The Crucial Roles of Asp-314 and Thr-315 in the Catalytic Activation of Molecular Oxygen by Neuronal Nitric-oxide Synthase

Nitric-oxide synthase (NOS) is a flavohemoprotein that has a cytochrome P450 (P450)-type heme active site and catalyzes the monooxygenation of L-Arg to NG-hydroxy-L-Arg (NHA) according to the normal P450-type reaction in the first step of NO synthesis. However, there is some controversy as to how the second step of the reaction, from NHA to NO and L-citrulline, occurs within the P450 domain of NOS. By referring to the heme active site of P450, it is conjectured that polar amino acid(s) such as Asp/Glu and Thr must be responsible for the activation of molecular oxygen in NOS. In this study, we have created Asp-314-->Ala and Thr-315-->Ala mutants of neuronal NOS, both of which had absorption maxima at 450 nm in the spectra of the CO-reduced complexes and studied NO formation rates and other kinetic parameters as well as the substrate binding affinity. The Asp-314-->Ala mutant totally abolished NO formation activity and markedly increased the rate of H2O2 formation by 20-fold compared with the wild type when L-Arg was used as the substrate. The NADPH oxidation and O2 consumption rates for the Asp-314-->Ala mutant were 60-65% smaller than for the wild type. The Thr-315-->Ala mutant, on the other hand, retained NO formation activity that was 23% higher than the wild type, but like the Asp-314-->Ala mutation, markedly increased the H2O2 formation rate. The NADPH oxidation and O2 consumption rates for the Thr-315-->Ala mutant were, respectively, 56 and 27% higher than for the wild type. When NHA was used as the substrate, similar values were obtained. Thus, we propose that Asp-314 is crucial for catalysis, perhaps through involvement in the stabilization of an oxygen-bound intermediate. An important role for Thr-315 in the catalysis is also suggested.