title: Mutational analysis of the tetrahydrobiopterin-binding site in inducible nitric-oxide synthase.
abstract: Inducible nitric-oxide synthase (iNOS) is a hemeprotein that requires tetrahydrobiopterin (H4B) for activity. The influence of H4B on iNOS structure-function is complex, and its exact role in nitric oxide (NO) synthesis is unknown. Crystal structures of the mouse iNOS oxygenase domain (iNOSox) revealed a unique H4B-binding site with a high degree of aromatic character located in the dimer interface and near the heme. Four conserved residues (Arg-375, Trp-455, Trp-457, and Phe-470) engage in hydrogen bonding or aromatic stacking interactions with the H4B ring. We utilized point mutagenesis to investigate how each residue modulates H4B function. All mutants contained heme ligated to Cys-194 indicating no deleterious effect on general protein structure. Ala mutants were monomers except for W457A and did not form a homodimer with excess H4B and Arg. However, they did form heterodimers when paired with a full-length iNOS subunit, and these were either fully or partially active regarding NO synthesis, indicating that preserving residue identities or aromatic character is not essential for H4B binding or activity. Aromatic substitution at Trp-455 or Trp-457 generated monomers that could dimerize with H4B and Arg. These mutants bound Arg and H4B with near normal affinity, but Arg could not displace heme-bound imidazole, and they had NO synthesis activities lower than wild-type in both homodimeric and heterodimeric settings. Aromatic substitution at Phe-470 had no significant effects. Together, our work shows how hydrogen bonding and aromatic stacking interactions of Arg-375, Trp-457, Trp-455, and Phe-470 influence iNOSox dimeric structure, heme environment, and NO synthesis and thus help modulate the multiple effects of H4B.
authors.affiliation: Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
authors.forename: S
authors.initials: S
authors.lastname: Ghosh
authors.forename: D
authors.initials: D
authors.lastname: Wolan
authors.forename: S
authors.initials: S
authors.lastname: Adak
authors.forename: B R
authors.initials: BR
authors.lastname: Crane
authors.forename: N S
authors.initials: NS
authors.lastname: Kwon
authors.forename: J A
authors.initials: JA
authors.lastname: Tainer
authors.forename: E D
authors.initials: ED
authors.lastname: Getzoff
authors.forename: D J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D019281:Dimerization; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D017354:Point Mutation; D013329:Structure-Activity Relationship
chemical_list: D001708:Biopterins; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C003402:sapropterin