hexacyanoferrate-iii and myxothiazol

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 redox reactions of the bis-heme cytochrome b of the ubiquinol:cytochrome c oxidoreductase complex (complex III, bc1 complex) were studied in bovine heart submitochondrial particles (SMP). It was shown that (i) when SMP were treated with the complex III inhibitor myxothiazol (or MOA-stilbene or stigmatellin) or with KCN and ascorbate to reduce the high potential centers of complex III (iron-sulfur protein and cytochromes c + c1), NADH or succinate reduced heme bL slowly and incompletely. In contrast, heme bH was reduced by these substrates completely and much more rapidly. Only when the complex III inhibitor was antimycin, and the high potential centers were in the oxidized state, NADH or succinate was able to reduce both bH and bL rapidly and completely. (ii) When NADH or succinate was added to SMP inhibited at complex III by antimycin and energized by ATP, the bis-heme cytochrome b was reduced only partially. Prereduction of the high potential centers was not necessary for this partial b reduction, but slowed down the reduction rate. Deenergization of SMP by uncoupling (or addition of oligomycin to inhibit ATP hydrolysis) resulted in further b reduction. Addition of ATP after b was reduced by substrate resulted in partial b oxidation, and the heme remaining reduced appeared to be mainly bL. Other experiments suggested that the redox changes of cytochrome b effected by energization and deenergization of SMP occurred via electronic communication with the ubiquinone pool. These results have been discussed in relation to current concepts regarding the mechanism of electron transfer by complex III.

Topics: Animals; Antifungal Agents; Antimycin A; Ascorbic Acid; Cattle; Cytochrome b Group; Electron Transport Complex III; Ferricyanides; Methacrylates; Models, Chemical; NAD; Oxidation-Reduction; Potassium Cyanide; Spectrophotometry, Atomic; Submitochondrial Particles; Succinates; Succinic Acid; Thiazoles