title: Catalytic reduction of a tetrahydrobiopterin radical within nitric-oxide synthase.
abstract: Nitric-oxide synthases (NOS) are catalytically self-sufficient flavo-heme enzymes that generate NO from arginine (Arg) and display a novel utilization of their tetrahydrobiopterin (H(4)B) cofactor. During Arg hydroxylation, H(4)B acts as a one-electron donor and is then presumed to redox cycle (i.e. be reduced back to H(4)B) within NOS before further catalysis can proceed. Whereas H(4)B radical formation is well characterized, the subsequent presumed radical reduction has not been demonstrated, and its potential mechanisms are unknown. We investigated radical reduction during a single turnover Arg hydroxylation reaction catalyzed by neuronal NOS to document the process, determine its kinetics, and test for involvement of the NOS flavoprotein domain. We utilized a freeze-quench instrument, the biopterin analog 5-methyl-H(4)B, and a method that could separately quantify the flavin and pterin radicals that formed in NOS during the reaction. Our results establish that the NOS flavoprotein domain catalyzes reduction of the biopterin radical following Arg hydroxylation. The reduction is calmodulin-dependent and occurs at a rate that is similar to heme reduction and fast enough to explain H(4)B redox cycling in NOS. These results, in light of existing NOS crystal structures, suggest a "through-heme" mechanism may operate for H(4)B radical reduction in NOS.
authors.affiliation: Department of Pathobiology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA. cwei@siue.edu
authors.forename: Chin-Chuan
authors.initials: CC
authors.lastname: Wei
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Wang
authors.forename: Jesús
authors.initials: J
authors.lastname: Tejero
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Yang
authors.forename: Craig
authors.initials: C
authors.lastname: Hemann
authors.forename: Russ
authors.initials: R
authors.lastname: Hille
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry; D002384:Catalysis; D018360:Crystallography, X-Ray / Q000379:methods; D004578:Electron Spin Resonance Spectroscopy; D005415:Flavins / Q000737:chemistry; D005609:Free Radicals; D007700:Kinetics; D009569:Nitric Oxide / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011622:Pterins / Q000737:chemistry; D051381:Rats
chemical_list: D005415:Flavins; D005609:Free Radicals; D011622:Pterins; D001708:Biopterins; D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase; C003402:sapropterin