XANTHINE-OXIDASE REACTION WITH NITRIC-OXIDE AND PEROXYNITRITE

Nitric oxide (. NO) and peroxynitrite (ONOO-) inhibit enzymes that dep end on metal cofactors or oxidizable amino acids for activity. Since x anthine 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 (less th an or equal to 1 mu M) and supraphysiological (less than or equal to 1 00 mu M) concentrations of dissolved . NO gas did not inhibit the cata lytic activity or alter the spectral characteristics of XO at 25 degre es C and pH 7.0, differing from reports showing XO inhibition by . NO. The apparent decrease in XO activity observed previously was the resu lt of depressed rates of uric acid accumulation in XO assay systems, d ue to ONOO--mediated oxidation of uric acid upon reaction of residual . NO with XO-derived superoxide (O-2(.-)). Nitric oxide derived from S -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 b y similar concentrations and rates of production of . NO. In contrast to . NO, ONOO- inhibited XO (0.2 mu M, 50 mU/ml) with an IC50 of 57 mu M (for 3 mu M/min infusion of ONOO-) or 120 mu M (for bolus addition of ONOO-). Addition of 1% bovine serum albumin, 50 mu M xanthine, or 1 0 mu M uric acid protected XO from inactivation by ONOO-. Thus, in the presence of purine substrates and other more readily oxidized compone nts of the biological milieu, XO should not be inhibited by either . N O or ONOO-. These observations reveal that . NO will not serve as an i ndirect antioxidant by inhibiting XO-derived production of reactive sp ecies and that the XO-derived products O-2(.-) and uric acid readily m odify the reactivities of . NO and ONOO-. (C) 1998 Academic Press.