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 (O₂/·^-) 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 O₂/·^- generation and enzyme activity of XO, respectively, by EPR spin trapping of O₂/·^- using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide and spectrophotometric assay. ONOO^- markedly inhibited both O₂/·^- generation and XO activity in dose-dependent manner, while NO· from NO· gas in concentrations up to 200 μM had no effect. Furthermore, we observed that NO· donors such as NOR-1 also inhibited O₂/·^- generation and XO activity; however, these effects were O₂/·^--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 downregulate XO activity and O₂/·^- generation, which in turn may serve to limit further ONOO^- formation.