ICE/Ced-3Proteases
Apoptosis
We
have measured the rates of superoxide anion
generation by cytochrome bc(1) complexes isolated
from bovine heart and yeast mitochondria and by cytochrome
bc(1) 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 bc(1)
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 bc(1)
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 bc(1)
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
c reductase activity. These results indicate that superoxide anion is formed by similar mechanisms in
mammalian and yeast bc(1) 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.
.