Post-translational modification in the gas phase: Mechanism of cysteine S-nitrosylation via ion-molecule reactions

The gas-phase mechanism of S-nitrosylation of thiols was studied in a quadrupole ion trap mass spectrometer. This was done via ion-molecule reactions of protonated cysteine and many of its derivatives and other thiol ions with neutral tert-butyl nitrite or nitrous acid. Our results showed that the presence of the carboxylic acid functional group, -COOH, in the vicinity of the thiol group is essential for the gas-phase nitrosylation of thiols. When the carboxyl proton is replaced by a methyl group (cysteine methyl ester) no nitrosylation was observed. Other thiols lacking a carboxylic acid functional group displayed no S-nitrosylation, strongly suggesting that the carboxyl hydrogen plays a key role in the nitrosylation process. These results are in excellent agreement with a solution-phase mechanism proposed by Stamler et al. (J. S. Stamler, E. J. Toone, S. A. Lipton, N. J. Sucher. Neuron 1997, 18, 691-696) who suggested a catalytic role for the carboxylic acid group adjacent to cysteine residues and with later additions by Ascenzi et al. (P. Ascenzi, M. Colasanti, T. Persichini, M. Muolo, F. Polticelli, G. Venturini, D. Bordo, M. Bolognesi. Biol. Chem. 2000, 381, 623-627) who postulated that the presence of the carboxyl in the cysteine microenvironment in proteins is crucial for S-nitrosylation. A concerted mechanism for the gas-phase S-nitrosylation was proposed based on our results and was further studied using theoretical calculations. Our calculations showed that this proposed pathway is exothermic by 44.0 kJ mol^-1. This is one of the few recent examples when a gas-phase mechanism matches one in solution.