Enhancement of hydrogen peroxide formation by protophores and ionophores in antimycin-supplemented mitochondria

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Abstract
Rat and pigeon heart mitochondria supplemented with antimycin produce 0.3–1.0nmol of H2O2/min per mg of protein. These rates are stimulated up to 13-fold by addition of protophores (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, carbonyl cyanide m-chloromethoxyphenylhydrazone and pentachlorophenol). Ionophores, such as valinomycin and gramicidin, and Ca2+ also markedly stimulated H2O2 production by rat heart mitochondria. The enhancement of H2O2 generation in antimycin-supplemented mitochondria and the increased O2 uptake of the State 4-to-State 3 transition showed similar protophore, ionophore and Ca2+ concentration dependencies. Thenoyltrifluoroacetone and N-bromosuccinimide, which inhibit succinate–ubiquinone reductase activity, also decreased mitochondrial H2O2 production. Addition of cyanide to antimycin-supplemented beef heart submitochondrial particles inhibited the generation of O2−, the precursor of mitochondrial H2O2. This effect was parallel to the increase in cytochrome c reduction and it is interpreted as indicating the necessity of cytochrome c13+ to oxidize ubiquinol to ubisemiquinone, whose autoxidation yields O2−. The effect of protophores, ionophores and Ca2+ is analysed in relation to the propositions of a cyclic mechanism for the interaction of ubiquinone with succinate dehydrogenase and cytochromes b and c1 [Wikstrom & Berden (1972) Biochim. Biophys. Acta 283, 403–420; Mitchell (1976) J. Theor. Biol. 62, 337–367]. A collapse in membrane potential, increasing the rate of ubisemiquinone formation and O2− production, is proposed as the molecular mechanism for the enhancement of H2O2 formation rates observed on addition of protophores, ionophores and Ca2+.