Biological formation

The cellular metabolism of S-nitrosothiols is complex and has not yet been fully elucidated. In general, the levels of S-nitrosothiols formed in vivo are determined by the NO flux, i.e., activity of NOS and availability of substrate; rate of formation of the S-nitrosating species; and competition of different thiols and other reactive motifs for reaction with the nitrosating species. We found that S-nitrosated proteins can form within endothelial cells from an exogenous S-nitrosothiol donor or from endogenous production of NO by endothelial NO synthase activity [25]. In comparison to CysNO, NO is a relatively poor nitrosating agent in cultured cells [22,26,27]. It is important to point out that the reaction of nitric oxide with thiols under anaerobic conditions does not yield an S-nitrosothiol, but disulfide and nitroxyl anion. Nitrosating intermediates are formed in the course of aerobic oxidation of NO. It is commonly assumed that nitrosation of thiols by NO in the presence of oxygen involves electrophilic substitution by N2O3. In contrast to NO auto-oxidation, the reaction between NO and superoxide is first order in both reactants, occurs at near diffusion limits [28-30], and greatly enhances protein S-nitrosation [31]. Nitrosation is maximal when the molar ratio of superoxide to NO is near-equivalent [32].

By confocal microscopy and in situ S-nitrosothiol fluorescence labeling, we found that S-nitrosoproteins exist mainly in the mitochondria, which is the primary superoxide source in resting cells [25]. PseudoRhoo cells, which are essentially devoid of mitochondria, demonstrate much less S-nitrosoprotein formation than do cells with a normal complement of mitochondria. Cellular protein S-nitrosothiol formation is also diminished when cells are treated with electron transport inhibitors to decrease superoxide generation. These studies suggest that superoxide anion reacts with endogenous NO or exogenous NO to form the nitrosating species peroxynitrite or N2O3. In addition, physiological concentrations of CO2 modestly enhance levels of NO2 formation via formation of nitrosoperoxocarbonate from peroxynitrite [32], thereby affecting nitrosation potential.

Other possible mechanisms for S-nitrosothiols formation include the role of metal ions [33] and dinitrosyl iron complexes [34]. Protein thiols may donate one electron to transition metals like copper and become thiyl radicals in the process, which then react with nitric oxide to form S-nitrosothiols [34-37]. The S-nitrosating agent NO+ may also form in the presence of transition metals [38]. Several proteins containing transition metals also catalyze the formation of S-nitrosothiols. Romeo suggested that superoxide dismutase targets NO from GSNO to Cys|393 of oxyhemoglobin [39], and ceruloplasmin showed potent RSNO-forming activity in cell culture [40]. S-nitrosothiols may also be formed from nitrite anion through heme protein-mediated nitrite reductase activity [41-43]. Such enzymatic nitrite reductase pathways are discussed in Chapters 14 and 15 of this volume. Xanthine oxidase also catalyzes anaerobic transformation of organic nitrates to nitric oxide and S-nitrosothiols [44].

S-nitrosothiol formation is also controlled by the competition between GSH and different proteins. Decreased intracellular GSH concentration markedly enhances protein S-nitrosothiol formation [25], while overexpression of metallothionein, which also exists in a very reduced apo-form in the cell [45], protects proteins from S-nitrosation [46].

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