Characterization of the effects of oxygen on xanthine oxidase-mediated nitric oxide formation.
Under anaerobic conditions, xanthine oxidase (XO)-catalyzed nitrite reduction can be an important source of nitric oxide (NO). However, questions remain regarding whether significant XO-mediated NO generation also occurs under aerobic conditions. Therefore, electron paramagnetic resonance, chemiluminescence NO-analyzer, and NO-electrode studies were performed to characterize the kinetics and magnitude of XO-mediated nitrite reduction as a function of oxygen tension. With substrates xanthine or 2,3-dihydroxybenz-aldehyde that provide electrons to XO at the molybdenum site, the rate of NO production followed Michaelis-Menten kinetics, and oxygen functioned as a competitive inhibitor of nitrite reduction. However, with flavin-adenine dinucleotide site-binding substrate NADH as electron donor, aerobic NO production was maintained at more than 70% of anaerobic levels, and binding of NADH to the flavin-adenine dinucleotide site seemed to prevent oxygen binding. Therefore, under aerobic conditions, NADH would be the main electron donor for XO-catalyzed NO production in tissues. Studies of the pH dependence of NO formation indicated that lower pH values decrease oxygen reduction but greatly increase nitrite reduction, facilitating NO generation. Isotope tracer studies demonstrated that XO-mediated NO formation occurs in normoxic and hypoxic heart tissue. Thus, XO-mediated NO generation occurs under aerobic conditions and is regulated by oxygen tension, pH, nitrite, and reducing substrate concentrations.
Li
Haitao
H
Center for Biomedical EPR Spectroscopy and Imaging, Davis Heart and Lung Research Institute and Division of Cardiovascular Medicine, Department of Internal Medicine, Ohio State University College of Medicine, Columbus, Ohio 43210, USA.
Samouilov
Alexandre
A
Liu
Xiaoping
X
Zweier
Jay L
JL
D000818:Animals; D001547:Benzaldehydes / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D001671:Biochemistry / Q000295:instrumentation; D002396:Catechols / Q000494:pharmacology; D043302:Cultured Milk Products / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004563:Electrochemistry / Q000379:methods; D004566:Electrodes; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D005415:Flavins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008163:Luminescent Measurements; D008280:Magnetics; D008297:Male; D008956:Models, Chemical; D008982:Molybdenum / Q000494:pharmacology; D009206:Myocardium / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry*; D010100:Oxygen / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014969:Xanthine Oxidase / Q000378:metabolism*
D001547:Benzaldehydes; D002396:Catechols; D005415:Flavins; C067077:2,3-dihydroxybenzaldehyde; D009569:Nitric Oxide; D008982:Molybdenum; D014969:Xanthine Oxidase; D010100:Oxygen