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 mu M), 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 mu M), 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. (C) 2009 Elsevier B.V. All rights reserved.