title: Superoxide induces endothelial nitric-oxide synthase protein thiyl radical formation, a novel mechanism regulating eNOS function and coupling.
abstract: An increase in production of reactive oxygen species resulting in a decrease in nitric oxide bioavailability in the endothelium contributes to many cardiovascular diseases, and these reactive oxygen species can oxidize cellular macromolecules. Protein thiols are critical reducing equivalents that maintain cellular redox state and are primary targets for oxidative modification. We demonstrate endothelial NOS (eNOS) oxidant-induced protein thiyl radical formation from tetrahydrobiopterin-free enzyme or following exposure to exogenous superoxide using immunoblotting, immunostaining, and mass spectrometry. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) followed by immunoblotting using an anti-DMPO antibody demonstrated the formation of eNOS protein radicals, which were abolished by superoxide dismutase and L-NAME, indicating that protein radical formation was due to superoxide generation from the eNOS heme. With tetrahydrobiopterin-reconstituted eNOS, eNOS protein radical formation was completely inhibited. Using mass spectrometric and mutagenesis analysis, we identified Cys-908 as the residue involved in protein radical formation. Mutagenesis of this key cysteine to alanine abolished eNOS thiyl radical formation and uncoupled eNOS, leading to increased superoxide generation. Protein thiyl radical formation leads to oxidation or modification of cysteine with either disulfide bond formation or S-glutathionylation, which induces eNOS uncoupling. Furthermore, in endothelial cells treated with menadione to trigger cellular superoxide generation, eNOS protein radical formation, as visualized with confocal microscopy, was increased, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Thus, eNOS protein radical formation provides the basis for a mechanism of superoxide-directed regulation of eNOS, involving thiol oxidation, defining a unique pathway for the redox regulation of cardiovascular function.
authors.affiliation: Davis Heart and Lung Research Institute and Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Chun-An
authors.initials: CA
authors.lastname: Chen
authors.affiliation: Davis Heart and Lung Research Institute and Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Cho-Hao
authors.initials: CH
authors.lastname: Lin
authors.affiliation: Davis Heart and Lung Research Institute and Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Druhan
authors.affiliation: Davis Heart and Lung Research Institute and Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Tse-Yao
authors.initials: TY
authors.lastname: Wang
authors.affiliation: Davis Heart and Lung Research Institute and Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Yeong-Renn
authors.initials: YR
authors.lastname: Chen
authors.affiliation: Davis Heart and Lung Research Institute and Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, Ohio 43210. Electronic address: Jay.Zweier@osumc.edu.
authors.forename: Jay L
authors.initials: JL
authors.lastname: Zweier
mesh_terms: D000818:Animals; D002417:Cattle; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis; D019331:NG-Nitroarginine Methyl Ester / Q000737:chemistry; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D019097:Spin Trapping / Q000379:methods; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013481:Superoxides / Q000737:chemistry* / Q000378:metabolism
chemical_list: D013438:Sulfhydryl Compounds; D013481:Superoxides; D006418:Heme; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D019331:NG-Nitroarginine Methyl Ester