Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions.
We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O(2)) conditions. The system measures the concentrations of O(2) and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O(2) concentration and electron turnover of the enzyme. At a high O(2) concentration (70 microM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O(2). At low O(2) (15 microM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O(2) consumption. At both high and low O(2) concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.
Unitt
David C
DC
Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, UK.
Hollis
Veronica S
VS
Palacios-Callender
Miriam
M
Frakich
Nanci
N
Moncada
Salvador
S
D001120:Arginine; D019069:Cell Respiration; D002478:Cells, Cultured; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010101:Oxygen Consumption
D009569:Nitric Oxide; D001120:Arginine; D052247:Nitric Oxide Synthase Type II; D003576:Electron Transport Complex IV; D010100:Oxygen