1. Pigeon heart mitochondria produce H2O2 at a maximal rate of about 20nmol/min per mg of protein. 2. Succinate–glutamate and malate–glutamate are substrates which are able to support maximal H2O2 production rates. With malate–glutamate, H2O2 formation is sensitive to rotenone. Endogenous substrate, octanoate, stearoyl-CoA and palmitoyl-carnitine are by far less efficient substrates. 3. Antimycin A exerts a very pronounced effect in enhancing H2O2 production in pigeon heart mitochondria; 0.26nmol of antimycin A/mg of protein and the addition of an uncoupler are required for maximal H2O2 formation. 4. In the presence of endogenous substrate and of antimycin A, ATP decreases and uncoupler restores the rates of H2O2 formation. 5. Reincorporation of ubiquinone-10 and ubiquinone-3 to ubiquinone-depleted pigeon heart mitochondria gives a system in which H2O2 production is linearly related to the incorporated ubiquinone. 6. The generation of H2O2 by pigeon heart mitochondria in the presence of succinate–glutamate and in metabolic state 4 has an optimum pH value of 7.5. In states 1 and 3u, and in the presence of antimycin A and uncoupler, the optimum pH value is shifted towards more alkaline values. 7. With increase of the partial pressure of O2 to the hyperbaric region the formation of H2O2 is markedly increased in pigeon heart mitochondria and in rat liver mitochondria. With rat liver mitochondria and succinate as substrate in state 4, an increase in the pO2 up to 1.97MPa (19.5atm) increases H2O2 formation 10–15-fold. Similar pO2 profiles were observed when rat liver mitochondria were supplemented either with antimycin A or with antimycin A and uncoupler. No saturation of the system with O2 was observed up to 1.97MPa (19.5atm). By increasing the pO2 to 1.97MPa (19.5atm), H2O2 formation in pigeon heart mitochondria with succinate as substrate increased fourfold in metabolic state 4, with antimycin A added the increase was threefold and with antimycin A and uncoupler it was 2.5-fold. In the last two saturation of the system with oxygen was observed, with an apparent Km of about 71kPa (0.7–0.8atm) and a Vmax. of 12 and 20nmol of H2O2/min per mg of protein. 8. It is postulated that in addition to the well-known flavin reaction, formation of H2O2 may be due to interaction with an energy-dependent component of the respiratory chain at the cytochrome b level.

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