antimycin and quinone

Using a stochastic simulation without any other hypotheses, we recently demonstrated the natural emergence of the modified Mitchell Q-cycle in the functioning of the bc(1) complex, with few short-circuits and a very low residence time of the reactive semiquinone species in the Q(o) site. However, this simple model fails to explain both the inhibition by antimycin of the bc(1) complex and the accompanying increase in ROS production. To obtain inhibition, we show that it is necessary to block the return of the electron from the reduced haem b(L) to Q(o). With this added hypothesis we obtain a sigmoid inhibition curve due to the fact that when only one antimycin is bound per bc(1) dimer, the electron of the inhibited monomer systematically crosses the dimer interface from b(L) to b(L) to reduce a quinone or a semiquinone species in the other (free) Q(i) site. Because this step is not limiting, the activity is unchanged (compared to the activity of the free dimer). Interestingly, this b(L)-b(L) pathway is almost exclusively taken in this half-bound antimycin dimer. In the free dimer, the natural faster pathway is b(L)-b(H) on the same monomer. The addition of the assumption of half-of-the-sites reactivity to the previous hypothesis leads to a transient activation in the antimycin titration curve preceding a quasi-complete inhibition at antimycin saturation.

Topics: Algorithms; Antimycin A; Benzoquinones; Binding Sites; Biocatalysis; Computer Simulation; Cytochromes b; Electron Transport; Electron Transport Complex III; Electrons; Iron-Sulfur Proteins; Kinetics; Models, Biological; Oxidation-Reduction; Protein Binding; Protein Multimerization; Reactive Oxygen Species; Stochastic Processes; Substrate Specificity

The cytochrome bc1 complex resides in the inner membrane of mitochondria and transfers electrons from ubiquinol to cytochrome c. This electron transfer is coupled to the translocation of protons across the membrane by the protonmotive Q cycle mechanism. This mechanism topographically separates reduction of quinone and reoxidation of quinol at sites on opposite sites of the membrane, referred to as center N (Qn site) and center P (Qp site), respectively. Both are located on cytochrome b, a transmembrane protein of the bc1 complex that is encoded on the mitochondrial genome. To better understand the parameters that affect ligand binding at the Qn site, we applied the Qn site inhibitor ilicicolin H to select for mutations conferring resistance in Saccharomyces cerevisiae. The screen resulted in seven different single amino acid substitutions in cytochrome b rendering the yeast resistant to the inhibitor. Six of the seven mutations have not been previously linked to inhibitor resistance. Ubiquinol-cytochrome c reductase activities of mitochondrial membranes isolated from the mutants confirmed that the differences in sensitivity toward ilicicolin H originated in the cytochrome bc1 complex. Comparative in vivo studies using the known Qn site inhibitors antimycin and funiculosin showed little cross-resistance, indicating different modes of binding of these inhibitors at center N of the bc1 complex.

Topics: Anthraquinones; Antifungal Agents; Antimycin A; Benzaldehydes; Benzoquinones; Chromosome Mapping; Culture Media; DNA, Mitochondrial; Electron Transport Complex III; Mitochondria; Models, Molecular; Mutation; Oxygen; Pyridones; Saccharomyces cerevisiae

Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions. The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA. Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone-10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q-cycle-type model.

Topics: Antimycin A; Bacteria; Bacterial Physiological Phenomena; Benzoquinones; Cytochrome b Group; Cytochrome c Group; Electron Transport Complex III; Electrons; Enzyme Inhibitors; Ferricyanides; Kinetics; Light; Methacrylates; Naphthoquinones; Oxidation-Reduction; Phenylenediamines; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Proteobacteria; Thiazoles; Time Factors; Titrimetry

The cytochrome b subunit (subunit I) of the ubiquinolcytochrome c reductase (bc1 complex) is thought to participate in the formation of two quinone/quinol reaction centers, an oxidizing center (Qo) and a reducing center, in accordance with the quinone cycle mechanism. Threonine 160 is a highly conserved residue in a segment of subunit I that was shown to bind quinone and is placed near the putative Qo site in current models of the bc1 complex. Rhodobacter sphaeroides cells expressing bc1 complexes with Ser or Tyr substituted for Thr160 grow photosynthetically at a reduced rate, and cells expressing the mutated complexes produce an "elevated" level of the bc1 complex. The Ser substitution also affects the interaction of subunit IV with subunit I. Replacement of Thr160 by Ser results in about a 70% loss of the activity in the purified complex, whereas substitution by Tyr lowers the activity by more than 80%. Both replacements lower the apparent Km for ubiquinol. Electron paramagnetic resonance (EPR) spectroscopy shows that in the Ser substituted complex, the environments of the Rieske iron-sulfur cluster in subunit III and the high potential cytochrome b (b562) in subunit I have been modified. The spectra of the Ser160 and Tyr160 iron-sulfur clusters have become redox-insensitive, with a line shape resembling that of the native complex in the fully reduced state. The EPR signal of b562 in the Ser160 complex is shifted from g = 3.50 to g = 3.52, but otherwise the line shape is very similar to the spectrum of the native complex. Most of these results are consistent with current ideas regarding the structure and function of Qo in the bc1 complex, except for the alteration of the b562 EPR feature, because this heme is not thought to be located in proximity to Qo. Immunoblotting analysis showed that the Ser or Tyr substituted complex contained significantly less than a stoichiometric amount of subunit IV. The enzymatic activity of mutated bc1 complex was found to be activable by the addition of purified subunit IV. These results indicate that Thr160 plays an important role in the structure and/or function of the bc1 complex.

Topics: Antimycin A; Base Sequence; Benzoquinones; Cytochrome b Group; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Iron-Sulfur Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Oligodeoxyribonucleotides; Protein Binding; Rhodobacter sphaeroides; Threonine

The results are presented of an electrochemical and high-resolution spectral analysis of the heme prosthetic groups in the bc1 complex from mouse cells. To study the long-range interactions between the Qo and Qi quinone redox sites and the b heme groups, we analyzed the effects on the proximal and distal b heme groups, and the c1 heme, of inhibitors that tightly and specifically bind to the Qi or Qo redox site. A number of results emerged from these studies. (1) There is inhomogeneous broadening of the b heme alpha band absorption spectra. Furthermore, contrary to the conclusion from low-resolution spectral analysis, the higher energy transition in the split-alpha band spectrum of the bL heme is more intense than the lower energy transition. (2) Inhibitors that bind at the Qi site have significant effects upon the electronic environment of the distal bL heme. Conversely, Qo site inhibitors induced changes in the electronic environment of the distal bH heme. (3) In contrast, inhibitor binding at either site has little effect upon the midpoint potential of the distal heme. (4) Experiments in which both a Qi and a Qo inhibitor are bound at the redox sites indicate that the long-range effects of one inhibitor are not blocked by the second inhibitor; enhanced effects are often observed. (5) In the double-inhibitor titrations involving the Qo inhibitor myxothiazol, there is evidence for two electrochemically and spectrally distinct species of the bL heme group, a phenomenon not observed previously. (6) The high-resolution deconvolutions of alpha band absorption spectra allow an interpretation of these inhibitor-induced changes in terms of homogeneous broadening, inhomogeneous broadening, and changes in x-y degeneracy. The general conclusion from these experiments is that when an inhibitor binds to a quinone redox site of the cytochrome b protein, it produces local conformational changes that, in turn, are transmitted to distal regions of the protein. The ligation of the bH and bL hemes between two parallel transmembrane helices provides a mechanism by which long-distance interactions can be propagated. The lack of long-range effects upon the midpoint potentials of the heme groups suggests, however, that protein conformational changes are unlikely to be a major control mechanism for the transmembrane electron- and proton-transfer steps of the Q cycle.

Topics: Animals; Anthraquinones; Antimycin A; Benzoquinones; Binding Sites; Cell Line; Chromatography, Ion Exchange; Cytochrome b Group; Cytochromes c1; Electrochemistry; Electron Transport Complex III; Fibroblasts; Heme; Methacrylates; Mice; Oxidation-Reduction; Polyenes; Spectrophotometry; Thiazoles

The steady-state reduction of exogenous ubiquinone-2 by duroquinol as catalysed by the ubiquinol: cytochrome c oxidoreductase was studied in bovine heart mitoplasts. The reduction of ubiquinone-2 by duroquinol proceeds both in the absence of inhibitors of the enzyme, in the presence of outside inhibitors, e.g., myxothiazol, and in the presence of inside inhibitors, e.g., antimycin, but not in the presence of both inside and outside inhibitors. It is concluded that both the Qin-binding domain and the Qout-binding domain may independently catalyse this reaction. The rate of the reduction of ubiquinone-2 by duroquinol via the Qin-binding domain is dependent on the type of outside inhibitor used. The maximal rate obtained for the reduction of ubiquinone-2 by DQH2 via the Qout-binding domain, measured in the presence of antimycin, is similar to that catalysed by the Qin-binding domain of the non-inhibited enzyme and depends on the redox state of the high-potential electron carriers of the respiratory chain. The reduction of ubiquinone-2 by DQH2 via the Qin-binding domain can be described by a mechanism in which duroquinol reduces the enzyme, upon which the reduced enzyme is rapidly oxidized by ubiquinone-2 yielding ubiquinol-2. By determination of the initial rate under various conditions and simulation of the time course of reduction of ubiquinone-2 using the integrated form of the steady-state rate equation the values of the various kinetic constants were calculated. During the course of reduction of ubiquinone-2 by duroquinol in the presence of outside inhibitors only cytochrome b-562 becomes reduced. At all stages during the reaction, cytochrome b-562 is in equilibrium with the redox potential of the ubiquinone-2/ubiquinol-2 couple but not with that of the duroquinone/duroquinol couple. At low pH values, cytochrome b-562 is reduced in a single phase; at high pH separate reduction phases are observed. In the absence of inhibitors three reduction phases of cytochrome b-562 are discernible at low pH values and two at high pH values. In the presence of antimyin cytochrome b becomes reduced in two phases. Cytochrome b-562 is reduced in the first phase and cytochrome b-566 in the second phase after substantial reduction of ubiquinone-2 to ubiquinol-2 has occurred. In ubiquinone-10 depleted preparations, titration of cytochrome b-562, in the presence of myxothiazol, with the duroquinone/duroquinol redox couple yields a value of napp = 2, both at low and high pH.(

Topics: Animals; Antimycin A; Benzoquinones; Binding Sites; Cattle; Cytochrome b Group; Electron Transport; Electron Transport Complex III; Hydrogen-Ion Concentration; Hydroquinones; Methacrylates; Myocardium; Oxidation-Reduction; Thiazoles; Ubiquinone

Stopped-flow experiments were performed to distinguish between two hypotheses, the Q-cycle and the SQ-cycle, each describing the pathway of electron transfer in the QH2:cytochrome c oxidoreductases. It was observed that, when mitochondrial membranes from the yeast Saccharomyces cerevisiae were poised at a low redox potential with appropriate amounts of sodium dithionite to completely reduce cytochrome b, the kinetics of oxidation of cytochrome b showed a lag period of maximally 100 ms. Under the same experimental conditions, the oxidation-reduction kinetics of cytochromes c + c1 showed transient behaviour. These results do not support the presence of a mobile species of semiquinone in the QH2:cytochrome c oxidoreductases, as envisaged in the SQ-cycle, but are consistent with a Q-cycle mechanism in which the two quinone-binding domains do not exchange electrons directly on the timescale of turnover of the enzyme.

Topics: Antimycin A; Benzoquinones; Cytochrome b Group; Cytochrome c Group; Cytochromes c1; Dithionite; Hydroquinones; Intracellular Membranes; Kinetics; Methacrylates; Mitochondria; Models, Biological; Oxidation-Reduction; Quinones; Saccharomyces cerevisiae; Succinates; Succinic Acid; Thiazoles

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