Identification of amino acid residues essential for reconstitutive activity of subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides

A region of subunit IV comprising residues 77-85 is identified as essential for interaction with the core complex to restore the bc(1) activity (reconstitutive activity). Recombinant subunit IV mutants with single or multiple alanine substitution at this region were generated and characterized to identify the essential amino acid residues. Residues 81-84, with sequence of YRYR, are required for reconstitutive activity of subunit IV, because a mutant with these four residues substituted with alanine has little activity, while a mutant with alanine substitution at residues 77-80 and 85 have the same reconstitutive activity as that of the wild-type IV. The positively charged group at R-82 and R-84 and both the hydroxyl group and aromatic group at Y-81 and Y-83 are essential. The interactions between these four residues of subunit IV and residues of core subunits are also responsible for the stability of the complex. However, these interactions are not essential for the incorporation of subunit IV into the bc(1) complex.     

Effect of subunit IV on superoxide generation by Rhodobacter sphaeroides cytochrome bc1 complex

Previous studies indicate that the three-subunit cytochrome bc₁ core complex of Rhodobacter sphaeroides contains a fraction of the electron transfer activity of the wild-type enzyme. Addition of subunit IV to the core complex increases electron transfer activity to the same level as that of the wild-type complex. This activity increase may result from subunit IV preventing electron leakage, from the low potential electron transfer chain, and reaction with molecular oxygen, producing superoxide anion. This suggestion is based on the following observations: (1) the extent of cytochrome b reduction in the three-subunit core complex, by ubiquinol, in the presence of antimycin A, never reaches the same level as that in the wild-type complex; (2) the core complex produces 4 times as much superoxide anion as does the wild-type complex; and (3) when the core complex is reconstituted with subunit IVs having varying reconstitutive activities, the activity increase in reconstituted complexes correlates with superoxide production decrease and extent of cytochrome b reduction increase.     

Subunit IV of cytochrome bc1 complex from Rhodobacter sphaeroides. Localization of regions essential for interaction with the three-subunit core complex

Recombinant subunit IV mutants which identify the regions essential for restoration of bc(1) activity to the three-subunit core complex of Rhodobacter sphaeroides were generated and characterized. Four C-terminal truncated mutants: IV(1-109), IV(1-85), IV(1-76), and IV(1-40) had 100, 0, 0, and 0% of reconstitutive activity of the wild-type IV, indicating that residues 86-109 are essential. IV(1-109) is associated with the core complex in the same manner as the wild-type IV while mutants IV(1-85), IV(1-76), and IV(1-40) do not associate with the core complex, indicating that subunit IV requires its transmembrane helix region (residues 86-109) for assembly into the bc(1) complex. Since GST-IV(86-109) fusion protein has little reconstitutive activity, some region(s) in residues 1-85 are required for bc(1) activity restoration after subunit IV is incorporated into the complex through the transmembrane helix, presumably by interaction with cytochrome b in the core complex. The interacting regions are identified as residues 41-53 and 77-85, since mutants IV(21-109), IV(41-109), IV(54-109), and IV(77-109) had 95, 98, 53, and 53% of the reconstitutive activity of the wild-type IV. These two interacting regions are on the cytoplasmic side of the chromatophore membrane and closed to the DE loop and helix G of cytochrome b, respectively.     

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.     

The NMR solution structure of subunit G (G61-101) of the eukaryotic V1VO ATPase from Saccharomyces cerevisiae

Subunit G is an essential stalk subunit of the eukaryotic proton pump V₁V_O ATPase. Previously the structure of the N-terminal region, G_1-59, of the 13 kDa subunit G was solved at higher resolution. Here solution NMR was performed to determine the structure of the recombinant C-terminal region (G_61-101) of subunit G of the Saccharomyces cerevisiae V₁V_O ATPase. The protein forms an extended α-helix between residues 64 and 100, whereby the first five- and the last residues of G_61-101 are flexible. The surface charge distribution of G_61-101 reveals an amphiphilic character at the C-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The hydrophobic surface pattern is mainly formed by alanine residues. The alanine residues 72, 74 and 81 were exchanged by a single cysteine in the entire subunit G. Cysteines at positions 72 and 81 showed disulfide formation. In contrast, no crosslink could be formed for the mutant Ala74Cys. Together with the recently determined NMR solution structure of G_1-59, the presented solution structure of G_61-101 enabled us to present a first structural model of the entire subunit G of the S. cerevisiae V₁V_O ATPase.     

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc₁ complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c₁ position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c₁ [S141C(ISP)/G180C(cyt c₁)]. The formation of a disulfide bond between ISP and cyt c₁ in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with β-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc₁ complex. Formation of the disulfide bond between ISP and cyt c₁ shortens the distance between the [2Fe-2S] cluster and heme c₁, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.     

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.     

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 bc₁ 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 bc₁ 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 °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 Å resolution.     

The Role of the Supernumerary Subunit of Rhodobactersphaeroides Cytochrome bc 1 Complex

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group,is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc ₁ complex.This subunit is involved in Q binding and the structural integrity of the complex. When thecytochrome bc ₁ complex is photoaffinity labeled with [³H]azido-Q derivative, radioactivity isfound in subunits IV and I (cytochrome b), indicating that these two subunits are responsiblefor Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R.sphaeroides chromosome, the resulting strain (RSIV) requires a period of adaptation beforethe start of photosynthetic growth. The cytochrome bc ₁ complex in adapted RSIVchromatophores is labile to detergent treatment (60–75% inactivation), and shows a four-fold increasein the K _m for Q₂H₂. The first two changes indicate a structural role of subunit IV; the thirdchange supports its Q-binding function. Tryptophan-79 is important for structural andQ-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GSTfusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunitIV is functionally active as it can restore the bc ₁ complex activity from the three-subunit corecomplex to the same level as that of wild-type or complement complex. Three regions in thesubunit IV sequence, residues 86–109, 77–85, and 41–55, are essential for interaction withthe core complex because deleting one of these regions yields a subunit completely or partiallyunable to restore cytochrome bc ₁ from the core complex.     

Conservation of lipid functions in cytochrome bc complexes

Lipid binding sites and properties are compared in two sub-families of hetero-oligomeric membrane protein complexes known to have similar functions in order to gain further understanding of the role of lipid in the function, dynamics, and assembly of these complexes. Using the crystal structure information for both complexes, we compared the lipid binding properties of the cytochrome b₆f and bc₁ complexes that function in photosynthetic and respiratory membrane energy transduction. Comparison of lipid and detergent binding sites in the b₆f complex with those in bc₁ shows significant conservation of lipid positions. Seven lipid binding sites in the cyanobacterial b₆f complex overlap three natural sites in the Chlamydomonas reinhardtii algal complex and four sites in the yeast mitochondrial bc₁ complex. The specific identity of lipids is different in b₆f and bc₁ complexes: b₆f contains sulfoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol, whereas cardiolipin, phosphatidylethanolamine, and phosphatidic acid are present in the yeast bc₁ complex. The lipidic chlorophyll a and β-carotene (β-car) in cyanobacterial b₆f, as well as eicosane in C. reinhardtii, are unique to the b₆f complex. Inferences of lipid binding sites and functions were supported by sequence, interatomic distance, and B-factor information on interacting lipid groups and coordinating amino acid residues. The lipid functions inferred in the b₆f complex are as follows: (i) substitution of a transmembrane helix by a lipid and chlorin ring, (ii) lipid and β-car connection of peripheral and core domains, (iii) stabilization of the iron-sulfur protein transmembrane helix, (iv) n-side charge and polarity compensation, and (v) β-car-mediated super-complex with the photosystem I complex.     

Thebc 1 complexes ofRhodobacter sphaeroides andRhodobacter capsulatus

Photosynthetic bacteria offer excellent experimental opportunities to explore both the structure and function of the ubiquinol-cytochromec oxidoreductase (bc ₁ complex). In bothRhodobacter sphaeroides andRhodobacter capsulatus, thebc ₁ complex functions in both the aerobic respiratory chain and as an essential component of the photosynthetic electron transport chain. Because thebc ₁ complex in these organisms can be functionally coupled to the photosynthetic reaction center, flash photolysis can be used to study electron flow through the enzyme and to examine the effects of various amino acid substitutions. During the past several years, numerous mutations have been generated in the cytochromeb subunit, in the Rieske iron-sulfur subunit, and in the cytochromec ₁ subunit. Both site-directed and random mutagenesis procedures have been utilized. Studies of these mutations have identified amino acid residues that are metal ligands, as well as those residues that are at or near either the quinol oxidase (Q_o) site or the quinol reductase (Q_i) site. The postulate that these two Q-sites are located on opposite sides of the membrane is supported by these studies. Current research is directed at exploring the details of the catalytic mechanism, the nature of the subunit interactions, and the assembly of this enzyme.     

Crystal structure of crotoxin reveals key residues involved in the stability and toxicity of this potent heterodimeric β-neurotoxin

The crystal structure of crotoxin, a potent presynaptic neurotoxin from Crotalus durissusterrificus, was solved at 1.35 Å resolution. It shows the architecture of the three disulfide-linked polypeptide chains (α, β, and γ) of the acidic subunit CA noncovalently complexed with the basic phospholipase A₂ (PLA₂) subunit CB. The unique structural scaffold of the association of the CA and CB subunits indicates that posttranslational cleavage of the pro-CA precursor is a prerequisite for the assembly of the CA-CB complex. These studies provide novel structural insights to explain the role of the CA subunit in the mechanism of action of crotoxin. The crystal structure of the highly toxic and stable CA₂CBb complex crystallized here allows us to identify key amino acid residues responsible for significant differences in the pharmacological activities of the two classes of crotoxin complexes. In particular, we show that critical residues Trp31 and Trp70 of the CBb subunit establish intermolecular polar contacts with Asp99 and Asp89, respectively, of the β-chain of CA₂ and contribute to the stability and toxicity of the CA₂CBb complex. These interactions also lead to decreased PLA₂ activity by partially blocking substrate access to the catalytic dyad and by masking several interfacial binding surface residues important for PLA₂ interaction with phospholipids.Identification of the binding interface between the CA subunits and the CB subunits of crotoxin is important for the structure-based design of antineurotoxic inhibitors. Since crotoxin displays numerous physiological functions, including antitumoral properties, knowledge of its three-dimensional structure will be useful for the understanding of these diverse effects.     

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.     

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.     

Redox-linked protonation state changes in cytochrome bc1 identified by Poisson-Boltzmann electrostatics calculations

Cytochrome bc₁ is a major component of biological energy conversion that exploits an energetically favourable redox reaction to generate a transmembrane proton gradient. Since the mechanistic details of the coupling of redox and protonation reactions in the active sites are largely unresolved, we have identified residues that undergo redox-linked protonation state changes. Structure-based Poisson-Boltzmann/Monte Carlo titration calculations have been performed for completely reduced and completely oxidised cytochrome bc₁. Different crystallographically observed conformations of Glu272 and surrounding residues of the cytochrome b subunit in cytochrome bc₁ from Saccharomyces cerevisiae have been considered in the calculations. Coenzyme Q (CoQ) has been modelled into the CoQ oxidation site (Q_o-site). Our results indicate that both conformational and protonation state changes of Glu272 of cytochrome b may contribute to the postulated gating of CoQ oxidation. The Rieske iron-sulphur cluster could be shown to undergo redox-linked protonation state changes of its histidine ligands in the structural context of the CoQ-bound Q_o-site. The proton acceptor role of the CoQ ligands in the CoQ reduction site (Q_i-site) is supported by our results. A modified path for proton uptake towards the Q_i-site features a cluster of conserved lysine residues in the cytochrome b (Lys228) and cytochrome c₁ subunits (Lys288, Lys289, Lys296). The cardiolipin molecule bound close to the Q_i-site stabilises protons in this cluster of lysine residues.     

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.     

Molecular modeling studies of the DCCD-treated cytochrome bc1 complex: predicted conformational changes and inhibition of proton translocation

Dicyclohexylcarbodiimide (DCCD) binds covalently to an acidic amino acid located in the cd loop connecting membrane-spanning helices C and D of cytochrome b resulting in an inhibition of proton translocation in the cytochrome bc ₁ complex with minimal effects on the steady state rate of electron transfer. Single turnover studies performed with the yeast cytochrome bc ₁ complex indicated that the initial phase of cytochrome b reduction was inhibited 25–45% in the DCCD-treated cytochrome bc ₁ complex, while the rate of cytochrome c ₁ reduction was unaffected. Simulations by molecular modeling predict that binding of DCCD to glutamate 163 located in the cd2 loop of cytochrome b of chicken liver mitochondria results in major conformational changes in the protein. The conformation of the cd loop and the end of helix C appeared twisted with a concomitant rearrangement of the amino acid residues of both cd1 and cd2 loops. The predicted rearrangement of the amino acid residues of the cd loop results in disruptions of the hydrogen bonds predicted to form between amino acid residues of the cd and ef loops. Simultaneously, two new hydrogen bonds are predicted to form between glutamate 272 and two residues, aspartate 253 and tyrosine 272. Formation of these new hydrogen bonds would restrict the rotation and protonation of glutamate 272, which may be necessary for the release of the second electrogenic proton obtained during ubiquinol oxidation in the bc₁ complex.     

A four-subunit cytochrome bc1 complex complements the respiratory chain of Thermus thermophilus

Several components of the respiratory chain of the eubacterium Thermus thermophilus have previously been characterized to various extent, while no conclusive evidence for a cytochrome bc₁ complex has been obtained. Here, we show that four consecutive genes encoding cytochrome bc₁ subunits are organized in an operon-like structure termed fbcCXFB. The four gene products are identified as genuine subunits of a cytochrome bc₁ complex isolated from membranes of T. thermophilus. While both the cytochrome b and the FeS subunit show typical features of canonical subunits of this respiratory complex, a further membrane-integral component (FbcX) of so far unknown function copurifies as a subunit of this complex. The cytochrome c₁ carries an extensive N-terminal hydrophilic domain, followed by a hydrophobic, presumably membrane-embedded helical region and a typical heme c binding domain. This latter sequence has been expressed in Escherichia coli, and in vitro shown to be a kinetically competent electron donor to cytochrome c₅₅₂, mediating electron transfer to the ba₃ oxidase. Identification of this cytochrome bc₁ complex bridges the gap between the previously reported NADH oxidation activities and terminal oxidases, thus, defining all components of a minimal, mitochondrial-type electron transfer chain in this evolutionary ancient thermophile.     

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.     

Cytochrome c1 exhibits two binding sites for cytochrome c in plants

In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c₁, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c₁ and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a “floating boat bridge” of cytochrome c molecules (between complexes III and IV) in plant respirasome.