which allows electrons to equilibrate between monomers.     

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 ∼ 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.     

Ubiquinol oxidation in the cytochrome bc1 complex: Reaction mechanism and prevention of short-circuiting

This review is focused on the mechanism of ubiquinol oxidation by the cytochrome bc₁ complex (bc₁). This integral membrane complex serves as a “hub” in the vast majority of electron transfer chains. The bc₁ oxidizes a ubiquinol molecule to ubiquinone by a unique “bifurcated” reaction where the two released electrons go to different acceptors: one is accepted by the mobile redox active domain of the [2Fe-2S] iron-sulfur Rieske protein (FeS protein) and the other goes to cytochrome b. The nature of intermediates in this reaction remains unclear. It is also debatable how the enzyme prevents short-circuiting that could happen if both electrons escape to the FeS protein. Here, I consider a reaction mechanism that (i) agrees with the available experimental data, (ii) entails three traits preventing the short-circuiting in bc₁, and (iii) exploits the evident structural similarity of the ubiquinone binding sites in the bc₁ and the bacterial photosynthetic reaction center (RC). Based on the latter congruence, it is suggested that the reaction route of ubiquinol oxidation by bc₁ is a reversal of that leading to the ubiquinol formation in the RC. The rate-limiting step of ubiquinol oxidation is then the re-location of a ubiquinol molecule from its stand-by site within cytochrome b into a catalytic site, which is formed only transiently, after docking of the mobile redox domain of the FeS protein to cytochrome b. In the catalytic site, the quinone ring is stabilized by Glu-272 of cytochrome b and His-161 of the FeS protein. The short circuiting is prevented as long as: (i) the formed semiquinone anion remains bound to the reduced FeS domain and impedes its undocking, so that the second electron is forced to go to cytochrome b; (ii) even after ubiquinol is fully oxidized, the reduced FeS domain remains docked to cytochrome b until electron(s) pass through cytochrome b; (iii) if cytochrome b becomes (over)reduced, the binding and oxidation of further ubiquinol molecules is hampered; the reason is that the Glu-272 residue is turned towards the reduced hemes of cytochrome b and is protonated to stabilize the surplus negative charge; in this state, this residue cannot participate in the binding/stabilization of a ubiquinol molecule.     

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

Hydroxy-naphthoquinones are competitive inhibitors of the cytochrome bc₁ 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 bc₁ 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 bc₁ complexes as surrogates to model the interaction of atovaquone with the bc₁ complexes of the target pathogens and human host. As a first step to identify new cytochrome bc₁ 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 bc₁ complex. Molecular modeling of these into the crystal structure of the yeast cytochrome bc₁ 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.     

Discrimination between two possible reaction sequences that create potential risk of generation of deleterious radicals by cytochrome bc1: Implications for the mechanism of superoxide production

In addition to its bioenergetic function of building up proton motive force, cytochrome bc₁ can be a source of superoxide. One-electron reduction of oxygen is believed to occur from semiquinone (SQ_o) formed at the quinone oxidation/reduction Q_o site (Q_o) as a result of single-electron oxidation of quinol by the iron-sulfur cluster (FeS) (semiforward mechanism) or single-electron reduction of quinone by heme b_L (semireverse mechanism). It is hotly debated which mechanism plays a major role in the overall production of superoxide as experimental data supporting either reaction exist. To evaluate a contribution of each of the mechanisms we first measured superoxide production under a broad range of conditions using the mutants of cytochrome bc₁ that severely impeded the oxidation of FeS by cytochrome c₁, changed density of FeS around Q_o by interfering with its movement, or combined these two effects together. We then compared the amount of generated superoxide with mathematical models describing either semiforward or semireverse mechanism framed within a scheme assuming competition between the internal reactions at Q_o and the leakage of electrons on oxygen. We found that only the model of semireverse mechanism correctly reproduced the experimentally measured decrease in ROS for the FeS motion mutants and increase in ROS for the mutants with oxidation of FeS impaired. This strongly suggests that this mechanism dominates in setting steady-state levels of SQ_o that present a risk of generation of superoxide by cytochrome bc₁. Isolation of this reaction sequence from multiplicity of possible reactions at Q_o helps to better understand conditions under which complex III might contribute to ROS generation in vivo.     

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 bc₁ complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc₁ 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 bc₁ 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 c₁ 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.     

Traffic within the Cytochrome b6f Lipoprotein Complex: Gating of the Quinone Portal

The cytochrome bc complexes b₆f and bc₁ catalyze proton-coupled quinol/quinone redox reactions to generate a transmembrane proton electrochemical gradient. Quinol oxidation on the electrochemically positive (p) interface of the complex occurs at the end of a narrow quinol/quinone entry/exit Q_p portal, 11 Å long in bc complexes. Superoxide, which has multiple signaling functions, is a by-product of the p-side quinol oxidation. Although the transmembrane core and the chemistry of quinone redox reactions are conserved in bc complexes, the rate of superoxide generation is an order of magnitude greater in the b₆f complex, implying that functionally significant differences in structure exist between the b₆f and bc₁ complexes on the p-side. A unique structure feature of the b₆f p-side quinol oxidation site is the presence of a single chlorophyll-a molecule whose function is unrelated to light harvesting. This study describes a cocrystal structure of the cytochrome b₆f complex with the quinol analog stigmatellin, which partitions in the Q_p portal of the bc₁ complex, but not effectively in b₆f. It is inferred that the Q_p portal is partially occluded in the b₆f complex relative to bc₁. Based on a discrete molecular-dynamics analysis, occlusion of the Q_p portal is attributed to the presence of the chlorophyll phytyl tail, which increases the quinone residence time within the Q_p portal and is inferred to be a cause of enhanced superoxide production. This study attributes a novel (to our knowledge), structure-linked function to the otherwise enigmatic chlorophyll-a in the b₆f complex, which may also be relevant to intracellular redox signaling.     

Superoxide anion generation by the cytochrome bc1 complex

We have measured the rates of superoxide anion generation by cytochrome bc₁ complexes isolated from bovine heart and yeast mitochondria and by cytochrome bc₁ complexes from yeast mutants in which the midpoint potentials of the cytochrome b hemes and the Rieske iron-sulfur cluster were altered by mutations in those proteins. With all of the bc₁ complexes the rate of superoxide anion production was greatest in the absence of bc₁ inhibitor and ranged from 3% to 5% of the rate of cytochrome c reduction. Stigmatellin, an inhibitor that binds to the ubiquinol oxidation site in the bc₁ complex, eliminated superoxide anion formation, while myxothiazol, another inhibitor of ubiquinol oxidation, allowed superoxide anion formation at a low rate. Antimycin, an inhibitor that binds to the ubiquinone reduction site in the bc₁ complex, also allowed superoxide anion formation and at a slightly greater rate than myxothiazol. Changes in the midpoint potentials of the cytochrome b hemes had no significant effect on the rate of cytochrome c reduction and only a small effect on the rate of superoxide anion formation. A mutation in the Rieske iron-sulfur protein that lowers its midpoint potential from +285 to +220 mV caused the rate of superoxide anion to decline in parallel with a decline in cytochrome c reductase activity. These results indicate that superoxide anion is formed by similar mechanisms in mammalian and yeast bc₁ complexes. The results also show that changes in the midpoint potentials of the redox components that accept electrons during ubiquinol oxidation have only small effects on the formation of superoxide anion, except to the extent that they affect the activity of the enzyme.     

Traffic within the Cytochrome b6f Lipoprotein Complex: Gating of the Quinone Portal

The cytochrome bc complexes b₆f and bc₁ catalyze proton-coupled quinol/quinone redox reactions to generate a transmembrane proton electrochemical gradient. Quinol oxidation on the electrochemically positive (p) interface of the complex occurs at the end of a narrow quinol/quinone entry/exit Q_p portal, 11 Å long in bc complexes. Superoxide, which has multiple signaling functions, is a by-product of the p-side quinol oxidation. Although the transmembrane core and the chemistry of quinone redox reactions are conserved in bc complexes, the rate of superoxide generation is an order of magnitude greater in the b₆f complex, implying that functionally significant differences in structure exist between the b₆f and bc₁ complexes on the p-side. A unique structure feature of the b₆f p-side quinol oxidation site is the presence of a single chlorophyll-a molecule whose function is unrelated to light harvesting. This study describes a cocrystal structure of the cytochrome b₆f complex with the quinol analog stigmatellin, which partitions in the Q_p portal of the bc₁ complex, but not effectively in b₆f. It is inferred that the Q_p portal is partially occluded in the b₆f complex relative to bc₁. Based on a discrete molecular-dynamics analysis, occlusion of the Q_p portal is attributed to the presence of the chlorophyll phytyl tail, which increases the quinone residence time within the Q_p portal and is inferred to be a cause of enhanced superoxide production. This study attributes a novel (to our knowledge), structure-linked function to the otherwise enigmatic chlorophyll-a in the b₆f complex, which may also be relevant to intracellular redox signaling.     

What information do inhibitors provide about the structure of the hydroquinone oxidation site of ubihydroquinone: Cytochromec oxidoreductase?

The Q cycle mechanism of thebc ₁ complex requires two quinone reaction centers, the hydroquinone oxidation (Q_P) and the quinone reduction (Q_N) center. These sites can be distinguished by the specific binding of inhibitors to either of them. A substantial body of information about the hydroquinone oxidation site has been provided by the analysis of the binding of Q_P site inhibitors to thebc ₁ complex in different redox states and to preparations depleted of lipid or protein components as well as by functional studies with mutantbc ₁ complexes selected for resistance toward the inhibitors. The reaction site is formed by at least five protein segments of cytochromeb and parts of the iron-sulfur protein. At least two different binding sites for Q_P site inhibitors could be detected, one for the methoxyacrylate-type inhibitors binding predominantly to cytochromeb, the other for the chromone-type inhibitors and hydroxyquinones binding predominantly to the iron-sulfur protein. The interactions with the protein segments, between different protein segments, and between protein and ligands (substrate, inhibitors) are discussed in detail and a working model of the Q_P pocket is proposed.     

Generation of semiquinone-[2Fe-2S]+ spin-coupled center at the Qo site of cytochrome bc1 in redox-poised,illuminated photosynthetic membranes from Rhodobacter capsulatus

One of the less understood parts of the catalytic cycle of cytochrome bc₁/b₆f complexes is the mechanism of electronic bifurcation occurring within the hydroquinone oxidation site (Q_o site). Several models describing this mechanism invoke a phenomenon of formation of an unstable semiquinone. Recent studies with isolated cytochrome bc₁ or b₆f revealed that a relatively stable semiquinone spin-coupled to the reduced Rieske cluster (SQ-FeS) is generated at the Q_o site during the oxidation of ubi- or plastohydroquinone analogs under conditions of continuous turnover. Here, we identified the EPR transition of SQ-FeS formed upon oxidation of ubihydroquinone in native photosynthetic membranes from purple bacterium Rhodobacter capsulatus. We observed a significant amount of SQ-FeS generated when the antimycin-inhibited enzyme experiences conditions of non-equilibrium caused by the continuous light activation of the reaction center. We also noted that SQ-FeS cannot be detected under equilibrium redox titrations in dark. The non-equilibrium redox titrations of SQ-FeS indicate that this center has a higher apparent redox midpoint potential when compared to the redox midpoint potential of the quinone pool. This suggests that SQ-FeS is stabilized, which corroborates a recently proposed mechanism in which the SQ-FeS state is metastable and functions to safely hold electrons at the local energy minimum during the oxidation of ubihydroquinone and limits superoxide formation. Our results open new possibilities to study the formation and properties of this state in cytochromes bc under close to physiological conditions in which non-equilibrium is attained by the light activation of bacterial reaction centers or photosystems.     

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 bc₁ 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.     

Flash-induced turnover of the cytochrome bc1 complex in chromatophores of Rhodobacter capsulatus: binding of Zn decelerates likewise the oxidation of cytochrome b, the reduction of cytochrome c1 and the voltage generation

The effect of Zn²⁺ on the rates of electron transfer and of voltage generation in the cytochrome bc₁ complex (bc₁) was investigated under excitation of Rhodobacter capsulatus chromatophores with flashing light. When added, Zn²⁺ retarded the oxidation of cytochrome b and allowed to monitor (at 561-570 nm) the reduction of its high potential heme b_h (in the absence of Zn²⁺ this reaction was masked by the fast re-oxidation of the heme). The effect was accompanied by the deceleration of both the cytochrome c₁ reduction (as monitored at 552-570 nm) and the generation of transmembrane voltage (monitored by electrochromism at 522 nm). At Zn²⁺ <100 μM the reduction of heme b_h remained 10 times faster than other reactions. The kinetic discrepancy was observed even after an attenuated flash, when bc₁ turned over only once. These observations (1) raise doubt on the notion that the transmembrane electron transfer towards heme b_h is the main electrogenic reaction in the cytochrome bc₁ complex, (2) imply an allosteric link between the site of heme b_h oxidation and the site of cytochrome c₁ reduction at the opposite side of the membrane, and (3) indicate that the internal redistribution of protons might account for the voltage generation by the cytochrome bc₁ complex.     

Reaction center and UQH2:cytc 2 oxidoreductase act as independent enzymes inRps. sphaeroides

Turnover of the ubiquinol oxidizing site of the UQH₂:cyt c₂ oxidoreductase (b/c ₁ complex) ofRps. sphaeroides can be assayed by measuring the rate of reduction of cytb ₅₆₁ in the presence of antimycin (AA). Oxidation of ubiquinol is a second-order process, with a value ofk ₂ of about 3 × 10⁵ M^–1. The reaction shows saturation at high quinol concentrations, with an apparentK _m of about 6–8 mM (with respect to the concentration of quinol in the membrane). When the quinone pool is oxidized before illumination, reduction of the complex shows a substantial lag (about 1 ms) after a flash, indicating that the quinol produced as a result of the photochemical reactions is not immediately available to the complex. We have suggested that the lag may be due to several factors, including the leaving time of the quinol from the reaction center, the diffusion time to the complex, and the time for the head group to cross the membrane. We have suggested aminimal value for the diffusion coefficient of ubiquinone in the membrane (assuming that the lag is due entirely to diffusion) of about 10^–9 cm^–2 sec^–1. The lag is reduced to about 100 µsec when the pool is significantly reduced, showing that quinol from the pool is more rapidly available to the complex than that from the reaction center. With the pool oxidized, similar kinetics are seen when the reduction of cytb ₅₆₁ occurs through the AA-sensitive site (with reactions at the quinol oxidizing site blocked by myxothiazol). These results show that there is no preferential reaction pathway for transfer of reducing equivalents from reaction center tob/c ₁ complex. Oxidation of cytb ₅₆₁ through the AA-sensitive site can be assayed from the slow phase of the carotenoid electrochromic change, and by comparison with the kinetics of cytb ₅₆₁. As long as the quinone pool is significantly oxidized, the reaction is not rate-determining for the electrogenic process. On reduction of the pool below 1 quinone per complex, a slowing of the electrogenic process occurs, which could reflect a dependence on the concentration of quinone. If the process is second-order, the rate constant must be about 2–5 times greater than that for quinol oxidation, since the effect on rate is relatively small compared with the effect seen at the quinol oxidizing site when the quinol concentration is changed over theE _h range where the first few quinols are produced on reductive titration. When the quinone pool is extracted (experiments in collaboration with G. Venturoli and B. A. Melandri), the slowing of the electrochromic change on reduction of the pool is not enhanced; we assume that this is due to the fact that a minimum of one quinone per active complex is produced by turnover of the quinol oxidizing site. Two lines of research lead us to revise our previous estimate for the minimal value of the quinone diffusion coefficient. These relate to the relation between the diffusion coefficient and the rate constants for processes involving the quinones: (a) The estimated rate constant for reaction of quinone at the AA-site approaches the calculated diffusion limited rate constant, implying an improbably efficient reaction. (b) From a preliminary set of experiments, the activation energy determined by measuring the variation of the rate constant for quinol oxidation with temperature, is about 8 kcal mol^–1. Although we do not know the contribution of entropic terms to the pre-exponential factor, the result is consistent with a considerably larger value for the diffusion coefficient than that previously suggested.     

Ascochlorin is a novel, specific inhibitor of the mitochondrial cytochrome bc1 complex

Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc₁ complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q_i site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q_o site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A₃ in a fashion similar to the Q_o site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q_i and the Q_o sites of the fungal cytochrome bc₁ complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc₁ complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q_o and Q_i sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc₁ complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q_i and Q_o sites. Moreover, crystal structure of chicken cytochrome bc₁ complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q_i and Q_o sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc₁ inhibitor that acts at both of the active sites of this enzyme.     

Proton pumping in the bc1 complex: A new gating mechanism that prevents short circuits

The Q-cycle mechanism of the bc₁ complex explains how the electron transfer from ubihydroquinone (quinol, QH₂) to cytochrome (cyt) c (or c₂ in bacteria) is coupled to the pumping of protons across the membrane. The efficiency of proton pumping depends on the effectiveness of the bifurcated reaction at the Q_o-site of the complex. This directs the two electrons from QH₂ down two different pathways, one to the high potential chain for delivery to an electron acceptor, and the other across the membrane through a chain containing heme b_L and b_H to the Q_i-site, to provide the vectorial charge transfer contributing to the proton gradient. In this review, we discuss problems associated with the turnover of the bc₁ complex that center around rates calculated for the normal forward and reverse reactions, and for bypass (or short-circuit) reactions. Based on rate constants given by distances between redox centers in known structures, these appeared to preclude conventional electron transfer mechanisms involving an intermediate semiquinone (SQ) in the Q_o-site reaction. However, previous research has strongly suggested that SQ is the reductant for O₂ in generation of superoxide at the Q_o-site, introducing an apparent paradox. A simple gating mechanism, in which an intermediate SQ mobile in the volume of the Q_o-site is a necessary component, can readily account for the observed data through a coulombic interaction that prevents SQ anion from close approach to heme b_L when the latter is reduced. This allows rapid and reversible QH₂ oxidation, but prevents rapid bypass reactions. The mechanism is quite natural, and is well supported by experiments in which the role of a key residue, Glu-295, which facilitates proton transfer from the site through a rotational displacement, has been tested by mutation.     

Monitoring the redox and protonation dependent contributions of cardiolipin in electrochemically induced FTIR difference spectra of the cytochrome bc1 complex from yeast

Biochemical studies have shown that cardiolipin is essential for the integrity and activity of the cytochrome bc₁ complex and many other membrane proteins. Recently the direct involvement of a bound cardiolipin molecule (CL) for proton uptake at center N, the site of quinone reduction, was suggested on the basis of a crystallographic study. In the study presented here, we probe the low frequency infrared spectroscopy region as a technique suitable to detect the involvement of the lipids in redox induced reactions of the protein. First the individual infrared spectroscopic features of lipids, typically present in the yeast membrane, have been monitored for different pH values in micelles and vesicles. The pK_a values for cardiolipin molecule have been observed at 4.7 ± 0.3 and 7.9 ± 1.3, respectively. Lipid contributions in the electrochemically induced FTIR spectra of the bc₁ complex from yeast have been identified by comparing the spectra of the as isolated form, with samples where the lipids were digested by lipase-A₂. Overall, a noteworthy perturbation in the spectral region typical for the protein backbone can be reported. Interestingly, signals at 1159, 1113, 1039 and 980 cm^− 1 have shifted, indicating the perturbation of the protonation state of cardiolipin coupled to the reduction of the hemes. Additional shifts are found and are proposed to reflect lipids reorganizing due to a change in their direct environment upon the redox reaction of the hemes. In addition a small shift in the alpha band from 559 to 556 nm can be seen after lipid depletion, reflecting the interaction with heme b_H and heme c. Thus, our work highlights the role of lipids in enzyme reactivity and structure.