Ubiquinol-cytochrome c oxidoreductase - The redox reactions of the bis-heme cytochrome b in ubiquinone-sufficient and ubiquinone-deficient systems

Antimycin and myxothiazol are stoichiometric inhibitors of complex III (ubiquinol-cytochrome c oxidoreductase), exerting their highest degree of inhibition at 1 mol each/mol of complex III monomer. Phenomenologically, however, they each inhibit three steps in the redox reaction of the bis-heme cytochrome b in submitochondrial particles (SMP), and all three inhibitions are incomplete to various extents. (i) In SMP, reduction of hemes b(H) and b(L) by NADH or succinate is inhibited when the particles are treated with both antimycin and myxothiazol. Each inhibitor alone allows reduced b(H) and b(L) to accumulate, indicating that each inhibits the reoxidation of these hemes, (E)-Methyl-3-methoxy-2-(4'trans-stilbenyl)acrylate in combination with antimycin or 2-n-heptyl-4-hydroxyquinoline-N-oxide in combination with myxothiazol causes less inhibition of b reduction than the combination of antimycin and myxothiazol. (ii) Reoxidation of reduced b(L) is inhibited by either antimycin or myxothiazol (or 2-n-heptyl-4-hydroxyquinoline-N-oxide, (E)-methyl-3-methoxy-2-(4'-trans-stilbenyl)acrylate, or stigmatellin). (iii) Reoxidation of reduced b(H) is also inhibited by any one of these reagents. These inhibitions are also incomplete, and reduced b(L) is oxidized through the leaks allowed by these inhibitors at least 10 times faster than reduced b(H). Heme b(H) can be reduced in SMP via cytochrome c(1) and the Rieske iron-sulfur protein by ascorbate and faster by ascorbate + TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine). Energization of SMP by the addition of ATP affords reduction of b(L) as well. Reverse electron transfer to b(H) and b(L) is inhibited partially by myxothiazol, much more by antimycin, Ascorbate + TMPD also reduce b(H) in ubiquinone-extracted SMP in which the molar ratio of ubiquinone to cytochrome b has been reduced 200-fold from 12.5 to similar to 0.06. Reconstitution of the extracted particles with ubiquinone-10 restores substrate oxidation but does not improve the rate or the extent of b(H) reduction by ascorbate + TMPD. These reagents also partially reduce cytochrome b in SMP from a ubiquinone-deficient yeast mutant. The above results are discussed in relation to the Q-cycle hypothesis.