The crucial roles of Asp-314 and Thr-315 in the catalytic activation of molecular oxygen by neuronal nitric-oxide synthase - A site-directed mutagenesis study

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 N-G-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 GO-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 O-2, 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 H,O, formation rate, The NADPH oxidation and O-2, 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.