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Papers In Press, published online ahead of print March 10, 2006
Internal Medicine, The Ohio State University, Columbus, OH 43210
Corresponding Author: yeong-renn.chen{at}osumc.edu
Mitochondrial superoxide (O2•-) production is an important mediator of oxidative cellular injury. Succinate-cytochrome c reductase (SCR) of the electron transport chain (ETC) 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 Qo 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/VIS 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 SOD, 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.
J. Biol. Chem, 10.1074/jbc.M513627200
Submitted on December 21, 2005
Revised on March 6, 2006
Accepted on March 10, 2006
Direct and indirect roles of cytochrome b in the mediation of superoxide generation and no catabolism by mitochondrial succinate-cytochrome c reductase
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