title: Distal Val346Ile mutation in inducible NO synthase promotes substrate-dependent NO confinement.
abstract: The function of inducible NO synthase (WT iNOS) depends on the release of NO from the ferric heme before the enzyme is reduced. Key parameters controlling ligand dynamics include the distal and proximal heme pocket amino acids, as well as the inner solvent molecules. In this work, we tested how a point mutation in the distal heme side of WT iNOS affected the geminate rebinding of NO by ultrafast kinetics and molecular dynamics simulations. The mutation sequestered much of the photodissociated NO close to the heme compared to WT iNOS, with a main picosecond phase accounting for 78% of the rebinding to the arginine-bound Val346Ile protein. Consequently, the probability of NO release from Val346Ile decreased as compared to that from WT iNOS, provided the substrate binding site is filled. These data are rationalized by a steric effect of the Ile methyl group inducing events mediated by the substrate, transmitted via the propionates to the NO and the protein. This model is consistent with the role of the H-bonding network involving the heme, the substrate, and the BH4 cofactor in controlling NO release, with a key role of the heme propionates [Gautier et al. (2006) Nitric Oxide 15, 312]. These data support the effect of Val346Ile mutation in decreasing NO release and slowing down NO synthesis compared to WT iNOS determined by single turnover catalysis [Wang et al. (2004) J. Biol. Chem. 279, 19018].
authors.affiliation: INSERM U696, Laboratory of Optics and Biosciences, CNRS UMR 7645, Ecole Polytechnique, 91128 Palaiseau, France.
authors.forename: Edward
authors.initials: E
authors.lastname: Beaumont
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Lambry
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Wang
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Martin
authors.forename: Anny
authors.initials: A
authors.lastname: Slama-Schwok
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D006860:Hydrogen Bonding; D007532:Isoleucine / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D014633:Valine / Q000737:chemistry* / Q000235:genetics
chemical_list: D007532:Isoleucine; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D014633:Valine