Dimeric Cytochrome Bc1 Complex Research Articles

Rcf proteins and their differential specificity for respiratory chain complexes: A unique role for Rcf2 on oxygen sensitive supercomplexes?

The respiratory chain, embedded in the inner mitochondrial membrane, is organized as a network of individual complexes, as well as large supercomplex structures. In the yeast S. cerevisiae, these supercomplexes consist of a dimeric cytochrome bc1-complex adjoined by one or two copies of cytochrome c oxidase. The formation of these complexes is a dynamic process and is regulated by various factors in order to adapt to environmental and metabolic changes. These adaptions occur at the level of enzyme regulation, complex assembly, as well as altered nuclear and mitochondrial transcription and translation. Members of the Rcf protein family (Rcf1, Rcf2 and Rcf3) are required for respiratory complex biogenesis and act mainly by regulating the assembly and enzyme activity of complex IV within supercomplexes. Rcf1 functions in the assembly process via the COX3 module, whereas Rcf2 and Rcf3 are responsible for enzymatic regulation. In this study, we have extended this knowledge to show that Rcf2 and Rcf3 can also associate with newly synthesized mitochondrial encoded proteins, such as Cox3, and therefore contribute to complex IV assembly. Since the Rcf proteins have overlapping regions of sequence similarities, we engineered novel fusion proteins of Rcf1 and Rcf3, with parts of Rcf2, to probe which of the Rcf protein domains can be attributed to their functions. The fusion proteins could compensate for the individual phenotypes of the complexIV assembly defect and the lack of complex IV regulation. Finally, the role of Rcf proteins for defined species of respiratory chain complexes in a hypoxic model was investigated, uncovering a unique association of Rcf2 with the hypoxic III2IV supercomplex. We therefore suggest an involvement of Rcf2 for adaption of the respiratory chain to altering oxygen levels.

X-ray structure of the dimeric cytochrome bc1 complex from the soil bacterium Paracoccus denitrificans at 2.7-Å resolution

The respiratory cytochrome bc1 complex is a fundamental enzyme in biological energy conversion. It couples electron transfer from ubiquinol to cytochrome c with generation of proton motive force which fuels ATP synthesis. The complex from the α-proteobacterium Paracoccus denitrificans, a model for the medically relevant mitochondrial complexes, lacked structural characterization. We show by LILBID mass spectrometry that truncation of the organism-specific, acidic N-terminus of cytochrome c1 changes the oligomerization state of the enzyme to a dimer. The fully functional complex was crystallized and the X-ray structure determined at 2.7-Å resolution. It has high structural homology to mitochondrial complexes and to the Rhodobacter sphaeroides complex especially for subunits cytochrome b and ISP. Species-specific binding of the inhibitor stigmatellin is noteworthy. Interestingly, cytochrome c1 shows structural differences to the mitochondrial and even between the two Rhodobacteraceae complexes. The structural diversity in the cytochrome c1 surface facing the ISP domain indicates low structural constraints on that surface for formation of a productive electron transfer complex. A similar position of the acidic N-terminal domains of cytochrome c1 and yeast subunit QCR6p is suggested in support of a similar function. A model of the electron transfer complex with membrane-anchored cytochrome c552, the natural substrate, shows that it can adopt the same orientation as the soluble substrate in the yeast complex. The full structural integrity of the P. denitrificans variant underpins previous mechanistic studies on intermonomer electron transfer and paves the way for using this model system to address open questions of structure/function relationships and inhibitor binding.

Ilicicolin Inhibition and Binding at Center N of the Dimeric Cytochrome bc1 Complex Reveal Electron Transfer and Regulatory Interactions between Monomers

We have determined the kinetics of ilicicolin binding and dissociation at center N of the yeast bc(1) complex and its effect on the reduction of cytochrome b with center P blocked. The addition of ilicicolin to the oxidized complex resulted in a non-linear inhibition of the extent of cytochrome b reduction by quinol together with a shift of the reduced b(H) heme spectrum, indicating electron transfer between monomers. The possibility of a fast exchange of ilicicolin between center N sites was excluded in two ways. First, kinetic modeling showed that fast movement of an inhibitor between monomers would result in a linear inhibition of the extent of cytochrome b reduction through center N. Second, we determined a very slow dissociation rate for ilicicolin (k = 1.2 x 10(-3) s(-1)) as calculated from its displacement by antimycin. Ilicicolin binding to the reduced bc(1) complex occurred in a single phase (k(on) = 1.5-1.7 x 10(5) m(-1) s(-1)) except in the presence of stigmatellin, where a second slower binding phase comprising approximately 50% of the spectral change was observed. This second kinetic event was weakly dependent on ilicicolin concentration, which suggests that binding of ilicicolin to one center N in the dimer transmits a slow (k = 2-3 s(-1)) conformational change that allows binding of the inhibitor in the other monomer. These results, together with the evidence for intermonomeric electron transfer, provide further support for a dimeric model of regulatory interactions between center P and center N sites in the bc(1) complex.

S15/3 Regulatory interactions in the dimeric cytochrome bc1 complex

S15/3 Regulatory interactions in the dimeric cytochrome bc1 complex

Proton translocation by the cytochrome bc1 complexes of phototrophic bacteria: introducing the activated Q-cycle

The cytochrome bc1 complexes are proton-translocating, dimeric membrane ubiquinol:cytochrome c oxidoreductases that serve as "hubs" in the vast majority of electron transfer chains. After each ubiquinol molecule is oxidized in the catalytic center P at the positively charged membrane side, the two liberated electrons head out, according to the Mitchell's Q-cycle mechanism, to different acceptors. One is taken by the [2Fe-2S] iron-sulfur Rieske protein to be passed further to cytochrome c1. The other electron goes across the membrane, via the low- and high-potential hemes of cytochrome b, to another ubiquinone-binding site N at the opposite membrane side. It has been assumed that two ubiquinol molecules have to be oxidized by center P to yield first a semiquinone in center N and then to reduce this semiquinone to ubiquinol. This review is focused on the operation of cytochrome bc1 complexes in phototrophic purple bacteria. Their membranes provide a unique system where the generation of membrane voltage by light-driven, energy-converting enzymes can be traced via spectral shifts of native carotenoids and correlated with the electron and proton transfer reactions. An "activated Q-cycle" is proposed as a novel mechanism that is consistent with the available experimental data on the electron/proton coupling. Under physiological conditions, the dimeric cytochrome bc1 complex is suggested to be continually primed by prompt oxidation of membrane ubiquinol via center N yielding a bound semiquinone in this center and a reduced, high-potential heme b in the other monomer of the enzyme. Then the oxidation of each ubiquinol molecule in center P is followed by ubiquinol formation in center N, proton translocation and generation of membrane voltage.

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.

Evidence for Electron Equilibrium between the Two Hemes bL in the Dimeric Cytochrome bc1 Complex

Structural analysis of the dimeric mitochondrial cytochrome bc1 complex suggests that electron transfer between inter-monomer hemes bL-bL may occur during bc1 catalysis. Such electron transfer may be facilitated by the aromatic pairs present between the two bL hemes in the two symmetry-related monomers. To test this hypothesis, R. sphaeroides mutants expressing His6-tagged bc1 complexes with mutations at three aromatic residues (Phe-195, Tyr-199, and Phe-203), located between two bL hemes, were generated and characterized. All three mutants grew photosynthetically at a rate comparable to that of wild-type cells. The bc1 complexes prepared from mutants F195A, Y199A, and F203A have, respectively, 78%, 100%, and 100% of ubiquinol-cytochrome c reductase activity found in the wild-type complex. Replacing the Phe-195 of cytochrome b with Tyr, His, or Trp results in mutant complexes (F195Y, F195H, or F195W) having the same ubiquinol-cytochrome c reductase activity as the wild-type. These results indicate that the aromatic group at position195 of cytochrome b is involved in electron transfer reactions of the bc1 complex. The rate of superoxide anion (O2*) generation, measured by the chemiluminescence of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one hydrochloride-O2* adduct during oxidation of ubiquinol, is 3 times higher in the F195A complex than in the wild-type or mutant complexes Y199A or F203A. This supports the idea that the interruption of electron transfer between the two bL hemes enhances electron leakage to oxygen and thus decreases the ubiquinol-cytochrome c reductase activity.

A concerted, alternating sites mechanism of ubiquinol oxidation by the dimeric cytochrome bc1 complex

A refinement of the protonmotive Q cycle mechanism is proposed in which oxidation of ubiquinol is a concerted reaction and occurs by an alternating, half-of-the-sites mechanism. A concerted mechanism of ubiquinol oxidation is inferred from the finding that there is reciprocal control between the high potential and low potential redox components involved in ubiquinol oxidation. The potential of the Rieske iron–sulfur protein controls the rate of reduction of the b cytochromes, and the potential of the b cytochromes controls the rate of reduction of the Rieske protein and cytochrome c 1. A concerted mechanism of ubiquinol oxidation reconciles the findings that the ubiquinol–cytochrome c reductase kinetics of the bc 1 complex include both a pH dependence and a dependence on Rieske iron–sulfur protein midpoint potential. An alternating, half-of-the-sites mechanism for ubiquinol oxidation is inferred from the finding that some inhibitory analogs of ubiquinol that block ubiquinol oxidation by binding to the ubiquinol oxidation site in the bc 1 complex inhibit the yeast enzyme with a stoichiometry of 0.5 per bc 1 complex. One molecule of inhibitor is sufficient to fully inhibit the dimeric enzyme, and 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. An alternating, half-of-the-sites mechanism implies that, at least under some conditions, only half of the sites in the dimeric enzyme are reactive at any one time. This provides a raison d'être for the dimeric structure of the enzyme, in that bc 1 activity may be regulated and capable of switching between a half-of-the-sites active and a fully active enzyme.

The cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supracomplex in yeast mitochondria.

The mitochondrial electron transport chain complexes are large multisubunit complexes embedded in the inner membrane. We report here that in the yeast Saccharomyces cerevisiae, the cytochrome bc(1) and cytochrome c oxidase complexes co-exist as a larger complex of approximately 1000 kDa in the mitochondrial membrane. Following solubilization with a mild detergent, the cytochrome bc(1)-cytochrome c oxidase complex remains stable. It was analyzed using the techniques of gel filtration and blue native-polyacrylamide gel electrophoresis. Direct physical association of subunits of the cytochrome bc(1) complex with those of the cytochrome c oxidase complex was verified by co-immunoprecipitation analysis. Our data indicate that the cytochrome bc(1) complex is exclusively in association with the cytochrome c oxidase complex in yeast mitochondria. We term this complex the cytochrome bc(1)-cytochrome c oxidase supracomplex.

Subunit 6 regulates half-of-the-sites reactivity of the dimeric cytochrome bc1 complex in Saccharomyces cerevisiae.

We have characterized the activities of the cytochrome bc1 complex in mitochondrial membranes from a yeast strain in which we deleted the nuclear gene (QCR6, COR3) which codes for the highly acidic subunit 6 of the bc1 complex. The chromosomal copy of QCR6 was replaced with a plasmid derived copy of QCR6, in which the entire coding region of QCR6 was replaced with the yeast LEU2 gene. The resulting deletion strain, MES8, contained no detectable mRNA for QCR6, and the cytochrome bc1 complex purified from the deletion strain lacked subunit 6. The deletion strain respired and grew on nonfermentable carbon sources such as ethanol and glycerol. Ubiquinol-cytochrome c reductase activity of mitochondria from the deletion strain was decreased 50% under conditions where the activity is zero order with respect to cytochrome c, and there was a similar decrease in the first-order rate constant for cytochrome c reduction. The loss of bc1 complex activities, observed at physiological ionic strengths, was reversible. Both the zero order rate and the first-order rate constant for cytochrome c reduction could be recovered to those of the parental strain by measuring these activities in mitochondrial membranes under conditions of low ionic strength. The zero order rate and first-order rate constant for cytochrome c reduction in membranes from the parent, wild-type yeast showed essentially no change coincident with this change in ionic strength. The 50% drop in both turnover number and first-order rate constant of ubiquinol-cytochrome c reductase activity indicates that half of the cytochrome bc1 complexes are inactive in the deletion strain at physiological ionic strengths. Inhibition by myxothiazol of cytochrome c reductase activity of mitochondrial membranes from the deletion strain showed an ionic strength-dependent lag in the titration curve that extended to the point where half of the inhibitor sites are filled. This lag was not observed with membranes from the wild-type, parent strain. This response to the inhibitor is consistent with half of the cytochrome bc1 complexes being inactive in mitochondria from the deletion strain at physiological ionic strength, but with both active and inactive complexes still able to bind inhibitor. The reversible, half-of-the-sites reactivity indicates that the bc1 complex must be dimeric in situ, in agreement with previous findings that the complexes isolated from fungal (Leonard, K., Wingfield, P., Arad, T., and Weiss, H. (1981) J. Mol. Biol. 149, 259-274) and mammalian (Nalecz, M. J., Bolli, R., and Azzi, A. (1985) Arch. Biochem. Biophys. 236, 619-628) mitochondria are structural dimers.(ABSTRACT TRUNCATED AT 400 WORDS)