title: Tetrahydrobiopterin redox cycling in nitric oxide synthase: evidence supports a through-heme electron delivery.
abstract: The nitric oxide synthases (NOS) catalyze a two-step oxidation of l-arginine (Arg) to generate NO. In the first step, O2activation involves one electron being provided to the heme by an enzyme-bound 6R-tetrahydro-l-biopterin cofactor (H4B), and the H4B radical must be reduced back to H4B in order for NOS to continue catalysis. Although an NADPH-derived electron is used to reduce the H4B radical, how this occurs is unknown. We hypothesized that the NOS flavoprotein domain might reduce the H4B radical by utilizing the NOS heme porphyrin as a conduit to deliver the electron. This model predicts that factors influencing NOS heme reduction should also influence the extent and rate of H4B radical reduction in kind. To test this, we utilized single catalytic turnover and stop-freeze methods, along with electron paramagnetic resonance spectroscopy, to measure the rate and extent of reduction of the 5-methyl-H4B radical formed in neuronal NOS (nNOS) during Arg hydroxylation. We used several nNOS variants that supported either a slower or faster than normal rate of ferric heme reduction. We found that the rates and extents of nNOS heme reduction correlated well with the rates and extents of 5-methyl-H4B radical reduction among the various nNOS enzymes. This supports a model where the heme porphyrin transfers an electron from the NOS flavoprotein to the H4B radical formed during catalysis, revealing that the heme plays a dual role in catalyzing O2activation or electron transfer at distinct points in the reaction cycle.
authors.affiliation: Department of Pathobiology, Lerner Research Institute, The Cleveland Clinic, OH, USA.
authors.forename: Somasundaram
authors.initials: S
authors.lastname: Ramasamy
authors.affiliation: Department of Pathobiology, Lerner Research Institute, The Cleveland Clinic, OH, USA.
authors.forename: Mohammad Mahfuzul
authors.initials: MM
authors.lastname: Haque
authors.affiliation: Department of Chemistry and Biochemistry, Kent State University, OH, USA.
authors.forename: Mahinda
authors.initials: M
authors.lastname: Gangoda
authors.affiliation: Department of Pathobiology, Lerner Research Institute, The Cleveland Clinic, OH, USA.
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004583:Electrons; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D000072417:Protein Domains; D051381:Rats
chemical_list: D005609:Free Radicals; D001708:Biopterins; D006418:Heme; D001120:Arginine; D052248:Nitric Oxide Synthase Type I; C003402:sapropterin
keywords: biopterin; electron paramagnetic resonance; electron transfer; flavoprotein; freeze quench; heme porphyrin; heme reduction; neuronal NOS