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

摘要

Ascorbate reacts with S-nitrosothiols generally, in the pH 3-13 by way of two distinct pathways, (a) at low [ascorbate], typially below approx 1 * 10~(-4) mol dm~(-3) which leads to the formation of NO and the disulfide, and (b) at higher [ascorbate] when the products are the thiol and NO. Reaction (a) is Cu~(2+)-dependent, and is completely cut out in the presence of EDTA, whereas reaction (b) is totally independent of [Cu~(2+)] and takes place readily whether EDTA is present or not. For S-nitrosoglutathione (GSNO) the two reactions can be made quite seaprate, although for some reactants the two reactions overlap. In reaction (a), ascorbate acts as a reducing agent, generating Cu~+ from Cu~(2+), which in turn racts with RSNO forming initially NO, Cu~(2+) adn RS~_. The latter can then play the orle of reducing ageanat for Cu~(2+), leading to disulfide formation. Ascorbate will initiate reaction when the free thiolatea has initially been reduced to a very low level by the synthesis of RSNO from a large excess of nitrous acid over the thioil. Reaction (b) is interpreted in terms of nucleophilic attack by ascorbate at the nitroso-nitrogen atom, leading to thiol and O-nitrosoascorbate which breaks up, by a free-radical pathway, to give dehydroascorbic acid and NO. A similar pathway is the accepted mechanism in the literature for the nitrosation of ascorbate by nitrous acid and alkyl nitrites. The rate constant for the CU~(2+)-independent pathway increases sharply with pH and analysis of the variation of the rate constant with pH identifies a reaction pathway via both the mono-and di-anion forms of ascrobate, with the latter being the more reactive. As expected the entropy of activation is large and negative. Some aspects of structure-reactivity trends are discussed.