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.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Ghosh, DK; Rashid, MB; Crane, B; Taskar, V; Mast, M; Misukonis, MA; Weinberg, JB; Eissa, NT

Published Date

  • August 28, 2001

Published In

Volume / Issue

  • 98 / 18

Start / End Page

  • 10392 - 10397

PubMed ID

  • 11517317

Pubmed Central ID

  • PMC56971

International Standard Serial Number (ISSN)

  • 0027-8424

Digital Object Identifier (DOI)

  • 10.1073/pnas.181251298


  • eng

Conference Location

  • United States