Abstract
Xanthine oxidase (XO) was shown to catalyze the reduction of nitrite to nitric oxide (NO), under anaerobic conditions, in the presence of either NADH or xanthine as reducing substrate. NO production was directly demonstrated by ozone chemiluminescence and showed stoichiometry of approximately 2:1 versus NADH depletion. With xanthine as reducing substrate, the kinetics of NO production were complicated by enzyme inactivation, resulting from NO-induced conversion of XO to its relatively inactive desulfo-form. Steady-state kinetic parameters were determined spectrophotometrically for urate production and NADH oxidation catalyzed by XO and xanthine dehydrogenase in the presence of nitrite under anaerobic conditions. pH optima for anaerobic NO production catalyzed by XO in the presence of nitrite were 7.0 for NADH and </=6.0 for xanthine. Involvement of the molybdenum site of XO in nitrite reduction was shown by the fact that alloxanthine inhibits xanthine oxidation competitively with nitrite. Strong preference for Mo=S over Mo=O was shown by the relatively very low NADH-nitrite reductase activity shown by desulfo-enzyme. The FAD site of XO was shown not to influence nitrite reduction in the presence of xanthine, although it was clearly involved when NADH was the reducing substrate. Apparent production of NO decreased with increasing oxygen tensions, consistent with reaction of NO with XO-generated superoxide. It is proposed that XO-derived NO fulfills a bactericidal role in the digestive tract.
Highlights
Xanthine dehydrogenase (XDH; EC 1.1.1.204), which predominates in vivo, and xanthine oxidase (XO; EC 1.1.3.22)
Stoichiometry of Nitric oxide (NO) Production and Enzyme Inactivation under Anaerobic Conditions—XO-catalyzed generation of NO in the presence of nitrite could be directly demonstrated by ozone chemiluminescence
The present study examines the kinetics and mechanism of nitrite reduction catalyzed by Xanthine oxidoreductase (XOR) with both NADH and xanthine as reducing substrates and addresses the effects of oxygen
Summary
(Received for publication, September 16, 1999, and in revised form, December 16, 1999). Xanthine oxidase (XO) was shown to catalyze the reduction of nitrite to nitric oxide (NO), under anaerobic conditions, in the presence of either NADH or xanthine as reducing substrate. We examine the kinetics and stoichiometry of NO production catalyzed by XOR in the presence of inorganic nitrite and both NADH and xanthine as reducing substrates under conditions of different oxygen tensions and provide evidence for the sites of action of the various substrates. Each subunit contains one molybdenum, one FAD, and two nonidentical ironsulfur redox centers [1, 2] It has broad specificities for both reducing and oxidizing substrates, its conventionally accepted role is in purine catabolism, where it catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid. § This work is in partial fulfillment of the requirements for a Ph.D. at the University of Bath for this author
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