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Human inducible nitric oxide synthase (iNOS) is active as a dimer of two identical subunits. Each subunit has an amino-terminal oxygenase domain that binds the substrate l-Arg and the cofactors heme and tetrahydrobiopterin and a carboxyl-terminal reductase domain that binds FMN, FAD, and NADPH. We previously demonstrated that a subdomain in the oxygenase domain encoded by exons 8 and 9 is important for dimer formation and NO synthesis. Further, we 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 H(2)O(2)-supported oxidation of N(omega)-hydroxy-l-Arg, we demonstrate that the iNOS mutants' inabilities to synthesize NO are due to selective defects in the oxygenase domain activity. Detailed characterization of the Asp-280-Ala mutant revealed that it retains a functional reductase domain, as measured by its ability to reduce cytochrome c. Gel permeation chromatography confirmed that the Asp-280-Ala mutant exists as a dimer, but, in contrast to wild-type iNOS, urea-generated monomers of the mutant fail to reassociate into dimers when incubated with l-Arg and tetrahydrobiopterin, suggesting inadequate subunit interaction. Spectral analysis reveals that the Asp-280-Ala mutant does not bind l-Arg. This indicates that, in addition to dimerization, proper subunit interaction is required for substrate binding. These data, by defining a critical role for Asp-280 in substrate binding and subunit interactions, give insights into the mechanisms of regulation of iNOS activity.

proceedings of the national academy of sciences of the united states of america

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