Activation of cytochrome c to a peroxidase compound I-type intermediate by H₂O₂: relevance to redox signalling in apoptosis

The release of cytochrome c from mitochondria during apoptosis results in the enhanced production of superoxide radicals, which are converted to H₂O₂ by Mn-superoxide dismutase. We have been concerned with the role of cytochrome c/H₂O₂ in the induction of oxidative stress during apoptosis. Our initial studies showed that cytochrome c is a potent catalyst of 2′,7′-dichlorofluorescin oxidation, thereby explaining the increased rate of production of the fluorophore 2′,7′-dichlorofluorescein in apoptotic cells. Although it has been speculated that the oxidizing species may be a ferryl-haem intermediate, no definitive evidence for the formation of such a species has been reported. Alternatively, it is possible that the hydroxyl radical may be generated, as seen in the reaction of certain iron chelates with H₂O₂. By examining the effects of radical scavengers on 2′,7′-dichlorofluorescin oxidation by cytochrome c/H₂O₂, together with complementary EPR studies, we have demonstrated that the hydroxyl radical is not generated. Our findings point, instead, to the formation of a peroxidase compound I species, with one oxidizing equivalent present as an oxo-ferryl haem intermediate and the other as the tyrosyl radical identified by Barr and colleagues [Barr, Gunther, Deterding, Tomer and Mason (1996) J. Biol. Chem. 271, 15498-15503]. Studies with spin traps indicated that the oxo-ferryl haem is the active oxidant. These findings provide a physico-chemical basis for the redox changes that occur during apoptosis. Excessive changes (possibly catalysed by cytochrome c) may have implications for the redox regulation of cell death, including the sensitivity of tumour cells to chemotherapeutic agents.