Regulation of xanthine oxidase by nitric oxide and peroxynitrite.

Xanthine oxidase (XO) is a central mechanism of oxidative injury as occurs following ischemia. During the early period of reperfusion, both nitric oxide (NO()) and superoxide (O-(2)) generation are increased leading to the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the presence and nature of the interactions of NO() or ONOO(-) with XO and the role of this process in regulating oxidant generation. Therefore, we determined the dose-dependent effects of NO() and ONOO(-) on the O-(2) generation and enzyme activity of XO, respectively, by EPR spin trapping of O-(2) using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide and spectrophotometric assay. ONOO(-) markedly inhibited both O-(2) generation and XO activity in dose-dependent manner, while NO() from NO() gas in concentrations up to 200 microM had no effect. Furthermore, we observed that NO() donors such as NOR-1 also inhibited O-(2) generation and XO activity; however, these effects were O-(2)-dependent and blocked by superoxide dismutase or ONOO(-) scavengers. Finally, we found that ONOO(-) totally abolished the Mo(V) EPR spectrum. These changes were irreversible, suggesting oxidative disruption of the critical molybdenum center of the catalytic site. Thus, ONOO(-) formed in biological systems can feedback and down-regulate XO activity and O-*(2) generation, which in turn may serve to limit further ONOO(-) formation.