The mechanism of formation of S-nitrosothiols (RSNO) in vivo
is an important factor in understanding the biological actions of NO. RSNO have been implicated in the regulation of many cellular processes, including apoptosis [1
], cell proliferation [2
], and hypoxic vascular responsiveness [3
]. While there are some reports of enzymatic involvement in S-nitrosothiol formation [4
], thiol nitrosation is largely thought to be driven through the intrinsic biological chemistry of NO.
Three mechanisms have been proposed for the formation of S-nitrosothiols, in vitro
, from a combination of nitric oxide, thiol and oxygen (the three pathways are illustrated in ). The first (pathway 1 in ) involves the formation of an intermediary nitrosating agent, N2
, from the reaction between NO and oxygen [6
]. The second (pathway 2 in ) involves the one electron oxidation of the thiol by NO2
followed by radical-radical combination of the thiyl radical and nitric oxide [7
]. The third (pathway 3 in ) involves the reduction of oxygen by a putative intermediate radical (RSNOH) formed from the direct combination of nitric oxide and thiol [10
]. Experimental evidence exists in the literature for all three mechanisms based on kinetic, stoichiometric and scavenger studies. Both pathway 1 and pathway 3 are relatively simple with clearly defined stoichiometries, whereas pathway 2 is more complex due to the fact that the formation of thiyl radical as an intermediate opens up a plethora of additional reaction possibilities.
Proposed pathways of the reaction between GSH, NO, and oxygen
An additional factor that has been proposed to be involved in S-nitrosothiol synthesis is the presence of hydrophobic microenvironments [3
]. As both NO and oxygen are hydrophobic it has been demonstrated that hydrophobic environments can enhance the rate of reaction between these species due to increases in their local concentration [11
]. It has been suggested that this effect may enhance S-nitrosation in the vicinity of hydrophobic environments [12
]. In extremity, it has been reported that NO oxidation and nitrosation can be localized internally to a single protein [13
], such that the interaction of NO with a specific protein molecule can S-nitrosate a thiol within that same protein. Although this mechanism would allow for a very directed mechanism of S-nitrosthiol formation and NO signaling, it would requires significant curtailment of normal diffusivity of gas molecules, and also unprecedented stabilization of reaction intermediates.
In this study we have performed a series of experiments and kinetic simulations to attempt to differentiate between these various mechanisms of GSNO formation using glutathione (GSH) as the target thiol and to assess the importance of hydrophobic protein environements to the nitrosation process. We conclude that both thiyl-radical and N2O3-medaited nitrosation (pathways 1 and 2) occur in this system and that pathway 3 has negligible importance. In addition we show that thiyl radical scavengers have little effect on the formation of GSNO, but instead switch the mechanism of GSNO formation from pathway 2 to pathway 1. In addition the use of phosphate to inhibit pathway 1 also inhibits pathway 2. Consequently in such complex reaction networks, the use of inhibitors to probe specific pathways can affect more than one pathway. In addition we have re-examined the ability of hydrophobic environments to enhance S-nitrosothiol formation and conclude that proteins inhibit rather than enhance S-nitrosation. These results clarify potential mechanisms of S-nitrosothiol formation in biological systems.