title: Gating NO release from nitric oxide synthase.
abstract: We have investigated the kinetics of NO escape from Geobacillus stearothermophilus nitric oxide synthase (gsNOS). Previous work indicated that NO release was gated at position 223 in mammalian enzymes; our kinetics experiments include mutants at that position along with measurements on the wild type enzyme. Employing stopped-flow UV-vis methods, reactions were triggered by mixing a reduced enzyme/N-hydroxy-l-arginine complex with an aerated buffer solution. NO release kinetics were obtained for wt NOS and three mutants (H134S, I223V, H134S/I223V). We have confirmed that wt gsNOS has the lowest NO release rate of known NOS enzymes, whether bacterial or mammalian. We also have found that steric clashes at positions 223 and 134 hinder NO escape, as judged by enhanced rates in the single mutants. The empirical rate of NO release from the gsNOS double mutant (H134/I223V) is nearly as rapid as that of the fastest mammalian enzymes, demonstrating that both positions 223 and 134 function as gates for escape of the product diatomic molecule.
authors.affiliation: Beckman Institute and Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: Charlotte A
authors.initials: CA
authors.lastname: Whited
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Warren
authors.forename: Katherine D
authors.initials: KD
authors.lastname: Lavoie
authors.forename: Emily E
authors.initials: EE
authors.lastname: Weinert
authors.forename: Theodor
authors.initials: T
authors.lastname: Agapie
authors.forename: Jay R
authors.initials: JR
authors.lastname: Winkler
authors.forename: Harry B
authors.initials: HB
authors.lastname: Gray
mesh_terms: D020134:Catalytic Domain; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009569:Nitric Oxide; D019001:Nitric Oxide Synthase