Evidence for the Role of a Peroxidase Compound I-type Intermediate in the Oxidation of Glutathione, NADH, Ascorbate, and Dichlorofluorescin by Cytochrome c/H2O2

Abstract

The release of cytochrome c from mitochondria is a crucial step in apoptosis, resulting in the activation of the caspase proteases. A further consequence of cytochrome c release is the enhanced mitochondrial production of superoxide radicals (O2.), which are converted to hydrogen peroxide by manganese-superoxide dismutase. Recently, we showed that cytochrome c is a potent catalyst of 2',7'-dichlorofluorescin oxidation to the fluorescent 2',7'-dichlorofluorescein by these species, leading to the conclusion that 2',7'-dichlorofluorescein fluorescence is a reflection of cytosolic cytochrome c concentration rather than "reactive oxygen species" levels (Burkitt, M. J., and Wardman, P. (2001) Biochem. Biophys. Res. Commun. 282, 329-333). The oxidant generated from cytochrome c has so far not been identified. Several authors have suggested that the hydroxyl radical (*OH) is generated, but others have discussed the possibility of a peroxidase compound I. By examining the effects of various antioxidants (glutathione, ascorbate, and NADH) and "hydroxyl radical scavengers" (ethanol and mannitol) on the rate of 2',7'-dichlorofluorescin oxidation by cytochrome c, together with complementary EPR spin-trapping studies, we demonstrate that the hydroxyl radical is not generated. Instead, our findings suggest the formation of a peroxidase compound I-type intermediate, in which one oxidizing equivalent is present as an oxoferryl heme species and the other as the protein tyrosyl radical previously identified (Barr, D. P., Gunther, M. R., Deterding, L. J., Tomer, K. B., and Mason, R. P. (1996) J. Biol. Chem. 271, 15498-15503). Competition studies involving spin traps indicated that the oxoferryl heme component is the active oxidant. These findings provide an improved understanding of the physicochemical basis of the redox changes that occur during apoptosis.