Nitric oxide (NO) is synthesized from L-Arg via N-G-hydroxyl-L-Arg (NHA) in the heme active site of nitric oxide synthase (NOS). According to the crystal structure of other NOS isoforms, the carboxylate group of L-Arg hydrogen bonds to the hydroxyl group of the conserved Tyr588 residue in the heme distal site of neuronal NOS (nNOS). Indeed, the nNOS mutations Tyr588His, Tyr588Ser, and Tyr588Phe markedly increased the dissociation constants for L-Arg and NHA by 2.2-8.2-fold and 1.5-3.9-fold, respectively. Similarly, Tyr588His and Tyr588Ser mutations markedly decreased the L-Arg-driven NO formation rates by 50 and 30% than that of the wild type, respectively. However, the catalytic activities of the same mutants using NHA were higher than that of the wild type by up to 136%. As a result, the turnover ratio of NHA to L-Arg was 4.12 for the Tyr588Ser mutant, compared with 1.07 for the wild-type enzyme. Intriguingly, heme reduction rates for the Tyr588 mutants were much lower than for wild type by two orders of magnitude.