N omega-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from L-arginine

Authentic N omega-hydroxy-L-arginine was synthesized and used to determine whether it is an intermediate in nitric oxide (.NO) synthesis from L-arginine by macrophage .NO synthase. The apparent Km (6.6 microM) and Vmax (99 nmol x min-1 x mg-1) observed with N omega-hydroxy-L-arginine were similar to those observed with L-arginine (Km = 2.3 microM; Vmax = 54 mumol x min-1 x mg-1). N omega-Hydroxy-D-arginine was not a substrate. Stable isotope studies showed that .NO synthase exclusively oxidized the hydroxylated nitrogen of N omega-hydroxy-L-arginine, forming .NO and L-citrulline. As with L-arginine, O2 was the source of the ureido oxygen in L-citrulline from N omega-hydroxy-L-arginine. In the presence of excess N omega-hydroxy-L-arginine, .NO synthase generated a metabolite of L-[14C]arginine that cochromatographed with authentic N omega-hydroxy-L-arginine. The labeled metabolite exhibited identical chromatographic behavior in three solvent systems and generated the same product (L-citrulline) upon alkaline hydrolysis as authentic N omega-hydroxy-L-arginine. Experiments were then run to identify which redox cofactor (NADPH or tetrahydrobiopterin) participated in the enzymatic synthesis of N omega-hydroxy-L-arginine. Both cofactors were required for synthesis of .NO from either N omega-hydroxy-L-arginine or L-arginine. However, with L-arginine, the synthesis of 1 mol of .NO was coupled to the oxidation of 1.52 +/- 0.02 mol of NADPH; whereas with N omega-hydroxy-L-arginine, only 0.53 +/- 0.04 mol of NADPH was oxidized per mol of .NO formed. These results support a mechanism in which N omega-hydroxy-L-arginine is generated as an intermediate in .NO synthesis through an NADPH-dependent hydroxylation of L-arginine.