title: 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.
abstract: 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.
authors.affiliation: Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA. dgx@acpub.duke.edu
authors.forename: D K
authors.initials: DK
authors.lastname: Ghosh
authors.forename: M B
authors.initials: MB
authors.lastname: Rashid
authors.forename: B
authors.initials: B
authors.lastname: Crane
authors.forename: V
authors.initials: V
authors.lastname: Taskar
authors.forename: M
authors.initials: M
authors.lastname: Mast
authors.forename: M A
authors.initials: MA
authors.lastname: Misukonis
authors.forename: J B
authors.initials: JB
authors.lastname: Weinberg
authors.forename: N T
authors.initials: NT
authors.lastname: Eissa
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry; D020134:Catalytic Domain; D002460:Cell Line; D004252:DNA Mutational Analysis; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D001224:Aspartic Acid; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II