title: Kinetics of NO ligation with nitric-oxide synthase by flash photolysis and stopped-flow spectrophotometry.
abstract: Nitric-oxide synthase (NOS) catalyzes conversion of L-arginine to nitric oxide, which subsequently stimulates a host of physiological processes. Prior work suggests that NOS is inhibited by NO, providing opportunities for autoregulation. This contribution reports that NO reacts rapidly (ka congruent with 2 x 10(7) M-1 s-1) with neuronal NOS in both its ferric and ferrous oxidation states. Association kinetics are almost unaffected by L-arginine or the cofactor tetrahydrobiopterin. There is no evidence for the distinct two phases previously reported for association kinetics of CO. Small amounts of geminate recombination of NO trapped in a protein pocket can be observed over nanoseconds, and a much larger amount is inferred to take place at picosecond time scales. Dissociation rates are also very fast from the ferric form, in the neighborhood of 50 s-1, when measured by extrapolating association rates to the zero NO concentration limit. Scavenging experiments give dissociation rate constants more than an order of magnitude slower: still quite fast. For the ferrous species, extrapolation is not distinguishable from zero, while scavenging experiments give a dissociation rate constant near 10(-4) s-1. Implications of these results for interactions near the heme binding site are discussed.
authors.affiliation: Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093-0358, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Scheele
authors.forename: E
authors.initials: E
authors.lastname: Bruner
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: B S
authors.initials: BS
authors.lastname: Masters
authors.forename: V S
authors.initials: VS
authors.lastname: Sharma
authors.forename: D
authors.initials: D
authors.lastname: Magde
mesh_terms: D000818:Animals; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010782:Photolysis; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013057:Spectrum Analysis
chemical_list: D011994:Recombinant Proteins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat