Ubiquinol:cytochrome c oxidoreductase -: Effects of inhibitors on reverse electron transfer from the iron-sulfur protein to cytochrome b

The effects of inhibitors on the reduction of the bis-heme cytochrome b of ubiquinol: cytochrome c oxidoreductase (complex III, bc(1) complex) has been studied in bovine heart submitochondrial particles (SMP) when cytochrome b was reduced by NADH and succinate via the ubiquinone (Q) pool or by ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine via cytochrome c(1) and the iron-sulfur protein of complex III (ISP). The inhibitors used were antimycin (an N-side inhibitor), beta-methoxyacrylate derivatives, stigmatellin (P-side inhibitors), and ethoxyformic anhydride, which modifies essential histidyl residues in ISP, In agreement with our previous findings, the following results were obtained: (i) When ISP/cytochrome c(1) were prereduced or SMP were treated with a P-side inhibitor, the high potential heme b(H) was fully and rapidly reduced by NADH or succinate, whereas the low potential heme b(L) was only partially reduced. (ii) Reverse electron transfer from ISP/c(1) to cytochrome b was inhibited more by antimycin than by the P-side inhibitors. This reverse electron transfer was unaffected when, instead of normal SMP, Q-extracted SMP containing 200-fold less Q (0.06 mol Q/mol cytochrome b or c(1)) were used. (iii) The cytochrome b reduced by reverse electron transfer through the leak of a P-side inhibitor was rapidly oxidized upon subsequent addition of antimycin. This antimycin-induced reoxidation did not happen when Q-extracted SMP were used. The implications of these results on the path of electrons in complex III, on oxidant-induced extra cytochrome b reduction, and on the inhibition of forward electron transfer to cytochrome b by a P-side plus an N-side inhibitor have been discussed.