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.     

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.     

Effect of Mutations of Arginine 94 on Proton Pumping,Electron Transfer,and Superoxide Anion Generation in Cytochrome b of the bc1 Complex from Rhodobacter sphaeroides

Proton transfer involving internal water molecules that provide hydrogen bonds and facilitate proton diffusion has been identified in some membrane proteins. Arg-94 in cytochrome b of the Rhodobacter sphaeroides bc₁ complex is fully conserved and is hydrogen-bonded to the heme propionate and a chain of water molecules. To further elucidate the role of Arg-94, we generated the mutations R94A, R94D, and R94N. The wild-type and mutant bc₁ complexes were purified and then characterized. The results show that substitution of Arg-94 decreased electron transfer activity and proton pumping capability and increased O₂^˙̄ production, suggesting the importance of Arg-94 in the catalytic mechanism of the bc₁ complex in R. sphaeroides. This also suggests that the transport of H⁺, O₂, and O₂^˙̄ in the bc₁ complex may occur by the same pathway.     

Spectral analysis of the bc1 complex components in situ: Beyond the traditional difference approach

The cytochrome (cyt) bc₁ complex (ubiquinol: cytochrome c oxidoreductase) is the central enzyme of mitochondrial and bacterial electron-transport chains. It is rich in prosthetic groups, many of which have significant but overlapping absorption bands in the visible spectrum. The kinetics of the cytochrome components of the bc₁ complex are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. This difference-wavelength (DW) approach has been used extensively in the development and testing of the Q-cycle mechanism of the bc₁ complex in Rhodobacter sphaeroides chromatophores. However, the DW approach does not fully compensate for spectral interference from other components, which can significantly distort both amplitudes and kinetics. Mechanistic elaboration of cyt bc₁ turnover requires an approach that overcomes this limitation. Here, we compare the traditional DW approach to a least squares (LS) analysis of electron transport, based on newly determined difference spectra of all individual components of cyclic electron transport in chromatophores. Multiple sets of kinetic traces, measured at different wavelengths in the absence and presence of specific inhibitors, were analyzed by both LS and DW approaches. Comparison of the two methods showed that the DW approach did not adequately correct for the spectral overlap among the components, and was generally unreliable when amplitude changes for a component of interest were small. In particular, it was unable to correct for extraneous contributions to the amplitudes and kinetics of cyt b_L. From LS analysis of the chromophoric components (RC, c_tot, b_H and b_L), we show that while the Q-cycle model remains firmly grounded, quantitative reevaluation of rates, amplitudes, delays, etc., of individual components is necessary. We conclude that further exploration of mechanisms of the bc₁ complex, will require LS deconvolution for reliable measurement of the kinetics of individual components of the complex in situ.     

Role of phospholipids in respiratory cytochrome bc1 complex catalysis and supercomplex formation

Specific protein-lipid interactions have been identified in X-ray structures of membrane proteins. The role of specifically bound lipid molecules in protein function remains elusive. In the current study, we investigated how phospholipids influence catalytic, spectral and electrochemical properties of the yeast respiratory cytochrome bc₁ complex and how disruption of a specific cardiolipin binding site in cytochrome c₁ alters respiratory supercomplex formation in mitochondrial membranes. Purified yeast cytochrome bc₁ complex was treated with phospholipase A₂. The lipid-depleted enzyme was stable but nearly catalytically inactive. The absorption maxima of the reduced b-hemes were blue-shifted. The midpoint potentials of the b-hemes of the delipidated complex were shifted from − 52 to − 82 mV (heme b_L) and from + 113 to − 2 mV (heme b_H). These alterations could be reversed by reconstitution of the delipidated enzyme with a mixture of asolectin and cardiolipin, whereas addition of the single components could not reverse the alterations. We further analyzed the role of a specific cardiolipin binding site (CL_i) in supercomplex formation by site-directed mutagenesis and BN-PAGE. The results suggested that cardiolipin stabilizes respiratory supercomplex formation by neutralizing the charges of lysine residues in the vicinity of the presumed interaction domain between cytochrome bc₁ complex and cytochrome c oxidase. Overall, the study supports the idea, that enzyme-bound phospholipids can play an important role in the regulation of protein function and protein-protein interaction.     

Subunit analysis of mitochondrial cytochrome c oxidase and cytochrome bc1 by reversed-phase high-performance liquid chromatography

A rapid separation of the ten nuclearly-encoded subunits of mitochondrial cytochrome c oxidase, and ten out of the eleven subunits of cytochrome bc₁, was achieved using a short, 50 mm C₁₈-reversed-phase column. The short column decreased the elution time 4–7 fold while maintaining the same resolution quality. Elution was similar to a previously published protocol, i.e., a water/acetonitrile elution gradient containing trifluoroacetic acid. Isolated subunits were identified by MALDI-TOF. The rapidity of the described method makes it extremely useful for determining the subunit composition of isolated mitochondrial complexes. The method can be used for both analytical and micro-preparative purposes.     

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.     

Spectral and kinetic resolution of the bc1 complex components in situ: A simple and robust alternative to the traditional difference wavelength approach

The kinetics of the cytochrome (cyt) components of the bc₁ complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The “traditional” set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c_tot (cyt c₁ + cyt c₂), cyt b_L, cyt b_H, and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c₁ and c₂ is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c₁, c₂, b_L, and b_H) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc₁ complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc₁ complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.     

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.     

Generation,characterization and crystallization of a cytochrome c1-subunit IV fused cytochrome bc1 complex from Rhodobacter sphaeroides

Cytochrome bc₁ complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c₂) and couples this reaction to proton translocation across the membrane. Crystallization of the Rhodobacter sphaeroides bc₁ complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c₁ with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV (c₁-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting X-rays to 5.5 Å resolution.     

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.     

Fast oxidation of the primary electron acceptor under anaerobic conditions requires the organization of the photosynthetic chain of Rhodobacter sphaeroides in supercomplexes

The kinetics of reoxidation of the primary acceptor Q_a has been followed by measuring the changes in the fluorescence yield induced by a series of saturating flashes in intact cells of Rhodobacter sphaeroides in anaerobic conditions. At 0 °C, about half of Q_a⁻ is reoxidized in about 200 ms while reoxidation of the remaining fraction is completed in several seconds to minutes. The fast phase is associated with the transfer of ubiquinone formed at site Q_o of the cytochrome bc₁ complex while the slowest phase is associated with the diffusion of ubiquinone present in the membrane prior to the flash excitation. The biphasic kinetics of Q_a⁻ oxidation is interpreted assuming that the electron chain is organized in supercomplexes that associate two RCs and one cyt bc₁ complex, which allows a fast transfer of quinone formed at the level of cyt bc₁ complex to the RCs. In agreement with this model, the fast phase of Q_a⁻ reoxidation is inhibited by myxothiazol, a specific inhibitor of cyt bc₁. The PufX-deleted mutant displays only the slowest phase of Q_a⁻ oxidation; it is interpreted by the lack of supramolecular organization of the photosynthetic chain that leads to a larger average distance between cyt bc₁ and RCs.     

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.     

Hybrid fusions show that inter-monomer electron transfer robustly supports cytochrome bc1 function in vivo

Electronic connection between Q_o and Q_i quinone catalytic sites of dimeric cytochrome bc₁ is a central feature of the energy-conserving Q cycle. While both the intra- and inter-monomer electron transfers were shown to connect the sites in the enzyme, mechanistic and physiological significance of the latter remains unclear. Here, using a series of mutated hybrid cytochrome bc₁-like complexes, we show that inter-monomer electron transfer robustly sustains the function of the enzyme in vivo, even when the two subunits in a dimer come from different species. This indicates that minimal requirement for bioenergetic efficiency is to provide a chain of cofactors for uncompromised electron flux between the catalytic sites, while the details of protein scaffold are secondary.     

Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c1 and its physiological redox partners

The transient electron transfer (ET) interactions between cytochrome c₁ of the bc₁-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c₅₅₂ and cytochrome c₅₅₀ have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c₁ were generated and used together with a soluble cytochrome c₅₅₂ module as a model system for interprotein ET reactions. Both c₁ fragments lack the membrane anchor; the c₁ core fragment (c_1CF) consists of only the hydrophilic heme-carrying domain, whereas the c₁ acidic fragment (c_1AF) additionally contains the acidic domain unique to P. denitrificans. In order to determine the ionic strength dependencies of the ET rate constants, an optimized stopped-flow protocol was developed to overcome problems of spectral overlap, heme autoxidation and the prevalent non-pseudo first order conditions. Cytochrome c₁ reveals fast bimolecular rate constants (10⁷ to 10⁸ M^− 1 s^− 1) for the ET reaction with its physiological substrates c₅₅₂ and c₅₅₀, thus approaching the limit of a diffusion-controlled process, with 2 to 3 effective charges of opposite sign contributing to these interactions. No direct involvement of the N-terminal acidic c₁-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c₅₅₀ over c₅₅₂ reacting with cyochrome c₁ was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans.     

Generation, characterization and crystallization of a highly active and stable cytochrome bc1 complex mutant from Rhodobacter sphaeroides

The availability of the three dimensional structure of mitochondrial enzyme, obtained by X-ray crystallography, allowed a significant progress in the understanding of the structure-function relation of the cytochrome bc(1) complex. Most of the structural information obtained has been confirmed by molecular genetic studies of the bacterial complex. Despite its small size and simple subunit composition, high quality crystals of the bacterial complex have been difficult to obtain and so far, only low resolution structural data has been reported. The low quality crystal observed is likely associated in part with the low activity and stability of the purified complex. To mitigate this problem, we recently engineered a mutant [S287R(cytb)/V135S(ISP)] from Rhodobacter sphaeroides to produce a highly active and more stable cytochrome bc(1) complex. The purified mutant complex shows a 40% increase in electron transfer activity as compared to that of the wild type enzyme. Differential scanning calorimetric study shows that the mutant is more stable than the wild type complex as indicated by a 4.3 degrees C increase in the thermo-denaturation temperature. Crystals formed from this mutant complex, in the presence of stigmatellin, diffract X-rays up to 2.9 Angstroms resolution.     

Bcs1p can rescue a large and productive cytochrome bc1 complex assembly intermediate in the inner membrane of yeast mitochondria

The yeast cytochrome bc₁ complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc₁ 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 bc₁ assembly and the formation of a functionally inactive bc₁ core structure of about 500-kDa. This immature bc₁ 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 bc₁ core structure leading to the formation of the functional homodimeric bc₁ complex. Following Bcs1p expression, the mature bc₁ complex was also progressively converted into two supercomplexes with the cytochrome c oxidase complex. The capability of restoring the bc₁ 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 bc₁ complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc₁ core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc₁ complex and gives new insights into the molecular mechanisms involved in the last steps of bc₁ assembly.     

Supramolecular organization of the photosynthetic chain in mutants of Rhodobacter capsulatus deleted in cytochrome c 2

Both the soluble cytochrome c₂ and the membrane-bound cytochrome c_y act as secondary electron carriers in photoinduced cyclic electron transfer chain of Rhodobacter capsulatus [Jenney and Daldal (1993) EMBO J 12: 1283–1292]. In this work, we have studied the kinetics of electron transfer between these secondary electron donors and the reaction center in intact cells of two mutants, MT-G4/S4 and MT-GS18 deleted in cytochrome c₂ and in cytochrome c₂ plus cytochrome bc₁ complex, respectively. In the MT-G4/S4 mutant, only about one third of the primary electron donor is reduced by cytochrome c_y in less than five ms. The remaining fraction is reduced in several seconds, although about 90% of the photoxidized cytochrome c_y is reduced in less than 10 ms by the cytochrome bc₁ complex. This implies that cytochrome c_y is not in thermodynamic equilibrium with the large fraction of primary donors which are slowly reduced. As shown by energy transfer measurements, the reaction centers connected to cytochrome c_y and the disconnected reaction centers are localized in the same membrane region. We propose that the movement of cyt c_y is restricted to a small membrane domain which includes a single cytochrome bc₁ complex. The kinetics of cytochrome c_y photooxidation in the MT-G4/S4 mutant in the presence of myxothiazol presents a fast phase (t^1/2 3 µs) followed by a slower phase (t^1/2 20 µs). In the case of the double mutant MT-GS18, the kinetics of electron transfer between cytochrome c_y and the reaction center is highly multiphasic and much slower than those observed for the MT-G4/S4 mutant. In particular, the amplitude of the fast phase is decreased by more than a factor 2 and the 20-µs phase is not observed. This implies an important structural role of the cytochrome bc₁ complex in the interaction between reaction center and cytochrome c_y, and their formation in supercomplex. The more problable stoichiometry of electron carriers in this supercomplex is 2 reaction centers, 2 cytochrome c_y and 1 cytochrome bc₁ complex.     

Regulatory interactions in the dimeric cytochrome bc1 complex: The advantages of being a twin

The dimeric cytochrome bc₁ 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 bc₁ 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.