Generation of superoxide anion by succinate-cytochrome c reductase from bovine heart mitochondria.
Production of superoxide anion (O-2), measured as the chemiluminescence of the 2-methyl-6-(p-methoxyphenyl)-3, 7-dihydroimidazo[1,2-a]pyrazin-3-one hydrochloride (MCLA)-O-2 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 production, suggesting that O-2 generation is caused by the membrane potential generated during electron transfer through the cytochrome bc1 complex. Production of O-2 was also observed during electron transfer from succinate to cytochrome c by antimycin-treated reductase, in which approximately 99.7% of the reductase activity was inhibited. The rate of O-2 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 production and vice versa. When the oxygen concentration in the system was decreased, the rate of O-2 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 production and cytochrome c reduction by antimycin-treated reductase increased. The rate of antimycin-insensitive cytochrome c reduction was sensitive to Qo site inhibitors such as 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole. These results indicate that generation of O-2 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 Qo 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 O-2. When succinate dehydrogenase was reconstituted with the bc1 particles to form succinate-cytochrome c reductase, the production of O-2 diminished. These results suggest that reduced FAD of succinate dehydrogenase is the electron donor for oxygen to produce O-2 in the absence of their immediate electron acceptor and in the presence of cytochrome c.
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Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
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Yu
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D000818:Animals; D002417:Cattle; D007093:Imidazoles; D007202:Indicators and Reagents; D007700:Kinetics; D008163:Luminescent Measurements; D008929:Mitochondria, Heart / Q000201:enzymology*; D008956:Models, Chemical; D010101:Oxygen Consumption; D011719:Pyrazines; D013384:Succinate Cytochrome c Oxidoreductase / Q000378:metabolism*; D013481:Superoxides / Q000032:analysis / Q000378:metabolism*
D007093:Imidazoles; D007202:Indicators and Reagents; D011719:Pyrazines; D013481:Superoxides; C063412:2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo(1,2-alpha)pyrazin-3-one; D013384:Succinate Cytochrome c Oxidoreductase