Inhibition of the yeast cytochrome bc1 complex by ilicicolin H, a novel inhibitor that acts at the Qn site of the bc1 complex

Ilicicolin H is an antibiotic isolated from the "imperfect" fungus Cylindrocladium iliciola strain MFC-870. Ilicicolin inhibits mitochondrial respiration by inhibiting the cytochrome bc(1) complex. In order to identify the site of ilicicolin action within the bc(1) complex we have characterized the effects of ilicicolin on the cytochrome bc(1) complex of Saccharomyces cerevisiae. Ilicicolin inhibits ubiquinol-cytochrome c reductase activity of the yeast bc(1) complex with an IC(50) of 3-5 nM, while 200-250 nM ilicicolin was required to obtain comparable inhibition of the bovine bc(1) complex. Ilicicolin blocks oxidation-reduction of cytochrome b through center N of the bc(1) complex and promotes oxidant-induced reduction of cytochrome b but has no effect on oxidation of ubiquinol through center P. These results indicate that ilicicolin binds to the Qn site of the bc(1) complex. Ilicicolin induces a blue shift in the absorption spectrum of ferro-cytochrome b, and titration of the spectral shift indicates binding of one inhibitor molecule per Qn site. The effects of ilicicolin on electron transfer reactions in the bc(1) complex are similar to those of antimycin, another inhibitor that binds to the Qn site of the bc(1) complex. However, because the two inhibitors have different effects on the absorption spectrum of cytochrome b, they differ in their mode of binding to the Qn site.     

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

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

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

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

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

We have previously used inhibitors interacting with the Qn site of the yeast cytochrome 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.     

Mutational analysis of the mouse mitochondrial cytochrome b gene

The protonmotive cytochrome b protein of the mitochondrial bc1 respiratory chain complex contains two reactions centers, designated Qo and Qi, which can be distinguished by the effects of different inhibitors. The nucleotide sequences have been determined of the mitochondrial cytochrome b genes from a series of mouse cell mutants selected for increased inhibitor resistance. Each mutant contains a single nucleotide change which results in an amino acid substitution. When the proximity of the altered amino acid residues to the histidines involved in heme ligation is considered, the results support a model for cytochrome b folding in which there are eight transmembrane domains rather than the nine of the Widger-Saraste model. Replacement of the Gly38 residue by valine results in resistance to the Qi inhibitors antimycin A and funiculosin but not 2-n-heptyl-hydroxyquinoline-N-oxide. Based upon sequence comparisons of mitochondrial and bacterial cytochrome b and chloroplast b6 proteins, the region of the molecule involved in antimycin binding is as highly conserved as those domains involved in heme ligation. It is suggested that the antimycin binding domain of cytochrome b is involved in forming the Qi reaction center. Alterations of the Gly142 and Thr147 residues result in resistance to myxothiazol and stimatellin, respectively. While both inhibitors block the Qo reaction center, the two mutations do not confer cross-resistance to each other. This region of cytochrome b is the most highly conserved during evolution and these inhibitor binding sites probably occur within the protein domain constituting the Qo reaction center. In addition, there is a less conserved region of the protein, defined by the Leu294 residue, which may function in binding the hydrophobic portions of Qo inhibitors.     

Glycine-231 residue of the mouse mitochondrial protonmotive cytochrome b: mutation to aspartic acid deranges electron transport

The mouse LA9 HQN-R11 cytochrome b mutant, in which the glycine residue at position 231 is replaced by aspartic acid, has increased resistance to all inhibitors of the Qn redox center. It is shown here that this single amino acid alteration has multiple and unexpectedly diverse effects upon the mitochondrial protonmotive bc1 complex. (1) The specific activities of both succinate- and ubiquinol-cytochrome c oxidoreductases in isolated mitochondria are reduced by approximately 65% in the mutant. The parallel reductions in both oxidoreductase activities are not compatible with simple Q pool kinetics for mitochondrial electron transport. (2) There is also a reduction in the relative concentration of cytochrome b in the mutant when calculated on the basis of mitochondrial protein; this decrease does not account for more than a small portion of the reduced catalytic fluxes. (3) The increased antimycin resistance of the mutant is lost upon solubilization by the detergent dodecyl maltoside of the bc1 complex from mitochondria. (4) In pre-steady-state assays of cytochrome b reduction by quinol, the mutant shows a reduced extent of reduction. It was observed in other experiments that there was less oxidant-induced extrareduction of cytochrome b in the mutant. These results could arise from a lowering of the midpoint potentials of both the cytochrome b-562 and cytochrome b-566 heme groups. Alternatively, these effects may reflect changes at the Qp and Qn quinone/quinol binding sites. (5) An unexplained observation for the mutant is the increased rate of cytochrome c1 reduction in the presence of myxothiazol. (6) These functional alterations in the LA9 HQN-R11 mutant are not accompanied by detectable changes in the spectral properties of the cytochrome b or c1 heme groups.     

Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex

Cytochrome bc(1) is an integral membrane protein complex essential for cellular respiration and photosynthesis; it couples electron transfer from quinol to cytochrome c to proton translocation across the membrane. Specific bc(1) inhibitors have not only played crucial roles in elucidating the mechanism of bc(1) function but have also provided leads for the development of novel antibiotics. Crystal structures of bovine bc(1) in complex with the specific Q(o) site inhibitors azoxystrobin, MOAS, myxothiazol, stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole were determined. Interactions, conformational changes and possible mechanisms of resistance, specific to each inhibitor, were defined. Residues and secondary structure elements that are capable of discriminating different classes of Q(o) site inhibitors were identified for the cytochrome b subunit. Directions in the displacement of the cd1 helix of cytochrome b subunit in response to various Q(o) site inhibitors were correlated to the binary conformational switch of the extrinsic domain of the iron-sulfur protein subunit. The new structural information, together with structures previously determined, provide a basis that, combined with biophysical and mutational data, suggest a modification to the existing classification of bc(1) inhibitors. bc(1) inhibitors are grouped into three classes: class P inhibitors bind to the Q(o) site, class N inhibitors bind to the Q(i) site and the class PN inhibitors target both sites. Class P contains two subgroups, Pm and Pf, that are distinct by their ability to induce mobile or fixed conformation of iron-sulfur protein.     

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.     

Inhibitory analogs of ubiquinol act anti-cooperatively on the Yeast cytochrome bc1 complex. Evidence for an alternating, half-of-the-sites mechanism of ubiquinol oxidation.

The cytochrome bc(1) complex is a dimeric enzyme that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is re-reduced at a second center, referred to as center N. To understand better the mechanism of ubiquinol oxidation, we have examined the interaction of several inhibitory analogs of ubiquinol with the yeast cytochrome bc(1) complex. Stigmatellin and methoxyacrylate stilbene, two inhibitors that block ubiquinol oxidation at center P, inhibit the yeast enzyme with a stoichiometry of 0.5 per bc(1) complex, indicating that one molecule of inhibitor is sufficient to fully inhibit the dimeric enzyme. This stoichiometry was obtained when the inhibitors were titrated in cytochrome c reductase assays and in reactions of quinol with enzyme in which the inhibitors block pre-steady state reduction of cytochrome b. As an independent measure of inhibitor binding, we titrated the red shift in the optical spectrum of ferrocytochrome b with methoxyacrylate stilbene and thus confirmed the results of the inhibition of activity titrations. The titration curves also indicate that the binding is anti-cooperative, in that a second molecule of inhibitor binds with much lower affinity to a dimer in which an inhibitor molecule is already bound. Because these inhibitors bind to the ubiquinol oxidation site in the bc(1) complex, we propose that the yeast cytochrome bc(1) complex oxidizes ubiquinol by an alternating, half-of-the-sites mechanism.     

Rapid electron transfer between monomers when the cytochrome bc1 complex dimer is reduced through center N

We have obtained evidence for electron transfer between cytochrome b subunits of the yeast bc(1) complex dimer by analyzing pre-steady state reduction of cytochrome b in the presence of center P inhibitors. The kinetics and extent of cytochrome b reduced by quinol in the presence of variable concentrations of antimycin decreased non-linearly and could only be fitted to a model in which electrons entering through one center N can equilibrate between the two cytochrome b subunits of the bc(1) complex dimer. The b(H) heme absorbance in a bc(1) complex inhibited at center P and preincubated with substoichiometric concentrations of antimycin showed a red shift upon the addition of substrate, which indicates that electrons from the uninhibited center N in one monomer are able to reach the b(H) heme at the antimycin-blocked site in the other. The extent of cytochrome b reduction by variable concentrations of menaquinol could only be fitted to a kinetic model that assumes electron equilibration between center N sites in the dimer. Kinetic simulations showed that non-rate-limiting electron equilibration between the two b(H) hemes in the dimer through the two b(L) hemes is possible upon reduction through one center N despite the thermodynamically unfavorable b(H) to b(L) electron transfer step. We propose that electron transfer between cytochrome b subunits minimizes the formation of semiquinone-ferrocytochrome b(H) complexes at center N and favors ubiquinol oxidation at center P by increasing the amount of oxidized cytochrome b.     

Mutational analysis of cytochrome b at the ubiquinol oxidation site of yeast complex III

The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.     

Molecular basis for atovaquone resistance in Pneumocystis jirovecii modeled in the cytochrome bc(1) complex of Saccharomyces cerevisiae

Atovaquone is a substituted hydroxynaphthoquinone that is widely used to prevent and clear Plasmodium falciparum malaria and Pneumocystis jirovecii pneumonia. Atovaquone inhibits respiration in target organisms by specifically binding to the ubiquinol oxidation site at center P of the cytochrome bc(1) complex. The failure of atovaquone treatment and mortality of patients with malaria and P. jirovecii pneumonia has been linked to the appearance of mutations in the cytochrome b gene. To better understand the molecular basis of atovaquone resistance, we have introduced seven of the mutations from atovaquone-resistant P. jirovecii into the cytochrome b gene of Saccharomyces cerevisiae and thus obtained cytochrome bc(1) complexes resistant to inhibition by atovaquone. In these enzymes, the IC(50) for atovaquone increases from 25 nm for the enzyme from wild-type yeast to >500 nm for some of the mutated enzymes. Modeling of the changes in cytochrome b structure and atovaquone binding with the mutated bc(1) complexes provides the first quantitative explanation for the molecular basis of atovaquone resistance.     

Anti-cooperative oxidation of ubiquinol by the yeast cytochrome bc1 complex

We have investigated the interaction between monomers of the dimeric yeast cytochrome bc(1) complex by analyzing the pre-steady and steady state activities of the isolated enzyme in the presence of antimycin under conditions that allow the first turnover of ubiquinol oxidation to be observable in cytochrome c(1) reduction. At pH 8.8, where the redox potential of the iron-sulfur protein is approximately 200 mV and in a bc(1) complex with a mutated iron-sulfur protein of equally low redox potential, the amount of cytochrome c(1) reduced by several equivalents of decyl-ubiquinol in the presence of antimycin corresponded to only half of that present in the bc(1) complex. Similar experiments in the presence of several equivalents of cytochrome c also showed only half of the bc(1) complex participating in quinol oxidation. The extent of cytochrome b reduced corresponded to two b(H) hemes undergoing reduction through one center P per dimer, indicating electron transfer between the two cytochrome b subunits. Antimycin stimulated the ubiquinol-cytochrome c reductase activity of the bc(1) complex at low inhibitor/enzyme ratios. This stimulation could only be fitted to a model in which half of the bc(1) dimer is inactive when both center N sites are free, becoming active upon binding of one center N inhibitor molecule per dimer, and there is electron transfer between the cytochrome b subunits of the dimer. These results are consistent with an alternating half-of-the-sites mechanism of ubiquinol oxidation in the bc(1) complex dimer.     

A novel electron transfer mechanism suggested by crystallographic studies of mitochondrial cytochrome bc1 complex.

The crystal structure of bovine mitochondrial cytochrome bc1 complex, an integral membrane protein complex of 11 different subunits with a total molecular mass of 242 kDa, demonstrated a tightly associated dimer consisting of three major regions: a matrix region primarily made of subunits core1, core2, 6, and 9; a transmembrane-helix region of 26 helices in the dimer contributed by cytochrome b, cytochrome c1, the Rieske iron-sulfur protein (ISP), subunits 7, 10, and 11; and an intermembrane-space region composed of extramembrane domains of ISP, cytochrome c1, and subunit 8. The structure also revealed the positions of and distances between irons of prosthetic groups, and two symmetry related cavities in the transmembrane-helix region upon dimerization of the bc1 complex. Extensive crystallographic studies on crystals of bc1 complexed with inhibitors of electron transfer identified binding pockets for both Qo and Qi site inhibitors. Discrete binding sites for subtypes of Qo site inhibitors have been mapped onto the Qo binding pocket, and bindings of different subtypes of Qo site inhibitors are capable of inducing dramatic conformational changes in the extramembrane domain of ISP. A novel electron transfer mechanism for the bc1 complex consistent with crystallographic observations is discussed.     

Deletion of subunit 9 of the Saccharomyces cerevisiae cytochrome bc1 complex specifically impairs electron transfer at the ubiquinol oxidase site (center P) in the bc1 complex.

Deletion of QCR9, the nuclear gene encoding subunit 9 of the mitochondrial cytochrome bc1 complex in Saccharomyces cerevisiae, results in inactivation of the bc1 complex and inability of the yeast to grow on non-fermentable carbon sources. The loss of bc1 complex activity is due to loss of electron transfer activity at the ubiquinol oxidase site (center P) in the complex. Electron transfer at the ubiquinone reductase site (center N), is unaffected by the loss of subunit 9, but the extent of cytochrome b reduction is diminished. This is the first instance in which a supernumerary polypeptide, lacking a redox prosthetic group, has been shown to be required for an electron transfer reaction within the cytochrome bc1 complex.     

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

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

Molecular basis for resistance to myxothiazol, mucidin (strobilurin A), and stigmatellin. Cytochrome b inhibitors acting at the center o of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae

The respiratory bc1 complex transfers the electrons from ubiquinol to cytochrome c oxidase. Myxothiazol, strobilurin A (mucidin), and stigmatellin are center o inhibitors preventing electron transfer at the ubiquinone redox site Qo, which is located closer to the outer side of the inner mitochondrial membrane. The cytochrome b gene is carried by the organelle DNA. Yeast mutants resistant to myxothiazol and mucidin have been previously isolated and mapped to specific loci of the cytochrome b gene. In the present work, stigmatellin-resistant mutants were isolated and genetically analyzed. The mutated amino acid residues from seven myxothiazol-, four mucidin-, and six stigmatellin-resistant mutants have been identified by sequencing the relevant segments of the resistant cytochrome b gene. A third myxothiazol-resistant locus and the first stigmatellin-resistant locus were identified. The mutated codons were found to be clustered in two regions of the cytochrome b protein which appeared to be responsible for the resistance to Qo site inhibitors. The first region is within the end of the first, the second, and the beginning of the third exon whereas the second region is within exon five and the beginning of the sixth exon.     

Natural resistance to inhibitors of the ubiquinol cytochrome c oxidoreductase of Rubrivivax gelatinosus: sequence and functional analysis of the cytochrome bc(1) complex

Biochemical analyses of Rubrivivax gelatinosus membranes have revealed that the cytochrome bc(1) complex is highly resistant to classical inhibitors including myxothiazol, stigmatellin, and antimycin. This is the first report of a strain exhibiting resistance to inhibitors of both catalytic Q(0) and Q(i) sites. Because the resistance to cytochrome bc(1) inhibitors is primarily related to the cytochrome b primary structure, the petABC operon encoding the subunits of the cytochrome bc(1) complex of Rubrivivax gelatinosus was sequenced. In addition to homologies to the corresponding proteins from other organisms, the deduced amino acid sequence of the cytochrome b polypeptide shows (i) an E303V substitution in the highly conserved PEWY loop involved in quinol/stigmatellin binding, (ii) other substitutions that could be involved in resistance to cytochrome bc(1) inhibitors, and (iii) 14 residues instead of 13 between the histidines in helix IV that likely serve as the second axial ligand to the b(H) and b(L) hemes, respectively. These characteristics imply different functional properties of the cytochrome bc(1) complex of this bacterium. The consequences of these structural features for the resistance to inhibitors and for the properties of R. gelatinosus cytochrome bc(1) are discussed with reference to the structure and function of the cytochrome bc(1) complexes from other organisms.     

Regulatory interactions between ubiquinol oxidation and ubiquinone reduction sites in the dimeric cytochrome bc1 complex

We have obtained evidence for conformational communication between ubiquinol oxidation (center P) and ubiquinone reduction (center N) sites of the yeast bc1 complex dimer by analyzing antimycin binding and heme bH reduction at center N in the presence of different center P inhibitors. When stigmatellin was occupying center P, concentration-dependent binding of antimycin occurred only to half of the center N sites. The remaining half of the bc1 complex bound antimycin with a slower rate that was independent of inhibitor concentration, indicating that a slow conformational change needed to occur before half of the enzyme could bind antimycin. In contrast, under conditions where the Rieske protein was not fixed proximal to heme bL at center P, all center N sites bound antimycin with fast and concentration-dependent kinetics. Additionally, the extent of fast cytochrome b reduction by menaquinol through center N in the presence of stigmatellin was approximately half of that observed when myxothiazol was bound at center P. The reduction kinetics of the bH heme by decylubiquinol in the presence of stigmatellin or myxothiazol were also consistent with a model in which fixation of the Rieske protein close to heme bL in both monomers allows rapid binding of ligands only to one center N. Decylubiquinol at high concentrations was able to abolish the biphasic binding of antimycin in the presence of stigmatellin but did not slow down antimycin binding rates. These results are discussed in terms of half-of-the-sites activity of the dimeric bc1 complex.