Identification of Cu+ as the effective reagent in nitric oxide formation from S-nitrosothiols (RSNO)

Abstract

Decomposition of S-nitrosothiols (RSNO) in aqueous solution at pH 7.4 is brought about by copper ions, either present as an impurity or specifically added. The primary products are nitric oxide and the disulfide. In the presence of the specific Cu⁺ chelator, neocuproine, reaction is progressively inhibited as the [neocuproine] is increased, the reaction eventually stopping completely. The characteristic UV–VIS spectrum of the Cu⁺ adduct can be obtained from the reaction solutions. This shows clearly that Cu⁺ and not Cu²⁺ is the effective catalyst. Two limiting kinetic conditions can be identified for a range of S-nitrosothiols at specific copper ion concentrations (a) a first-order dependence and (b) a zero-order dependence upon [RSNO]. Normally both situations also have a short induction period. This induction period can be removed by the addition of the corresponding thiol RSH. A mechanism is proposed in which Cu⁺ is formed by reduction of Cu²⁺ by thiolate anion via an intermediate, possibly RSCu⁺. Loss of nitric oxide from RSNO is then brought about by Cu⁺, probably via another intermediate in which Cu⁺ is bound to the nitrogen atom of the NO group and another electron-rich atom (such as nitrogen from an amino group, or oxygen from a carboxylate group) involving a six-membered ring. As well as NO this produces both RS^– and Cu²⁺ which then are part of the cycle regenerating Cu⁺. Thiolate ion is oxidised to RS˙ which dimerizes to give the disulfide. Depending on the structure (and hence reactivity) of RSNO either Cu⁺ formation or its reaction with RSNO can be rate-limiting. Computer modelling of the reaction scheme allows the generation of absorbance time plots of the same forms as those generated experimentally, i.e. first- or zero-order, both with or without induction periods. We suggest that the thiolate ion necessary to bring about Cu²⁺ reduction is either present as a thiol impurity or is generated in small quantities by partial hydrolysis of the nitrosothiol, which results in an induction period. Addition of small quantities of thiol removes the induction period and leads to catalysis but larger quantities bring about a rate reduction by, it is suggested, complexation of the Cu²⁺. For two very unreactive substrates, S-nitrosoglutathione and S-nitroso-N-acetylcysteine very large induction periods were observed, typically three hours. This results, we suggest, from competitive re-oxidation of Cu⁺ to Cu²⁺ by the dissolved oxygen. Experiments carried out anaerobically confirm this, since there is then no induction period. Addition of hydrogen peroxide extends the induction period ever further. The results are discussed in terms of the biological properties of S-nitrosothiols which are related to nitric oxide release.