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 O2 transfer. Human ferric Mb binds •NO to yield nitrosyl-heme 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 × 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 kforward = 110 ± 3 and kreverse = 16 ± 3 M-1·s-1 for S-nitrosoglutathione and kforward = 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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.