title: X-ray absorption spectroscopic analysis of the high-spin ferriheme site in substrate-bound neuronal nitric-oxide synthase.
abstract: Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to citrulline and nitric oxide through two stepwise oxygenation reactions involving N(omega)-hydroxy-L-arginine, an enzyme-bound intermediate. The N(omega)-hydroxy-L-arginine- and arginine-bound NOS ferriheme centers show distinct, high-spin electron paramagnetic resonance signals. Iron X-ray absorption spectroscopy (XAS) has been used to examine the structure of the ferriheme site in the N(omega)-hydroxy-L-arginine-bound full-length neuronal NOS in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin. Iron XAS shows that the high-spin ferriheme sites in the N(omega)-hydroxy-L-arginine- and arginine-bound forms are strikingly similar, both being coordinated by the heme and an axial thiolate ligand, with an Fe-S distance of ca. 2.29 A. Cu(2+) inhibition slightly affects the spin-state equilibrium, but causes no XAS-detectable changes in the immediate ferriheme coordination environment of neuronal NOS. The structure and ligand geometry of the high-spin ferriheme in arginine-bound neuronal NOS are essentially identical to those of the N(omega)-hydroxy-L-arginine-bound form and only slightly affected by the divalent cation inhibitor of constitutive NOS.
authors.affiliation: Department of Chemistry, University of Georgia, Athens, GA 30602-2556, USA.
authors.forename: N J
authors.initials: NJ
authors.lastname: Cosper
authors.forename: R A
authors.initials: RA
authors.lastname: Scott
authors.forename: H
authors.initials: H
authors.lastname: Hori
authors.forename: T
authors.initials: T
authors.lastname: Nishino
authors.forename: T
authors.initials: T
authors.lastname: Iwasaki
mesh_terms: D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000494:pharmacology; D003300:Copper / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D013057:Spectrum Analysis / Q000379:methods*; D014965:X-Rays
chemical_list: D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D008024:Ligands; D001708:Biopterins; D009569:Nitric Oxide; D006418:Heme; C068309:N(omega)-hydroxyarginine; D003300:Copper; D001120:Arginine; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; C003402:sapropterin