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

Production of superoxide anion (O-2(radical anion), measured as the chemiluminescence of the 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[ 1,2-a]pyrazin-3-one hydrochloride (MCLA)-O-2(radical anion) 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 O-2(radical anion) production, suggesting that O-2(radical anion) generation is caused by the membrane potential generated during electron transfer through the cytochrome bc(1) complex. Production of O-2(radical anion) was also observed during electron transfer from succinate to cytochrome c by antimycin-treated reductase, in which similar to 99.7% of the reductase activity was inhibited. The rate of O-2(radical anion) production was closely related to the rate of antimycin-insensitive cytochrome c reduction. Factors affecting antimycin-insensitive reduction of cytochrome c also affected O-2(radical anion) production and vice versa. When the oxygen concentration in the system was decreased, the rate of O-2(radical anion) production and cytochrome c reduction by antimycin-treated reductase decreased. When the concentrations of MCLA and cytochrome c were increased, the rate of O-2(radical anion) 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 O-2(radical anion) during the oxidation of ubiquinol by the cytochrome be, 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 b(566) or ubisemiquinone of the Q(o) site because addition of MCLA to antimycin-treated cytochrome be, 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 O-2(radical anion). When succinate dehydrogenase was reconstituted with the be, particles to form succinate-cytochrome c reductase, the production of O-2(radical anion) diminished. These results suggest that reduced FAD of succinate dehydrogenase is the electron donor for oxygen to produce O-2(radical anion) in the absence of their immediate electron acceptor and in the presence of cytochrome c.