Reduction of nitrite to nitric oxide catalyzed by xanthine oxidoreductase

Xanthine oxidase (XO) was shown to catalyze the reduction of nitrite to nit ric 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 ve rsus NADH depletion. With xanthine as reducing substrate, the kinetics of N O production were complicated by enzyme inactivation, resulting from NO-ind uced conversion of XO to its relatively inactive desulfo-form. Steady-state kinetic parameters were determined spectrophotametrically for urate produc tion and NADH oxidation catalyzed by XO and xanthine dehydrogenase in the p resence of nitrite under anaerobic conditions. pH optima for anaerobic NO p roduction catalyzed by XO in the presence of nitrite were 7.0 for NADH and less than or equal to 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 activ ity 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 invo lved when NADH was the reducing substrate, Apparent production of NO decrea sed with increasing oxygen tensions, consistent with reaction of NO with XO -generated superoxide, It is proposed that XO-derived NO fulfills a bacteri cidal role in the digestive tract.