Characterization of key residues in the subdomain encoded by exons 8 and 9of human inducible nitric oxide synthase: A critical role for Asp-280 in substrate binding and subunit interactions

Human inducible nitric oxide synthase (iNOS) is active as a dimer of two id entical subunits. Each subunit has an amino-terminal oxygenase domain that binds the substrate L-Arg and the cofactors heme and tetrahydrobiopterin an d a carboxyl-terminal reductase domain that binds FMN, FAD, and NADPH. We p reviously demonstrated that a subdomain in the oxygenase domain encoded by exons 8 and 9 is important for dimer formation and NO synthesis. Further, w e identified Trp-260, Asn-261, Tyr-267, and Asp-280 as key residues in that subdomain. In this study, using an Escherichia coli expression system, we produced, purified, and characterized wild-type iNOS and iNOS-Ala mutants. Using H2O2-supported oxidation of N-omega-hydroxy-L-Arg, we demonstrate tha t the NOS mutants' inabilities to synthesize NO are due to selective defect s in the oxygenase domain activity. Detailed characterization of the Asp-28 0-Ala mutant revealed that it retains a functional reductase domain, as mea sured by its ability to reduce cytochrome c. Gel permeation chromatography confirmed that the Asp-280-Ala mutant exists as a dimer, but, in contrast t o wild-type NOS, urea-generated monomers of the mutant fail to reassociate into dimers when incubated with L-Arg and tetrahydrobiopterin, suggesting i nadequate subunit interaction. Spectral analysis reveals that the Asp-280-A la mutant does not bind L-Arg. This indicates that, in addition to dimeriza tion, proper subunit interaction is required for substrate binding. These d ata, by defining a critical role for Asp-280 in substrate binding and subun it interactions, give insights into the mechanisms of regulation of NOS act ivity.