Xanthine Oxidase Reaction with Nitric Oxide and Peroxynitrite

Nitric oxide (·NO) and peroxynitrite (ONOO−) inhibit enzymes that depend on metal cofactors or oxidizable amino acids for activity. Since xanthine oxidase (XO) is a 2(2Fe2S) enzyme having essential sulfhydryl groups linked with Mo–pterin cofactor function, the influence of ·NO and ONOO−on purified bovine XO was determined. Physiological (≤1 μM) and supraphysiological (≤100 μM) concentrations of dissolved ·NO gas did not inhibit the catalytic activity or alter the spectral characteristics of XO at 25°C and pH 7.0, differing from reports showing XO inhibition by ·NO. The apparent decrease in XO activity observed previously was the result of depressed rates of uric acid accumulation in XO assay systems, due to ONOO−-mediated oxidation of uric acid upon reaction of residual ·NO with XO-derived superoxide (O•−2). Nitric oxide derived fromS-nitrosoglutathione also did not inhibit cultured vascular endothelial cell XO activity. In contrast, purified and vascular endothelial cell catalase, a heme enzyme reversibly inhibited by ·NO, was inhibited by similar concentrations and rates of production of ·NO. In contrast to ·NO, ONOO−inhibited XO (0.2 μM, 50 mU/ml) with an IC50of 57 μM (for 3 μM/min infusion of ONOO−) or 120 μM (for bolus addition of ONOO−). Addition of 1% bovine serum albumin, 50 μM xanthine, or 10 μM uric acid protected XO from inactivation by ONOO−. Thus, in the presence of purine substrates and other more readily oxidized components of the biological milieu, XO should not be inhibited by either ·NO or ONOO−. These observations reveal that ·NO will not serve as an indirect antioxidant by inhibiting XO-derived production of reactive species and that the XO-derived products O•−2and uric acid readily modify the reactivities of ·NO and ONOO−.