Superoxide anion generation by the cytochrome bc1 complex

We have measured the rates of superoxide anion generation by cytochrome be, complexes isolated from bovine heart and yeast mitochondria, and by cytochrome be, complexes from yeast mutants in which the midpoint potentials of the cytochrome b hemes and the Rieske iron-sulfur cluster were altered by mutations in those proteins. With all of the be, complexes the rate of superoxide anion production was greatest in the absence of bc(1) inhibitor and ranged from 3% to 5% of the rate of cytochrome c reduction. Stigmatellin, an inhibitor that binds to the ubiquinol oxidation site in the be, complex, eliminated superoxide anion formation, while myxothiazol, another inhibitor of ubiquinol oxidation, allowed superoxide anion formation at a low rate. Antimycin, an inhibitor that binds to the ubiquinone reduction site in the be, complex, also allowed superoxide anion formation and at a slightly greater rate than myxothiazol. Changes in the midpoint potentials of the cytochrome b hemes had no significant effect on the rate of cytochrome c reduction and only a small effect on the rate of superoxide anion formation. A mutation in the Rieske iron-sulfur protein that lowers its midpoint potential from +285 to +220mV caused the rate of superoxide anion to decline in parallel with a decline in cytochrome e reductase activity. These results indicate that superoxide anion is formed by similar mechanisms in mammalian and yeast be, complexes. The results also show that changes in the midpoint potentials of the redox components that accept electrons during ubiquinol oxidation have only small effects on the formation of superoxide anion, except to the extent that they affect the activity of the enzyme. (C) 2003 Elsevier Inc. All rights reserved.