Direct and indirect roles of cytochrome b in the mediation of superoxide generation and NO catabolism by mitochondrial succinate-cytochrome c reductase.
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 Q(o*-) in the mediation of O2*- generation. Addition of cyanide significantly decreased (approximately 70%) O2*- production, indicating the involvement of the heme component. UV-visible spectral analysis revealed that oxidation of ferrocytochrome b was accompanied by cytochrome c(1) 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.
Chen
Yeong-Renn
YR
Davis Heart & Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, Ohio State University, Columbus, Ohio 43210; Department of Molecular and Cellular Biochemistry, College of Medicine, Ohio State University, Columbus, Ohio 43210. Electronic address: yeong-renn.chen@osumc.edu.
Chen
Chwen-Lih
CL
Davis Heart & Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, Ohio State University, Columbus, Ohio 43210.
Yeh
Alexander
A
Davis Heart & Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, Ohio State University, Columbus, Ohio 43210.
Liu
Xiaoping
X
Davis Heart & Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, Ohio State University, Columbus, Ohio 43210.
Zweier
Jay L
JL
Davis Heart & Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, Ohio State University, Columbus, Ohio 43210; Department of Molecular and Cellular Biochemistry, College of Medicine, Ohio State University, Columbus, Ohio 43210.
D000818:Animals; D002417:Cattle; D045303:Cytochromes b / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology; D009569:Nitric Oxide / Q000378:metabolism*; D011758:Pyrroles; D019097:Spin Trapping; D013384:Succinate Cytochrome c Oxidoreductase / Q000378:metabolism*; D019802:Succinic Acid / Q000737:chemistry / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
C510912:(diethoxyphosphoryl)-5-methylpyrroline N-oxide; D011758:Pyrroles; D013481:Superoxides; D009569:Nitric Oxide; D045303:Cytochromes b; D019802:Succinic Acid; D013384:Succinate Cytochrome c Oxidoreductase