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.
Lee
C I
CI
Molecular and Cellular Biophysics Laboratories, Department of Medicine, Division of Cardiology and the Electron Paramagnetic Resonance Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21224, USA.
Liu
X
X
Zweier
J L
JL
D004578:Electron Spin Resonance Spectroscopy; D008982:Molybdenum / Q000378:metabolism; D009566:Nitrates / Q000494:pharmacology*; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D014969:Xanthine Oxidase / Q000737:chemistry / Q000378:metabolism*
D009566:Nitrates; D020030:Nitric Oxide Donors; D013481:Superoxides; C039642:peroxynitric acid; D009569:Nitric Oxide; D008982:Molybdenum; D014969:Xanthine Oxidase