Mitochondrial superoxide (O2-.) production is an important mediator of oxidative cellular injury. Succinate-cytochrome c reductase (SCR) of the electron transport chain has been implicated as an essential part of the mediation of O2-. generation and an alternative target of nitric oxide (NO) in the regulation of mitochondrial respiration. The Q cycle mechanism plays a central role in controlling both events. In the present work, O2-. generation by SCR was measured with the EPR spin-trapping technique using DEPMPO (5- diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide) as the spin trap. In the presence of succinate, O2-. generation from SCR was detected as the spin adduct DEPMPO/.OOH. Inhibitors of the Q o site only marginally reduced (20-30%) this O2 -. production, suggesting a secondary role of Qo -. in the mediation of O2-. generation. Addition of cyanide significantly decreased (∼70%) O2 -. production, indicating the involvement of the heme component. UV-visible spectral analysis revealed that oxidation of ferrocytochrome b was accompanied by cytochrome c1 reduction, and the reaction was mediated by the formation of an O2-. intermediate, indicating a direct role for cytochrome b in O2-. generation. In the presence of NO, DEPMPO/.OOH production was progressively diminished, implying that NO interacted with SCR or trapped the O2-.. The consumption of NO by SCR was investigated by electrochemical detection using an NO electrode. In the presence of succinate, SCR-mediated NO consumption was observed and inhibited by the addition of superoxide dismutase, suggesting the involvement of O2-.. Under the conditions of argon saturation, the NO consumption rate was not enhanced by succinate, suggesting a direct role for O2-. in the mediation of NO consumption. In the presence of succinate, oxidation of the ferrocytochrome b moiety of SCR was accelerated by the addition of NO, and was inhibited by argon saturation, indicating an indirect role for cytochrome b in the mediation of NO consumption.
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