S-nitrosation

N-Nitrosamines have been shown to be inhibitors of cysteine-containing enzymes. For example, dephostatin and other N-methyl-N-nitrosoanilines (1) were found to be inhibitors of the protein tyrosin phosphatases, papain and caspase [90,91]. Inhibition results from the S-nitrosation of the critical cysteine residues in the active sites of the enzymes by the nitrosamines. Compounds 6 and 7 have been found to inhibit thrombus formation in arterioles and venules of rats [92], while N-nitrosamide 9 exhibited vasodilation and mutagenicity as a result of NO release [93]. 

However, this reaction can and has led to errors in the measurement of RSNOs in biological fluids when the samples are improperly buffered to avoid the HN02 -route to S-nitrosation (Tsikas, 2003). 

N203-dependent S-nitrosation has been demonstrated in the case of semm albumin (Nedospasov et al, 2000 Rafikova et al., 2002), in membranes (Liu et al., 1998) and in the protein-disulfide isomerase dependent transfer of NO-equivalents from extracellular RSNOs to the cytosol (Ramachandran et al., 2001). 

Intracellular and extracellular RSNO metabolism has been studied in LPS activated macrophages (Zhang and Hogg, 2003). This study showed that 0.02% of the NO produced in response to LPS, (detected as N02 ) was converted to cytosolic RSNOs and that all of the RSNOs detected were in the large molecular weight (>3K) protein fraction and were very stable to denitrosation (t1(f2 3h). These authors also showed that the molecular specie(s) responsible for S-nitrosation is freely diffusible and has to be transported to the cell surface before internal S-nitrosation could take place. 

Perhaps the most physiologically important metalloprotein to be S-nitrosated by NO is hemoglobin (Hb). The S-nitrosation of Hb-Cys 93 by NO was first demonstrated by Stamler and coworkers in 1996 (Jia et al., 1996 Stamler et al., 1997). To this day HbSNO remains controversial with respect to its mechanism of formation and physiological relevance. 

The wealth of data on HbSNO formation has given rise to two broad interpretations that either support the hypothesis that the S-nitrosation of Hb-Cys 93 is redox catalyzed by the heme and is under allosteric control or that it is N2 03 or N02 -mediated without the involvement of the hemes. 

In their initial report, Stamler and co-workers (Jiaetal, 1996) showed that exposure of Hb to NO resulted in the S-nitrosation of C93 in the p-chains. The reported yield upon exposure of Hb to 10-fold molar excess of NO, was lmol S-NO per tetramer. Given that there are 2 C 93/tetramer this is a 50% yield. Hb-SNO formation was accompanied by the conversion ofoxyHb (HbFe(II)02) to metHb (HbFe(III)). 

Hbp C93 S-nitrosation could also be accomplished by exposure to RSNOs (GSNO or CysNO). The rates RSNO-dependent Hb-S-nitrosation was 10-fold larger in oxy-Hb than in deoxy-Hb. Conversely, the rate of spontaneous decay of deoxy-Hb-SNO was -20-fold larger than oxy-Hb-SNO. An explanation for this differential reactivity was presented in a subsequent study (Stamler et al, 1997) where protein modeling data based on the X-ray structures of Hb in T and R states indicated that in OxyHb the SNO of Cys (1 93 is protected from solvent. In contrast, in deoxyHb the SNO is highly exposed to solvent. The implication was that the NO+ on Cys (193-S-NO could be transferred to thiols in RBC and eventually effluxed to induce vasodilation under conditions of low 02 saturation. 

Thiols, protein or small molecular weight, can be S-nitrosated either by reaction with N O-oxidation products (Scheme 4.1) in hydrophobic domains of plasma proteins or transnitrosated at the cell-plasma interface of NO-producing cells (endothelial cells) or NO-storing cells (RBCs). Efforts to determine the true levels in plasma and in the various cellular compartments are complicated owing to thermal sensitivity and photosensitivity (Sexton et al., 1994 Alpert et al., 1997 Mutus et al., 1999) of the RS-NO bond. Techniques currently employed for the detection and quantification of RSNOs have been summarized in recent reviews (Martinez Riuz and Lamas, 2004 Rasaf et al., 2004). 

The number of enzymes and functional proteins that are reportedly regulated by S-nitrosation is on the rise. For example, a search of PUBMED with the key word S-nitrosation revealed some 70 reports of in vitro regulation of enzymes, proteins and cellular processes that are affected by S-nitrosation. Some of these processes that have been well characterized include, nuclear regulatory proteins the NMDA receptor and the ertrocyte anion exchange protein 1 (AE1) (see review by Gaston, 2003). 

Butler et al. [32] also developed a series of O-acylated-S-nitrosated thiosugars (Fig. 8.5). Partial de-acylation in tissue should give a mixture of hydrophilic and hydrophobic groups. These compounds can be delivered transdermally and result in greatly enhanced subcutaneous blood flow [33]. Unfortunately, these compounds are not very stable and cannot be isolated. The alcoholic solution, in which they are stored and administered, must be kept on ice during use. 

There appears to be little reported work on S-nitrosation reactions of simple thioke-tones. Thiocamphor when treated with /50-amyl nitrite in fact gives the oxime58 (formerly called a isonitroso compounds), presumably via the tautomeric form of the thione, i.e. the enethiol. In this respect the reaction is very similar to the reactions of ketones59 which give oximes or C-nitroso compounds via the enol intermediates60. 

At higher acidities the S-nitrosation reaction of thiourea leads to the formation of urea64 (equation 28) via, it is believed, the intermediate formation of the S-nitroso species. The reaction can also be brought about by nitrosamines or alkyl nitrites as the carriers of NO+. Reaction is thought to involve nucleophilic attack of the intermediate by water or the elimination of HSNO giving a carbodiimide, which is then hydrated. 

There is indirect kinetic evidence that S-nitrosation of a sulphide occurs70, followed by a S to IV rearrangement of the nitroso group, leading finally to deamination (equation 31). The evidence is based on the much higher reaction rate when the sulphur atom is present. 

S-nitrosated and disulfide derivatives using CZE was developed. S -Nitroso thiols were selectively detected at 320 nm606. 

Giustarini D, Milzani A, Aldini G, Carini M, Rossi R, Dalle-Donne I. 2005. S-nitrosation versus S-glutathionylation of protein sulfhydryl groups by S-nitroso-glutathione. Antioxid Redox Signal 7 930-939. 

S-Nitrosation and S-nitroso compounds 418 10 Nitration and nitrosation 422 Acknowledgements 424... 

Until recently most of the mechanistic studies on nitrosation have been concerned with N-nitrosation reactions of amines, including the diazotisation reactions of primary amines. Now, work has been extended to include both O- and S-nitrosation, so that comparisons can be made. Mechanistic studies have also been extended in recent years to include reactions of nitrogen oxides, nitrosamines, alkyl nitrites, thionitrites and transition metal nitrosyl complexes. Many of these reactions have been used preparatively for a long time, but little has been known about their detailed reaction mechanisms. 

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