title: Human S-nitroso oxymyoglobin is a store of vasoactive nitric oxide.
abstract: Nitric oxide (.NO) regulates vascular function, and myoglobin (Mb) is a heme protein present in skeletal, cardiac, and smooth muscle, where it facilitates O(2) transfer. Human ferric Mb binds .NO to yield nitrosylheme and S-nitroso (S-NO) Mb (Witting, P. K., Douglas, D. J., and Mauk, A. G. (2001) J. Biol. Chem. 276, 3991-3998). Here we show that human ferrous oxy-myoglobin (oxyMb) oxidizes .NO, with a second order rate constant k = 2.8 +/- 0.1 x 10(7) M(-1).s(-1) as determined by stopped-flow spectroscopy. Mixtures containing oxyMb and S-nitrosoglutathione or S-nitrosocysteine added at 1.5-2 moles of S-nitrosothiol/mol oxyMb yielded S-NO oxyMb through trans-nitrosation equilibria as confirmed with mass spectrometry. Rate constants for the equilibrium reactions were k(forward) = 110 +/- 3 and k(reverse) = 16 +/- 3 M(-1).s(-1) for S-nitrosoglutathione and k(forward) = 293 +/- 5 and k(reverse) = 20 +/- 2 M(-1).s(-1) for S-nitrosocysteine. Incubation of S-NO oxyMb with Cu(2+) ions stimulated .NO release as measured with a .NO electrode. Similarly, Cu(2+) released .NO from Mb immunoprecipitated from cultured human vascular smooth muscle cells (VSMCs) that were pre-treated with diethylaminenonoate. No .NO release was observed from VSMCs treated with vehicle alone or immunoprecipitates obtained from porcine aortic endothelial cells with and without diethylaminenonoate treatment. Importantly, pre-constricted aortic rings relaxed in the presence of S-NO oxyMb in a cyclic GMP-dependent process. These data indicate that human oxyMb rapidly oxidizes .NO and that biologically relevant S-nitrosothiols can trans-(S)nitrosate human oxyMb. Furthermore, S-NO oxyMb can be isolated from cultured human VSMCs exposed to an exogenous .NO donor at physiologic concentration. The potential biologic implications of S-NO oxyMb acting as a source of .NO are discussed.
authors.affiliation: Centre for Vascular Research and Biomedical Mass Spectrometry Unit, University of New South Wales, Sydney 2052, New South Wales, Australia.
authors.forename: Benjamin S
authors.initials: BS
authors.lastname: Rayner
authors.forename: Ben-Jing
authors.initials: BJ
authors.lastname: Wu
authors.forename: Mark
authors.initials: M
authors.lastname: Raftery
authors.forename: Roland
authors.initials: R
authors.lastname: Stocker
authors.forename: Paul K
authors.initials: PK
authors.lastname: Witting
mesh_terms: D000818:Animals; D001011:Aorta / Q000473:pathology; D002478:Cells, Cultured; D003300:Copper / Q000737:chemistry; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D004566:Electrodes; D004583:Electrons; D004730:Endothelium, Vascular / Q000166:cytology; D006801:Humans; D047468:Immunoprecipitation; D007700:Kinetics; D008956:Models, Chemical; D009130:Muscle, Smooth / Q000166:cytology; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D009584:Nitrogen / Q000737:chemistry*; D010100:Oxygen / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D026422:S-Nitrosoglutathione / Q000737:chemistry; D026403:S-Nitrosothiols / Q000737:chemistry; D013053:Spectrophotometry; D013997:Time Factors
chemical_list: D009211:Myoglobin; D011994:Recombinant Proteins; D026403:S-Nitrosothiols; C017595:oxymyoglobin; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D003300:Copper; C021315:S-nitrosocysteine; D003545:Cysteine; D009584:Nitrogen; D010100:Oxygen