Disease-related mutations in cytochrome c oxidase studied in yeast and bacterial models.

Mitochondrial cytochrome c oxidase is a key protonmotive component of the respiratory chain. Mutations in the mitochondrially-encoded subunits of the complex have been reported in association with a range of diseases. In this work we used yeast and bacterial mutants to assess the effect of human mutations in subunit 1 (L196I) and subunit 3 (G78S, A200T, Delta F94-F98, F251L and W249Stop). While the stop mutation at the C-terminus of subunit 3 and the short deletion were highly deleterious and abolished the assembly of the mitochondrial enzyme, the four missense mutations caused little or no effect on the respiratory function. Detailed analysis of G78S, A200T and Delta F94-F98 in Rhodobacter sphaeroides confirmed and extended these observations. We show in this study that the combination of yeast and bacterial models is a useful tool to elucidate the effect of mutations in the catalytic core of cytochrome oxidase. The yeast enzyme is highly similar to the human enzyme and provides a good model to assess the deleterious effect of reported mutations. The bacterial system allows detailed biochemical analysis of the effect of the mutations on the function and assembly of the catalytic core of the enzyme.     

Disruption of the interaction between the Rieske iron-sulfur protein and cytochrome b in the yeast bc1 complex owing to a human disease-associated mutation within cytochrome b.

The mitochondrial cytochrome b missense mutation, G167E, has been reported in a patient with cardiomyopathy. The residue G167 is located in an extramembranous helix close to the hinge region of the iron-sulfur protein. In order to characterize the effects of the mutation on the structure and function of the bc(1) complex, we introduced G167E into the highly similar yeast cytochrome b. The mutation had a severe effect on the respiratory function, with the activity of the bc(1) complex decreased to a few per cent of the wild type. Analysis of the enzyme activity indicated that the mutation affected its stability, which could be the result of an altered binding of the iron-sulfur protein on the complex. G167E had no major effect on the interaction between the iron-sulfur protein headgroup and the quinol oxidation site, as judged by the electron paramagnetic resonance signal, and only a minor effect on the rate of cytochrome b reduction, but it severely reduced the rate of cytochrome c(1) reduction. This suggested that the mutation G167E could hinder the movement of the iron-sulfur protein, probably by distorting the structure of the hinge region. The function of bc(1) was partially restored by mutations (W164L and W166L) located close to the primary change, which reduced the steric hindrance caused by G167E. Taken together, these observations suggest that the protein-protein interaction between the n-sulfur protein hinge region and the cytochrome b extramembranous cd2 helix is important for maintaining the structure of the hinge region and, by consequence, the movement of the headgroup and the integrity of the enzyme.     

Aromatic amino acids in the Rieske iron-sulfur protein do not form an obligatory conduit for electron transfer from the iron-sulfur cluster to the heme of cytochrome c1 in the cytochrome bc1 complex

We have changed nine conserved aromatic amino acids by site-directed mutagenesis of the cloned iron-sulfur protein gene to determine if any of these residues form an obligatory conduit for electron transfer within the iron-sulfur protein of the yeast cytochrome bc1 complex. The residues include W111, F117, W152, F173, W176, F177, H184, Y205 and F207. Greater than 70% of the catalytic activity was retained for all of the mutated iron-sulfur proteins, except for those containing a W152L and a W176L-F177L double mutation, for which the activity was approximately 45%. The crystal structures of the bc1 complex indicate that F177 and H184 are at the surface of the iron-sulfur protein near the surface of cytochrome c1, but not directly in a linear pathway between the iron-sulfur cluster and the c1 heme. The pre-steady-state rates of reduction of cytochromes b and c1 in mutants in which F177 and H184 were changed to non-aromatic residues were approximately 70-85% of the wild-type rates. There was a large decrease in iron-sulfur protein levels in mitochondrial membranes resulting from the W152L mutation and the W176L-F177L double mutation, and a small decrease for the Y205L, W176L and F177L mutations. This indicates that the decreases in activity resulting from these amino acid changes are due to instability of the altered proteins. These results show that these aromatic amino acids are unnecessary for electron transfer, but several are required for structural stability.     

Failure to insert the iron-sulfur cluster into the Rieske iron-sulfur protein impairs both center N and center P of the cytochrome bc1 complex

Mutation of a serine that forms a hydrogen bond to the iron-sulfur cluster of the Rieske iron-sulfur protein to a cysteine results in a respiratory-deficient yeast strain due to formation of iron-sulfur protein lacking the iron-sulfur cluster. The Rieske apoprotein lacking the iron-sulfur cluster is inserted into both monomers of the dimeric cytochrome bc(1) complex and processed to mature size, but the protein lacking iron-sulfur cluster is more susceptible to proteolysis. In addition, the protein environment of center P in one half of the dimer is affected by failure to insert the iron-sulfur cluster as indicated by the fact that only one molecule of myxothiazol can be bound to the cytochrome bc(1) dimer. Although the bc(1) complex lacking the Rieske iron-sulfur cluster cannot oxidize ubiquinol through center P, rates of reduction of cytochrome b by menaquinol through center N are normal. However, less cytochrome b is reduced through center N, and only one molecule of antimycin can be bound at center N in the bc(1) dimer lacking iron-sulfur cluster. These results indicate that failure to insert the [2Fe-2S] cluster impairs assembly of the Rieske protein into the bc(1) complex and that this interferes with proper assembly of both center P and center N in one half of the dimeric enzyme.     

Point mutation in cytochrome b of yeast ubihydroquinone:cytochrome-c oxidoreductase causing myxothiazol resistance and facilitated dissociation of the iron-sulfur subunit.

Cytochrome-c reductase was isolated from Saccharomyces cerevisiae GM50-3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDS/PAGE. Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Q0 center (Q, ubiquinone) of cytochrome-c reductase, were analysed. The single amino acid substitution in the cytochrome-b subunit, N256Y in the mutant Myx-119 and G137R in the mutant Myx-118, caused a general resistance to all methoxyacrylate inhibitors to about fivefold higher concentrations. The kinetic measurements with the substrate analogue nonylbenzohydroquinone revealed a decrease in the Km by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The Km of the G137R mutant was not affected and the Vmax was 50% higher. Cytochrome-c reductase was isolated from mutants to allow determination of the Kd values of methoxyacrylate-stilbene and myxothiazol by means of fluorescence-quench and red-shift titration. Changes in the structure of the multisubunit complex due to a single amino acid exchange became obvious during the purification procedure. SDS/PAGE of the purified enzyme revealed that the substitution N256Y in cytochrome b led to a loss of the iron-sulfur protein and the fifth small subunit with no change in the pattern of the remaining eight subunits. The subunit pattern of the G137R mutant was identical to the wild type. This is the first report of a single amino acid exchange in the catalytic subunit of cytochrome b, greatly affecting the iron-sulfur protein, the second important catalytic subunit of the Q0 center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron-sulfur subunit.     

Cytochrome b mutation Y268S conferring atovaquone resistance phenotype in malaria parasite results in reduced parasite bc1 catalytic turnover and protein expression

Atovaquone is an anti-malarial drug used in combination with proguanil (e.g. Malarone(TM)) for the curative and prophylactic treatment of malaria. Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation (Q(o)) site of the mitochondrial cytochrome bc(1) complex. Inhibition of this enzyme results in the collapse of the mitochondrial membrane potential, disruption of pyrimidine biosynthesis, and subsequent parasite death. Resistance to atovaquone in the field is associated with point mutations in the Q(o) pocket of cytochrome b, most notably near the conserved Pro(260)-Glu(261)-Trp(262)-Tyr(263) (PEWY) region in the ef loop). The effect of this mutation has been extensively studied in model organisms but hitherto not in the parasite itself. Here, we have performed a molecular and biochemical characterization of an atovaquone-resistant field isolate, TM902CB. Molecular analysis of this strain reveals the presence of the Y268S mutation in cytochrome b. The Y268S mutation is shown to confer a 270-fold shift of the inhibitory constant (K(i)) for atovaquone with a concomitant reduction in the V(max) of the bc(1) complex of ～40% and a 3-fold increase in the observed K(m) for decylubiquinol. Western blotting analyses reveal a reduced iron-sulfur protein content in Y268S bc(1) suggestive of a weakened interaction between this subunit and cytochrome b. Gene expression analysis of the TM902CB strain reveals higher levels of expression, compared with the 3D7 (atovaquone-sensitive) control strain in bc(1) and cytochrome c oxidase genes. It is hypothesized that the observed differential expression of these and other key genes offsets the fitness cost resulting from reduced bc(1) activity.     

Modeling the Qo site of crop pathogens in Saccharomyces cerevisiae cytochrome b.

Saccharomyces cerevisiae has been used as a model system to characterize the effect of cytochrome b mutations found in fungal and oomycete plant pathogens resistant to Q(o) inhibitors (QoIs), including the strobilurins, now widely employed in agriculture to control such diseases. Specific residues in the Q(o) site of yeast cytochrome b were modified to obtain four new forms mimicking the Q(o) binding site of Erysiphe graminis, Venturia inaequalis, Sphaerotheca fuliginea and Phytophthora megasperma. These modified versions of cytochrome b were then used to study the impact of the introduction of the G143A mutation on bc(1) complex activity. In addition, the effects of two other mutations F129L and L275F, which also confer levels of QoI insensitivity, were also studied. The G143A mutation caused a high level of resistance to QoI compounds such as myxothiazol, axoxystrobin and pyraclostrobin, but not to stigmatellin. The pattern of resistance conferred by F129L and L275F was different. Interestingly G143A had a slightly deleterious effect on the bc(1) function in V. inaequalis, S. fuliginea and P. megasperma Q(o) site mimics but not in that for E. graminis. Thus small variations in the Q(o) site seem to affect the impact of the G143A mutation on bc(1) activity. Based on this observation in the yeast model, it might be anticipated that the G143A mutation might affect the fitness of pathogens differentially. If so, this could contribute to observed differences in the rates of evolution of QoI resistance in fungal and oomycete pathogens.     

A pathogenic cytochrome b mutation reveals new interactions between subunits of the mitochondrial bc1 complex

Energy transduction in mitochondria involves five oligomeric complexes embedded within the inner membrane. They are composed of catalytic and noncatalytic subunits, the role of these latter proteins often being difficult to assign. One of these complexes, the bc1 complex, is composed of three catalytic subunits including cytochrome b and seven or eight noncatalytic subunits. Recently, several mutations in the human cytochrome b gene have been linked to various diseases. We have studied in detail the effects of a cardiomyopathy generating mutation G252D in yeast. This mutation disturbs the biogenesis of the bc1 complex at 36 degrees C and decreases the steady-state level of the noncatalytic subunit Qcr9p. In addition, the G252D mutation and the deletion of QCR9 show synergetic defects that can be partially bypassed by suppressor mutations at position 252 and by a new cytochrome b mutation, P174T. Altogether, our results suggest that the supernumerary subunit Qcr9p enhances or stabilizes the interactions between the catalytic subunits, this role being essential at high temperature.     

Analysis of suppressor mutation reveals long distance interactions in the bc(1) complex of Saccharomyces cerevisiae

Four totally conserved glycines are involved in the packing of the two cytochrome b hemes, b(L) and b(H), of the bc(1) complex. The conserved glycine 131 is involved in the packing of heme b(L) and is separated by only 3 A from this heme in the bc(1) complex structure. The cytochrome b respiratory deficient mutant G131S is affected in the assembly of the bc(1) complex. An intragenic suppressor mutation was obtained at position 260, in the ef loop, where a glycine was replaced by an alanine. This respiratory competent revertant exhibited a low bc(1) complex activity and was affected in the electron transfer at the Q(P) site. The k(min) for the substrate DBH(2) was diminished by an order of magnitude and EPR spectra showed a partially empty Q(P) site. However, the binding of the Q(P) site inhibitors stigmatellin and myxothiazol remained unchanged in the suppressor strain. Optical spectroscopy revealed that heme b(L) is red shifted by 0.8 nm and that the E(m) of heme b(L) was slightly increased (+20 mV) in the revertant strain as compared to wild type strain values. Addition of a methyl group at position 260 is thus sufficient to allow the assembly of the bc(1) complex and the insertion of heme b(L) despite the presence of the serine at position 131. Surprisingly, reversion at position 260 was located 13 A away from the original mutation and revealed a long distance interaction in the yeast bc(1) complex.     

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.     

Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. III. Import, protease processing, and assembly into the cytochrome bc1 complex of iron-sulfur protein lacking the iron-sulfur cluster.

We have used site-directed mutagenesis of the Saccharomyces cerevisiae Rieske iron-sulfur protein gene (RIP 1) to convert cysteines 159, 164, 178, and 180 to serines, and to convert histidines 161 and 181 to arginines. These 4 cysteines and 2 histidines are conserved in all Rieske proteins sequenced to date, and 4 of these 6 residues are thought to ligate the iron-sulfur cluster to the apoprotein. We have also converted histidine 184 to arginine. This histidine is conserved only in respiring organisms. The site-directed mutations of the six fully conserved putative iron-sulfur cluster ligands result in an inactive iron-sulfur protein, lacking iron-sulfur cluster, and failure of the yeast to grow on nonfermentable carbon sources. In contrast, when histidine 184 is replaced by arginine, the iron-sulfur cluster is assembled properly and the yeast grow on nonfermentable carbon sources. The site-directed mutations of the 6 fully conserved residues do not prevent post-translational import of iron-sulfur protein precursor into mitochondria, nor do the mutations prevent processing of iron-sulfur protein precursor to mature size protein by mitochondrial proteases. Optical spectra of mitochondria from the six mutants indicate that cytochrome b is normal, in contrast to the deranged spectrum of cytochrome b which results when the iron-sulfur protein gene is deleted. In addition, mature size iron-sulfur apoprotein is associated with cytochrome bc1 complex purified from a site-directed mutant in which iron-sulfur cluster is not inserted. These results indicate that mature size iron-sulfur apoprotein, lacking iron-sulfur cluster, is inserted into the cytochrome bc1 complex, where it interacts with and preserves the optical properties of cytochrome b. Insertion of the iron-sulfur cluster is not an obligatory prerequisite to processing of the protein to its final size. Either the processing protease cannot distinguish between iron-sulfur protein with or without the iron-sulfur cluster, or insertion of the iron-sulfur cluster occurs after the protein is processed to its mature size, possibly after it is assembled in the cytochrome bc1 complex.     

Aspartate-186 in the head group of the yeast iron-sulfur protein of the cytochrome bc1 complex contributes to the protein conformation required for efficient electron transfer

Two conserved charged amino acids, aspartate-186 and arginine-190, localized in the aqueous head region of the iron-sulfur protein of the cytochrome bc(1) complex of yeast mitochondria, were mutated to alanine, glutamate, or asparagine and isoleucine, respectively. The R190I mutation resulted in the complete loss of antimycin- and myxothiazol-sensitive cytochrome c reductase activity due to loss of more than 60% of the iron-sulfur protein in the complex. Mitochondria isolated from the D186A mutant had a 50% decrease in cytochrome c reductase activity but no loss of the iron-sulfur protein or the [2Fe-2S] cluster. The midpoint potential of the [2Fe-2S] cluster of the D186A mutant was decreased from 281 to 178 mV. The D186E and D186N mutations did not result in a loss of cytochrome c reductase activity or content of iron-sulfur protein; however, the redox potential of the [2Fe-2S] cluster of D186N was decreased from 281 to 241 mV. Molecular modeling/dynamics studies predicted that substituting an alanine for Asp-186 causes global structural changes in the head group of the iron-sulfur protein resulting in changes in the orientation of the [2Fe-2S] cluster and consequently a lowered redox potential. The rate of electrogenic proton pumping in the bc(1) complex isolated from mutant D186A reconstituted into proteoliposomes decreased 64%; however, the H(+)/2e(-) ratio of 1.9 was identical in the mutant and the wild-type complexes. The carboxyl binding reagent, N-(ethoxycarbonyl)-2-ethoxyl-1,2-dihydroquinoline (EEDQ) blocked electrogenic proton pumping in the bc(1) complex reconstituted into proteoliposomes without affecting electron transfer resulting in a decrease in the H(+)/2e(-) ratio to 1.2 and 1.1, respectively. EEDQ was bound to the iron-sulfur protein and core protein II in both the wild type and the D186A mutant, indicating that Asp-186 of the iron-sulfur protein is not required for proton translocation in the bc(1) complex.     

Analysis of the nucleus-encoded and chloroplast-targeted rieske protein by classic and site-directed mutagenesis of Chlamydomonas.

Three mutants of the alga Chlamydomonas reinhardtii affected in the nuclear PETC gene encoding the Rieske iron-sulfur protein 2Fe-2S subunit of the chloroplast cytochrome b(6)f complex have been characterized. One has a stable deletion that eliminates the protein; two others carry substitutions Y87D and W163R that result in low accumulation of the protein. Attenuated expression of the stromal protease ClpP increases accumulation and assembly into b(6)f complexes of the Y87D and W163R mutant Rieske proteins in quantities sufficient for analysis. Electron-transfer kinetics of these complexes were 10- to 20-fold slower than those for the wild type. The deletion mutant was used as a recipient for site-directed mutant petC alleles. Six glycine residues were replaced by alanine residues (6G6A) in the flexible hinge that is critical for domain movement; substitutions were created near the 2Fe-2S cluster (S128 and W163); and seven C-terminal residues were deleted (G171och). Although the 6G6A and G171och mutations affect highly conserved segments in the chloroplast Rieske protein, photosynthesis in the mutants was similar to that of the wild type. These results establish the basis for mutational analysis of the nuclear-encoded and chloroplast-targeted Rieske protein of photosynthesis.     

The functional role of conserved acidic residues of the Qcr7 protein of the cytochrome bc(1) complex in Saccharomyces cerevisiae

The 14-kDa Qcr7 protein represents one of the 10 subunits that are components of a functional cytochrome bc(1) complex in Sacharomyces cerevisiae. Previous studies have shown that the N-terminus of the Qcr7 protein may be involved in the assembly of the cytochrome bc(1) complex and its C-terminus by interacting with cytochrome b and QCR8 proteins. It has also been suggested that Qcr7 protein may be involved in proton pumping. The coding sequence for two highly conserved aspartate residues, D46 and D47, in the QCR7 gene was altered by site-directed mutagenesis and the mutated genes expressed in cells lacking a functional QCR7 gene. Mutants D46E, D46G, D46N, and D47E were comparable to wild type in growth phenotype on nonfermentable carbon sources. Mutants D47G and D47N were respiratory deficient and analysis of complex components by immunoblotting and spectral analysis of cytochrome b suggests defective assembly. Despite being respiratory competent and having normal electron transport rates in broken mitochondria, the mutant D46G had markedly reduced ATP synthesis from electron transport reactions catalyzed by complexes II plus III of the respiratory chain. This suggests that the geometry of proton uptake by the bc(1) complex is disturbed by the mutation in D46.     

Cytochrome b mutations that modify the ubiquinol-binding pocket of the cytochrome bc1 complex and confer anti-malarial drug resistance in Saccharomyces cerevisiae

Atovaquone is a new anti-malarial agent that specifically targets the cytochrome bc1 complex and inhibits parasite respiration. A growing number of failures of this drug in the treatment of malaria have been genetically linked to point mutations in the mitochondrial cytochrome b gene. To better understand the molecular basis of atovaquone resistance in malaria, we introduced five of these mutations, including the most prevalent variant found in Plasmodium falciparum (Y268S), into the cytochrome b gene of the budding yeast Saccharomyces cerevisiae and thus obtained cytochrome bc1 complexes resistant to inhibition by atovaquone. By modeling the variations in cytochrome b structure and atovaquone binding with the mutated bc1 complexes, we obtained the first quantitative explanation for the molecular basis of atovaquone resistance in malaria parasites.     

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

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

Photoinduced electron transfer between the Rieske iron-sulfur protein and cytochrome c(1) in the Rhodobacter sphaeroides cytochrome bc(1) complex. Effects of pH,temperature, and driving force

Electron transfer from the Rieske iron-sulfur protein to cytochrome c(1) (cyt c(1)) in the Rhodobacter sphaeroides cytochrome bc(1) complex was studied using a ruthenium dimer complex, Ru(2)D. Laser flash photolysis of a solution containing reduced cyt bc(1), Ru(2)D, and a sacrificial electron acceptor results in oxidation of cyt c(1) within 1 micros, followed by electron transfer from the iron-sulfur center (2Fe-2S) to cyt c(1) with a rate constant of 80,000 s(-1). Experiments were carried out to evaluate whether the reaction was rate-limited by true electron transfer, proton gating, or conformational gating. The temperature dependence of the reaction yielded an enthalpy of activation of +17.6 kJ/mol, which is consistent with either rate-limiting conformational gating or electron transfer. The rate constant was nearly independent of pH over the range pH 7 to 9.5 where the redox potential of 2Fe-2S decreases significantly due to deprotonation of His-161. The rate constant was also not greatly affected by the Rieske iron-sulfur protein mutations Y156W, S154A, or S154A/Y156F, which decrease the redox potential of 2Fe-2S by 62, 109, and 159 mV, respectively. It is concluded that the electron transfer reaction from 2Fe-2S to cyt c(1) is controlled by conformational gating.     

The iron-sulfur protein of cytochrome bc1 complex. Its occurrence in the mitochondrial inner membrane in excess of the amount constituting the complex

Radioimmunoassay and quantitative immunoblot analysis have been developed for quantitation of the iron-sulfur protein of cytochrome bc1 complex in order to compare its content in isolated cytochrome bc1 complex with that in electron transport particles. The result by radioimmunoassay indicated that the content of the iron-sulfur protein/mol of cytochrome b is higher by approximately 30%, on the average, in electron transport particles than in cytochrome bc1 complex. This observation was supported by the data of immunoblot analysis. Since approximately 1/3 of cytochrome b in electron transport particles is not attributed to cytochrome bc1 complex, but to succinate-ubiquinone oxidoreductase complex (Davis, K.A., Hatefi, Y., Poff, K. L., and Butler, W. L. (1973) Biochim. Biophys. Acta 325, 341-356), the ratio of the iron-sulfur protein detectable by radioimmunoassay in electron transport particles to that in cytochrome bc1 complex is calculated to be approximately 2 on the basis of the content of 2 mol of b-type heme/mol of the complex. Therefore, it appears that the mitochondrial inner membrane contains approximately two times as much of the immunoreactive iron-sulfur protein as what is expected from the stoichiometry of one iron-sulfur center and two b-type hemes for cytochrome bc1 complex. This finding affords an interesting aspect in the study of biogenesis of cytochrome bc1 complex.     

Conformationally linked interaction in the cytochrome bc(1) complex between inhibitors of the Q(o) site and the Rieske iron-sulfur protein

The modified Q cycle mechanism accounts for the proton and charge translocation stoichiometry of the bc(1) complex, and is now widely accepted. However the mechanism by which the requisite bifurcation of electron flow at the Q(o) site reaction is enforced is not clear. One of several proposals involves conformational gating of the docking of the Rieske ISP at the Q(o) site, controlled by the stage of the reaction cycle. Effects of different Q(o)-site inhibitors on the position of the ISP seen in crystals may reflect the same conformational mechanism, in which case understanding how different inhibitors control the position of the ISP may be a key to understanding the enforcement of bifurcation at the Q(o) site (Table?1). Here we examine the available structures of cytochrome bc(1) with different Q(o)-site inhibitors and different ISP positions to look for clues to this mechanism. The effect of ISP removal on binding affinity of the inhibitors stigmatellin and famoxadone suggest a "mutual stabilization" of inhibitor binding and ISP docking, however this thermodynamic observation sheds little light on the mechanism. The cd(1) helix of cytochrome b moves in such a way as to accommodate docking when inhibitors favoring docking are bound, but it is impossible with the current structures to say whether this movement of α-cd(1) is a cause or result of ISP docking. One component of the movement of the linker between E and F helices also correlates with the type of inhibitor and ISP position, and seems to be related to the H-bonding pattern of Y279 of cytochrome b. An H-bond from Y279 to the ISP, and its possible modulation by movement of F275 in the presence of famoxadone and related inhibitors, or its competition with an alternate H-bond to I269 of cytochrome b that may be destabilized by bound famoxadone, suggest other possible mechanisms. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

Effects of mutations in mitochondrial cytochrome b in yeast and man. Deficiency, compensation and disease.

The mitochondrial cytochrome bc(1) complex is a key protonmotive component of eukaryotic respiratory chains. The mitochondrially encoded cytochrome b forms, with cytochrome c(1) and the iron--sulfur protein, the catalytic core of this multimeric enzyme. Mutations of cytochrome b have been reported in association with human diseases. In the highly homologous yeast cytochrome b, several mutations that impair the respiratory function, and reversions that correct the defect, have been described. In this paper, we re-examine the mutations in the light of the atomic structure of the complex, and discuss the possible effect, at enzyme level, of the human cytochrome b mutations and the correcting effect of the reversions.