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.
ASJC Scopus subject areas