Human S-nitroso oxymyoglobin is a store of vasoactive nitric oxide.

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.