Generation of superoxide anion by succinate-cytochrome c reductase from bovine heart mitochondria

Abstract

Production of superoxide anion (O2/(·-)), measured as the chemiluminescence of the 2-methyl-6-(p-methoxyphenyl)-3,7- dihydroimidazo[1,2-α]pyrazin-3-one hydrochloride (MCLA)-O2/(·-) adduct, was observed during electron transfer from succinate to cytochrome c by reconstituted succinate-cytochrome c reductase-phospholipid vesicles replenished with succinate dehydrogenase. Addition of carbonyl cyanide p- trifluoromethoxyphenylhydrazone or detergent to the reconstituted reductase- phospholipid vesicles abolished O2/(·-) production, suggesting that O2/(·-) generation is caused by the membrane potential generated during electron transfer through the cytochrome bc1 complex. Production of O2/(·- ) was also observed during electron transfer from succinate to cytochrome c by antimycin-treated reductase, in which ~99.7% of the reductase activity was inhibited. The rate of O2/(·-) production was closely related to the rate of antimycin-insensitive cytochrome c reduction. Factors affecting antimycin-insensitive reduction of cytochrome c also affected O2/(·-) production and vice versa. When the oxygen concentration in the system was decreased, the rate of O2/(·-) production and cytochrome c reduction by antimycin treated reductase decreased. When the concentrations of MCLA and cytochrome c were increased, the rate of O2/(·-) production and cytochrome c reduction by antimycin-treated reductase increased. The rate of antimycin- insensitive cytochrome c reduction was sensitive to Q(o) site inhibitors such as 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole. These results indicate that generation of 02/(·-) during the oxidation of ubiquinol by the cytochrome bc1 complex results from a leakage of the second electron of ubiquinol from its Q cycle electron transfer pathway to interact with oxygen. The electron- leaking site is located at the reduced cytochrome b566 or ubisemiquinone of the Q(o) site because addition of MCLA to antimycin-treated cytochrome bc1 complex, in the presence of catalytic amounts of succinate-cytochrome c reductase, delayed cytochrome b reduction by succinate. In the presence of oxidized cytochrome c, purified succinate dehydrogenase also catalyzed oxidation of succinate to generate O2/(·-). When succinate dehydrogenase was reconstituted with the bc1 particles to form succinate-cytochrome c reductase, the production of O2/(·-) diminished. These results suggest that reduced FAD of succinate dehydrogenase is the electron donor for oxygen to produce O2/(·-) in the absence of their immediate electron acceptor and in the presence of cytochrome c.