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 degre e of inhibition at 1 mol each/mol of complex III monomer. Phenomenolog ically, however, they each inhibit three steps in the redox reaction o f the bis-heme cytochrome b in submitochondrial particles (SMP), and a ll three inhibitions are incomplete to various extents. (i) In SMP, re duction of hemes b(H) and b(L) by NADH or succinate is inhibited when the particles are treated with both antimycin and myxothiazol. Each in hibitor alone allows reduced b(H) and b(L) to accumulate, indicating t hat each inhibits the reoxidation of these hemes, (E)-Methyl-3-methoxy -2-(4'trans-stilbenyl)acrylate in combination with antimycin or 2-n-he ptyl-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 a ntimycin or myxothiazol (or 2-n-heptyl-4-hydroxyquinoline-N-oxide, )-m ethyl-3-methoxy-2-(4'-trans-stilbenyl)acrylate, or stigmatellin). (iii ) Reoxidation of reduced b(H) is also inhibited by any one of these re agents. These inhibitions are also incomplete, and reduced b(L) is oxi dized 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 cytochro me c(1) and the Rieske iron-sulfur protein by ascorbate and faster by ascorbate + TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine). Energizat ion of SMP by the addition of ATP affords reduction of b(L) as well. R everse electron transfer to b(H) and b(L) is inhibited partially by my xothiazol, much more by antimycin, Ascorbate + TMPD also reduce b(H) i n ubiquinone-extracted SMP in which the molar ratio of ubiquinone to c ytochrome b has been reduced 200-fold from 12.5 to similar to 0.06. Re constitution of the extracted particles with ubiquinone-10 restores su bstrate oxidation but does not improve the rate or the extent of b(H) reduction by ascorbate + TMPD. These reagents also partially reduce cy tochrome b in SMP from a ubiquinone-deficient yeast mutant. The above results are discussed in relation to the Q-cycle hypothesis.