antimycin and ubiquinol

Topics: Antimycin A; Bacterial Chromatophores; Binding Sites; Biological Transport; Electron Transport; Electron Transport Complex III; Mitochondria; Models, Chemical; Rhodobacter sphaeroides; Ubiquinone

We previously proposed that the dimeric cytochrome bc(1) complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc(1) monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289-22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc(1) complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10-16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c(1) by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.

Topics: Animals; Antimycin A; Binding Sites; Chromatography, Affinity; Electron Transport Complex III; Enzyme Activation; Horses; Kinetics; Ligands; Mutagenesis; Mutant Proteins; Operon; Oxidation-Reduction; Paracoccus denitrificans; Protein Multimerization; Titrimetry; Ubiquinone

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.

Topics: Antimycin A; Binding Sites; Cytochromes b; Dimerization; Dose-Response Relationship, Drug; Electron Transport Complex III; Heme; Iron-Sulfur Proteins; Kinetics; Models, Biological; Protein Binding; Protein Conformation; Saccharomyces cerevisiae; Time Factors; Ubiquinone

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.

Topics: Antimycin A; Cytochromes b; Cytochromes c; Dimerization; Electron Transport Complex III; Fungal Proteins; Heme; Hydrogen-Ion Concentration; Iron-Sulfur Proteins; Kinetics; Mutation; Oxidation-Reduction; Oxygen; Spectrophotometry; Time Factors; Ubiquinone; Ultraviolet Rays

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.

Topics: Antimycin A; Binding Sites; Cytochrome b Group; Dimerization; Electron Transport; Electron Transport Complex III; Hydrogen-Ion Concentration; Iron-Sulfur Proteins; Kinetics; Models, Molecular; NADH Dehydrogenase; Proton-Motive Force; Thermodynamics; Ubiquinone; Vitamin K 2

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.

Topics: Anti-Bacterial Agents; Antimycin A; Electron Transport Complex III; Fungal Proteins; Oxidation-Reduction; Polyenes; Saccharomyces cerevisiae; Stilbenes; Ubiquinone

Crystal structures of the cytochrome bc1 complex indicate that the catalytic domain of the Rieske iron-sulfur protein, which carries the [2Fe-2S] cluster, is connected to a transmembrane anchor by a flexible linker region. This flexible linker allows the catalytic domain to move between two positions, proximal to cytochrome b and cytochrome c1. Addition of an alanine residue to the flexible linker region of the Rieske protein lowers the ubiquinol-cytochrome c reductase activity of the mitochondrial membranes by one half and causes the apparent Km for ubiquinol to decrease from 9.3 to 2.6 microM. Addition of two alanine residues lowers the activity by 90% and the apparent Km decreases to 1.9 microM. Deletion of an alanine residue lowers the activity by approximately 40% and the apparent Km decreases to 5.0 microM. Addition or deletion of an alanine residue also causes a pronounced decrease in efficacy of inhibition of ubiquinol-cytochrome c reductase activity by stigmatellin, which binds analogous to reaction intermediates of ubiquinol oxidation. These results indicate that the length of the flexible linker region is critical for interaction of ubiquinol with the bc1 complex, consistent with electron transfer mechanisms in which ubiquinol must simultaneously interact with the iron-sulfur protein and cytochrome b.

Topics: Alanine; Amino Acid Sequence; Antimycin A; Aspartic Acid; Blotting, Western; Catalysis; Crystallography, X-Ray; Electron Transport Complex III; Electrons; Intracellular Membranes; Iron-Sulfur Proteins; Kinetics; Mitochondria; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; NADH Dehydrogenase; Polyenes; Protein Structure, Tertiary; Saccharomyces cerevisiae; Sequence Homology, Amino Acid; Ubiquinone

This article is a study of the relationship between lipid peroxidation and protein modification in beef heart submitochondrial particles, and the protective effect of endogenous ubiquinol (reduced coenzyme Q) against these effects. ADP-Fe3+ and ascorbate were used to initiate lipid peroxidation and protein modification, which were monitored by measuring TBARS and protein carbonylation, respectively. Endogenous ubiquinone was reduced by the addition of succinate and antimycin. The parameters investigated included extraction and reincorporation of ubiquinone, and comparison of the effect of ubiquinol with those of various antioxidant compounds and enzymes, as well as the iron chelator EDTA. Under all conditions employed there was a close correlation between lipid peroxidation and protein carbonylation, and the inhibition of these effects by endogenous ubiquinol. SDS-PAGE analysis revealed a differential effect on individual protein components and its prevention by ubiquinol. Conceivable mechanisms behind the observed oxidative modifications of membrane phospholipids and proteins and of the role of ubiquinol in preventing these effects are considered.

Topics: Animals; Antimycin A; Antioxidants; Ascorbic Acid; Cattle; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Kinetics; Lipid Peroxidation; Mitochondria, Heart; Proteins; Submitochondrial Particles; Succinates; Succinic Acid; Thiobarbituric Acid Reactive Substances; Ubiquinone

The cytochrome bc1 complexes from the nonphotosynthetic strain R126 of Rhodobacter capsulatus and from its revertant MR126 were purified. Between both preparations, no difference could be observed in the stoichiometries of the cytochromes, in their spectral properties, and in their midpoint redox potentials. Both also showed identical polypeptide patterns after electrophoresis on polyacrylamide gels in the presence of sodium dodecylsulfate. The ubiquinol: cytochrome c oxidoreductase activity was strongly inhibited in the complex from the mutant compared to the one from the revertant. So was the oxidant-induced extra reduction of cytochrome b. Both preparations, however, showed an antimycin-induced red shift of cytochrome b, as well as antimycin-sensitive reduction of cytochrome b by ubiquinol. In accordance with a preceding study of chromatophores (Robertson et al. (1986). J. Biol. Chem. 261, 584-591), it is concluded that the mutation affects specifically the ubiquinol oxidizing site, leaving the ubiquinol reducing site unchanged.

Topics: Antimycin A; Catalysis; Centrifugation, Density Gradient; Electron Transport Complex III; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Methacrylates; Mutation; Oxidation-Reduction; Rhodobacter capsulatus; Spectrum Analysis; Thermodynamics; Thiazoles; Ubiquinone

A non-photosynthetic mutant (Ps-) of Rhodopseudomonas capsulata, designated R126, was analyzed for a defect in the cyclic electron transfer system. Compared to a Ps+ strain MR126, the mutant was shown to have a full complement of electron transfer components (reaction centers, ubiquinone-10, cytochromes b, c1, and c2, the Rieske 2-iron, 2-sulfur (Rieske FeS) center, and the antimycin-sensitive semiquinone). Functionally, mutant R126 failed to catalyze complete cytochrome c1 + c2 re-reduction or cytochrome b reduction following a short (10 microseconds) flash of actinic light. Evidence (from flash-induced carotenoid band shift) was characteristic of inhibition of electron transfer proximal to cytochrome c1 of the ubiquinol-cytochrome c2 oxidoreductase. Three lines of evidence indicate that the lesion of R126 disrupts electron transfer from quinol to Rieske FeS: 1) the degree of cytochrome c1 + c2 re-reduction following a flash is indicative of electron transfer from Rieske FeS to cytochrome c1 + c2 without redox equilibration with an additional electron from a quinol; 2) inhibitors that act at the Qz site and raise the Rieske FeS midpoint redox potential (Em), namely 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole or 3-alkyl-2-hydroxy-1,4-napthoquinone, have no effect on cytochrome c1 + c2 oxidation in R126; 3) the Rieske FeS center, although it exhibits normal redox behavior, is unable to report the redox state of the quinone pool, as metered by its EPR line shape properties. Flash-induced proton binding in R126 is indicative of normal functional primary (QA) and secondary (QB) electron acceptor activity of the photosynthetic reaction center. The Qc functional site of cytochrome bc1 is intact in R126 as measured by the existence of antimycin-sensitive, flash-induced cytochrome b reduction.

Topics: Antimycin A; Benzoquinones; Cytochrome c Group; Electron Spin Resonance Spectroscopy; Electron Transport; Electron Transport Complex III; Methacrylates; Multienzyme Complexes; Mutation; Oxidation-Reduction; Photolysis; Quinone Reductases; Quinones; Rhodopseudomonas; Thiazoles; Ubiquinone

Complex III of beef heart mitochondria was separated into the iron-sulfur protein and the complex devoid of it as described previously (Shimomura, Y., Nishikimi, M., and Ozawa, T. (1984) J. Biol. Chem. 259, 14059-14063). From the latter preparation, cytochrome c1 was subsequently purified by detergent-exchange chromatography on a phenyl-Sepharose column and DEAE-Sepharose column chromatography. In the former chromatography, the resolution of the iron-sulfur protein-depleted complex was achieved by changes of detergents on the surface of the complex; nearly homogeneous cytochrome c1 was eluted from the column with dodecyl octaethylene glycol mono-ether after dissociation of core proteins and subunit VI with guanidine and cholate. The purified cytochrome c1 consists of a single polypeptide as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 39 nmol of heme/mg of protein. The isolated iron-sulfur protein catalyzes reduction of cytochrome c by ubiquinol, which is insensitive to antimycin, at a rate of 0.03 mumol of cytochrome c reduced/min/nmol of protein, while the purified cytochrome c1 has no such catalytic activity. When cytochrome c1 and the iron-sulfur protein form a complex, the rate of cytochrome c reduction increases to 0.12 mumol/min/nmol of the iron-sulfur protein. In this reaction, cytochrome c1 mediates antimycin-insensitive electron transfer from the iron-sulfur protein to cytochrome c, thereby constituting a pathway of electrons: ubiquinol----iron-sulfur protein----cytochrome c1----cytochrome c. The complex formation between the iron-sulfur protein and cytochrome c1 was verified by binding of cytochrome c1 to a column of protein A-Sepharose to which the iron-sulfur protein was linked with immobilized anti-iron-sulfur protein antibody. The electron-transfer activity of the mixture is at a comparable level to that of antimycin-inhibited Complex III, and both activities are partially sensitive to superoxide dismutase. Thus, the above-described coupling of the iron-sulfur protein and cytochrome c1 is considered as reconstitution of the antimycin-insensitive pathway of electrons in Complex III.

Topics: Animals; Antimycin A; Cattle; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Cytochrome c Group; Electron Transport; Electron Transport Complex III; Electrophoresis, Polyacrylamide Gel; Iron-Sulfur Proteins; Macromolecular Substances; Metalloproteins; Mitochondria, Heart; Molecular Weight; Multienzyme Complexes; Quinone Reductases; Ubiquinone