Differential effects of mutations in human endothelial nitric oxide synthase at residues Tyr-357 and Arg-365 on l-arginine hydroxylation and [formula omitted]-hydroxy- l-arginine oxidation

Abstract

Biosynthesis of nitric oxide (NO) is catalyzed by NO synthase (NOS) through a two-step oxidation of l-arginine (Arg) with formation of an intermediate, GN -hydroxy- l-Arg (NHA). In this study we have employed mutagenesis to investigate how residues Y357 and R365 which interact primarily with the substrate Arg and (6R)-5,6,7,8-tetrahydro- l-biopterin (H 4B) modulate these two steps of the NOS reaction. Mutant Y357F preserved most wild-type heme characteristics and NADPH oxidation ability. However, mutation of this residue markedly increased the dissociation constants for both Arg and NHA by 20-fold and decreased the NO synthesis from Arg by 85% compared to that of wild type. Mutation of Y357 had less effect on the rate of NO generated from NHA. Mutant R365L purified in the presence of Arg had a normal heme environment and retained 9 and 55% of the wild-type NO formation rate from Arg and NHA, respectively. When Arg was removed from buffer, R365L instantly became a low-spin state (Soret peak at 418 nm) with the resultant loss of H 4B and instability of the heme–CO complex. The low-spin R365L exhibited an NADPH oxidation rate higher than that of wild type. Its Arg-driven NO formation was decreased to near the limit of detection, whereas the rate of NHA-driven NO synthesis was one third that of wild type. This NHA-driven NO formation completely relied on H 4B and was not sensitive to superoxide dismutase or catalase but was inhibited by imidazole. The wild-type eNOS required 14 μM NHA and 0.39 μM H 4B to reach the half-maximal NHA-driven NO formation rate (EC 50), while R365L needed 59 μM NHA and 0.73 μM H 4B to achieve EC 50. The differential effect of mutation on Arg and NHA oxidation suggests that distinct heme-based active oxidants are responsible for each step of NO synthesis.