title: Role of an isoform-specific substrate access channel residue in CO ligand accessibilities of neuronal and inducible nitric oxide synthase isoforms.
abstract: The rates of the bimolecular CO rebinding to the oxygenase domains of inducible and neuronal NOS proteins (iNOSoxy and nNOSoxy, respectively) after photolytic dissociation have been determined by laser flash photolysis. The following mutants at the isoform-specific sites (murine iNOSoxy N115L and rat nNOSoxy L337N, L337F) have been constructed to investigate role of the residues in the CO ligand accessibilities of the NOS isoforms. These residues are in the NOS distal substrate access channel. The effect of the (6R)-5,6,7,8-tetrahydrobiopterin (H(4)B) cofactor and l-arginine (Arg) substrate on the rates of CO rebinding have also been assessed. Addition of l-Arg to the iNOSoxy N115L mutant results in much faster CO rebinding rates, compared to the wild type. The results indicate that modifications to the iNOS channel in which the hydrophilic residue N115 is replaced by leucine (to resemble its nNOS cognate) open the channel somewhat, thereby improving access to the axial heme ligand binding position. On the other hand, introduction of a hydrophilic residue (L337N) or a bulky rigid aromatic residue (L337F) in the nNOS isoform does not significantly affect the kinetics profile, suggesting that the geometry of the substrate access pocket is not greatly altered. The bimolecular CO rebinding rate data indicate that the opening of the substrate access channel in the iNOS N115L mutant may be due to more widespread structural alterations induced by the mutation.
authors.affiliation: College of Pharmacy, University of New Mexico, Albuquerque, NM 87131, USA. cfeng@salud.unm.edu
authors.forename: Changjian
authors.initials: C
authors.lastname: Feng
authors.forename: Weihong
authors.initials: W
authors.lastname: Fan
authors.forename: Dipak K
authors.initials: DK
authors.lastname: Ghosh
authors.forename: Gordon
authors.initials: G
authors.lastname: Tollin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D051379:Mice; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry* / Q000235:genetics; D010105:Oxygenases / Q000737:chemistry; D010782:Photolysis; D051381:Rats
chemical_list: D008024:Ligands; D001120:Arginine; D010105:Oxygenases; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse