title: Rapid kinetic studies link tetrahydrobiopterin radical formation to heme-dioxy reduction and arginine hydroxylation in inducible nitric-oxide synthase.
abstract: To understand how heme and (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)B) participate in nitric-oxide synthesis, we followed ferrous-dioxy heme (Fe(II)O(2)) formation and disappearance, H(4)B radical formation, and Arg hydroxylation during a single catalytic turnover by the inducible nitric-oxide synthase oxygenase domain (iNOSoxy). In all cases, prereduced (ferrous) enzyme was rapidly mixed with an O(2)-containing buffer to start the reaction. A ferrous-dioxy intermediate formed quickly (53 s(-1)) and then decayed with concurrent buildup of ferric iNOSoxy. The buildup of the ferrous-dioxy intermediate preceded both H(4)B radical formation and Arg hydroxylation. However, the rate of ferrous-dioxy decay (12 s(-1)) was equivalent to the rate of H(4)B radical formation (11 s(-1)) and the rate of Arg hydroxylation (9 s(-1)). Practically all bound H(4)B was oxidized to a radical during the reaction and was associated with hydroxylation of 0.6 mol of Arg/mol of heme. In dihydrobiopterin-containing iNOSoxy, ferrous-dioxy decay was much slower and was not associated with Arg hydroxylation. These results establish kinetic and quantitative links among ferrous-dioxy disappearance, H(4)B oxidation, and Arg hydroxylation and suggest a mechanism whereby H(4)B transfers an electron to the ferrous-dioxy intermediate to enable the formation of a heme-based oxidant that rapidly hydroxylates Arg.
authors.affiliation: Department of Immunology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: C C
authors.initials: CC
authors.lastname: Wei
authors.forename: Z Q
authors.initials: ZQ
authors.lastname: Wang
authors.forename: Q
authors.initials: Q
authors.lastname: Wang
authors.forename: A L
authors.initials: AL
authors.lastname: Meade
authors.forename: C
authors.initials: C
authors.lastname: Hemann
authors.forename: R
authors.initials: R
authors.lastname: Hille
authors.forename: D J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D004789:Enzyme Activation; D005609:Free Radicals / Q000378:metabolism; D006418:Heme / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D006900:Hydroxylation; D007700:Kinetics; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D019163:Reducing Agents / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: D005609:Free Radicals; D016877:Oxidants; D011994:Recombinant Proteins; D019163:Reducing Agents; D001708:Biopterins; D009569:Nitric Oxide; D006418:Heme; D001120:Arginine; D010105:Oxygenases; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C003402:sapropterin; D010100:Oxygen