Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction: Evaluation of its role in nitric oxide generation in anoxic tissues

Haitao Li, Alexandre Samouilov, Xiaoping Liu, Jay L. Zweier

Research output: Contribution to journalArticle

Abstract

Xanthine oxidase (XO)-catalyzed nitrite reduction with nitric oxide (NO) production has been reported to occur under anaerobic conditions, but questions remain regarding the magnitude, kinetics, and biological importance of this process. To characterize this mechanism and its quantitative importance in biological systems, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and NO electrode studies were performed. The XO reducing substrates xanthine, NADH, and 2,3-dihydroxybenz-aldehyde triggered nitrite reduction to NO, and the molybdenum-binding XO inhibitor oxypurinol inhibited this NO formation, indicating that nitrite reduction occurs at the molybdenum site. However, at higher xanthine concentrations, partial inhibition was seen, suggesting the formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. Studies of the pH dependence of NO formation indicated that XO-mediated nitrite reduction occurred via an acid-catalyzed mechanism. Nitrite and reducing substrate concentrations were important regulators of XO-catalyzed NO generation. The substrate dependence of anaerobic XO-catalyzed nitrite reduction followed Michaelis-Menten kinetics, enabling prediction of the magnitude of NO formation and delineation of the quantitative importance of this process in biological systems. It was determined that under conditions occurring during no-flow ischemia, myocardial XO and nitrite levels are sufficient to generate NO levels comparable to those produced from nitric oxide synthase. Thus, XO-catalyzed nitrite reduction can be an important source of NO generation under ischemic conditions.

Original languageEnglish (US)
Pages (from-to)24482-24489
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number27
DOIs
StatePublished - Jul 6 2001

ASJC Scopus subject areas

  • Biochemistry

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