Reaction of ascorbic acid with S-nitrosothiols: clear evidence for two distinct reaction pathways

Ascorbate reacts with S-nitrosothiols generally, in the pH range 3-13 by wa y of two distinct pathways, (a) at low [ascorbate], typically below approxi mate to 1x10(-4) mol dm(-3) which leads to the formation of NO and the disu lfide, and (b) at higher [ascorbate] when the products are the thiol and NO . Reaction (a) is Cu2+-dependent, and is completely cut out in the presence of EDTA, whereas reaction (b) is totally independent of [Cu2+] and takes p lace readily whether EDTA is present or not. For S-nitrosoglutathione (GSNO ) the two reactions can be made quite separate, although for some reactants the two reactions overlap. In reaction (a), ascorbate acts as a reducing a gent, generating Cu+ from Cu2+, which in turn reacts with RSNO forming init ially NO, Cu2+ and RS-. The latter can then play the role of reducing agent for Cu2+, leading to disulfide formation. Ascorbate will initiate reaction when the free thiolate has initially been reduced to a very low level by t he synthesis of RSNO from a large excess of nitrous acid over the thiol. Re action (b) is interpreted in terms of nucleophilic attack by ascorbate at t he nitroso-nitrogen atom, leading to thiol and O-nitrosoascorbate which bre aks up, by a free-radical pathway, to give dehydroascorbic acid and NO. A s imilar pathway is the accepted mechanism in the literature for the nitrosat ion of ascorbate by nitrous acid and alkyl nitrites. The rate constant for the Cu2+ -independent pathway increases sharply with pH and analysis of the variation of the rate constant with pH identifies a reaction pathway via b oth the mono- and di-anion forms of ascorbate, with the latter being the mo re reactive. As expected the entropy of activation is large and negative. S ome aspects of structure-reactivity trends are discussed.