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J. Biol. Chem., Vol. 280, Issue 11, 9985-9993, March 18, 2005

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Human S-Nitroso Oxymyoglobin Is a Store of Vasoactive Nitric Oxide^*

Benjamin S. Rayner, Ben-Jing Wu, Mark Raftery¶, Roland Stocker, and Paul K. Witting||

From the Centre for Vascular Research and ^¶Biomedical Mass Spectrometry Unit, University of New South Wales, Sydney 2052, New South Wales, Australia and Vascular Biology Group, ANZAC Research Institute, Concord Repatriation General Hospital, Concord 2139, New South Wales, Australia

Nitric oxide (·NO) regulates vascular function, and myoglobin (Mb) is a heme protein present in skeletal, cardiac, and smooth muscle, where it facilitates O₂ 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 10⁷ 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 Cu²⁺ ions stimulated ·NO release as measured with a ·NO electrode. Similarly, Cu²⁺ 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.

Received for publication, September 14, 2004 , and in revised form, January 6, 2005.

^* This work was supported by Australian Research Fellowship DP034325 (to P. K. W.) from the Australian Research Council and Program Grant 222722 and Senior Principal Research Fellowship 151602 (to R. S.) from the National Health and Medical Research Council of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: ANZAC Research Institute, Hospital Rd., Concord Repatriation General Hospital, Concord 2139, New South Wales, Australia. Tel.: 61-2-9767-9103; Fax: 61-2-9767-9101; E-mail: pwitting{at}anzac.edu.au.

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