In addition to
nitric oxide (NO) generation from specific NO synthases, NO is also formed during
anoxia from
nitrite reduction, and
xanthine oxidase (XO) catalyzes this process. While in tissues and blood high
nitrate levels are present, questions remain regarding whether
nitrate is also a source of NO and if XO-mediated
nitrate reduction can be an important source of NO in biological systems. To characterize the kinetics, magnitude, and mechanism of XO-mediated
nitrate reduction under anaerobic conditions, EPR, chemiluminescence NO-analyzer, and NO-
electrode studies were performed. Typical XO reducing substrates,
xanthine,
NADH, and 2,3-dihydroxybenz-aldehyde, triggered
nitrate reduction to
nitrite and NO. The rate of
nitrite production followed Michaelis-Menten kinetics, while NO generation rates increased linearly following the accumulation of
nitrite, suggesting stepwise-reduction of
nitrate to
nitrite then to NO. The
molybdenum-binding XO inhibitor,
oxypurinol, inhibited both
nitrite and NO production, indicating that
nitrate reduction occurs at the
molybdenum site. At higher
xanthine concentrations, partial inhibition was seen, suggesting formation of a substrate-bound reduced
enzyme complex with
xanthine blocking the
molybdenum site. The pH dependence of
nitrite and NO formation indicate that XO-mediated
nitrate reduction occurs via an
acid-catalyzed mechanism. With conditions occurring during
ischemia, myocardial xanthine oxidoreductase and
nitrate levels were determined to generate up to 20 microM
nitrite within 10-20 min that can be further reduced to NO with rates comparable to those of maximally activated NOS. Thus, XOR catalyzed
nitrate reduction to
nitrite and NO occurs and can be an important source of NO production in ischemic tissues.