title: Kinetics of CO binding to the haem domain of murine inducible nitric oxide synthase: differential effects of haem domain ligands.
abstract: The binding of CO to the murine inducible nitric oxide synthase (iNOS) oxygenase domain has been studied by laser flash photolysis. The effect of the (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)) cofactor L-arginine and several Type I L-arginine analogues/ligands on the rates of CO rebinding has been evaluated. The presence of BH(4) in the iNOS active site has little effect on the rebinding of protein-caged haem-CO pairs (geminate recombination), but decreases the bimolecular association rates 2-fold. Addition of L-arginine to the BH(4)-bound complex completely abolishes geminate recombination and results in a further 80-fold decrease in the overall rate of bimolecular association. Three of the Type I ligands, S-ethylisothiourea, L-canavanine and 2,5-lutidine, displaced the CO from the haem iron upon addition to the iNOS oxygenase domain. The Type I ligands significantly decreased the rate of bimolecular binding of CO to the haem iron after photolysis. Most of these ligands also completely abolished geminate recombination. These results are consistent with a relatively open distal pocket that allows CO to bind unhindered in the active site of murine iNOS in the absence of L-arginine or BH(4). In the presence of BH(4) and L-arginine, however, the enzyme adopts a more closed structure that can greatly reduce ligand access to the haem iron. These observations are discussed in terms of the known structure of iNOS haem domain and solution studies of ligand binding in iNOS and neuronal NOS isoenzymes.
authors.affiliation: Biological NMR Centre, Medical Sciences Building, University of Leicester, Leicester LE1 9HN, UK.
authors.forename: T H
authors.initials: TH
authors.lastname: Stevenson
authors.forename: A F
authors.initials: AF
authors.lastname: Gutierrez
authors.forename: W K
authors.initials: WK
authors.lastname: Alderton
authors.forename: L
authors.initials: L
authors.lastname: Lian
authors.forename: N S
authors.initials: NS
authors.lastname: Scrutton
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry; D002248:Carbon Monoxide / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D008027:Light; D051379:Mice; D008956:Models, Chemical; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010782:Photolysis; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic; D013053:Spectrophotometry; D013997:Time Factors
chemical_list: D008024:Ligands; D020033:Protein Isoforms; D011994:Recombinant Proteins; D006418:Heme; D002248:Carbon Monoxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse