Abstract
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.
Highlights
The redox reactions of the bis-heme cytochrome b of the ubiquinol:cytochrome c oxidoreductase complex were studied in bovine heart submitochondrial particles (SMP)
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
This reaction was inhibited more strongly by antimycin than by myxothiazol. (iii) Ascorbate or ascorbate plus TMPD could partially reduce b in ubiquinone-depleted bovine heart SMP, in which the molar ratio of Q10 to complex III monomer had been reduced 200-fold from 12.5 to 0.06, or in SMP from a Q-deficient yeast mutant. These results agreed with the finding of others that in bovine or yeast complex III the removal of Q by extraction did not impair the oxidation of b via iron-sulfur protein (ISP)/c1 by ferricyanide [7, 8]. (iv) Our results showed that antimycin and myxothiazol, which exert their maximal inhibition at concentrations stoichiometric to complex III monomer, each inhibited three reactions of the bis-heme cytochrome b, all incompletely
Summary
THE REDOX REACTIONS OF THE BIS-HEME CYTOCHROME b IN UNENERGIZED AND ENERGIZED SUBMITOCHONDRIAL PARTICLES*. (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. (ii) Cytochrome b of complex III could be partially reduced via cytochrome c1 and the Rieske iron-sulfur protein (ISP) by ascorbate, or faster and to a greater extent by ascorbate plus TMPD This reaction was inhibited more strongly by antimycin than by myxothiazol (see Ref. 6). The strongest effect by either reagent was inhibition of electron transfer from heme bH to heme bL; was inhibition of the reoxidation of bL via ISP/c1; and least was inhibition of substrate (NADH or succinate) reduction of b, which is known to require the combined actions of both antimycin and myxothiazol [1, 2, 4].
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