title: Kinetics of CO ligation with nitric-oxide synthase by flash photolysis and stopped-flow spectrophotometry.
abstract: Interaction of CO with hemeproteins has physiological importance. This is especially true for nitric-oxide synthases (NOS), heme/flavoenzymes that produce .NO and citrulline from L-arginine (Arg) and are inhibited by CO in vitro. The kinetics of CO ligation with both neuronal NOS and its heme domain module were determined in the presence and absence of tetrahydrobiopterin and Arg to allow comparison with other hemeproteins. Geminate recombination in the nanosecond time domain is followed by bimolecular association in the millisecond time domain. Complex association kinetics imply considerable heterogeneity but can be approximated with two forms, one fast (2-3 x 10(6) M-1 s-1) and another slow (2-4 x 10(4) M-1 s-1). The relative proportions of the two forms vary with conditions. For the heme domain, fast forms dominate except in the presence of both tetrahydrobiopterin and Arg. In the holoenzyme, slow forms dominate except when both reagents are absent. Geminate recombination is substantial, approximately 50%, only when fast forms predominate. Stopped-flow mixing found dissociation constants near 0.3 s-1. These data imply an equilibrium constant such that very little CO should bind at physiological conditions unless large CO concentrations are present locally.
authors.affiliation: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Scheele
authors.forename: V G
authors.initials: VG
authors.lastname: Kharitonov
authors.forename: P
authors.initials: P
authors.lastname: Martásek
authors.forename: L J
authors.initials: LJ
authors.lastname: Roman
authors.forename: V S
authors.initials: VS
authors.lastname: Sharma
authors.forename: B S
authors.initials: BS
authors.lastname: Masters
authors.forename: D
authors.initials: D
authors.lastname: Magde
mesh_terms: D000818:Animals; D001120:Arginine / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010782:Photolysis; D051381:Rats; D013053:Spectrophotometry
chemical_list: D006418:Heme; D002248:Carbon Monoxide; D001120:Arginine; D019001:Nitric Oxide Synthase