Peroxynitrite Chemistry: Formation, Decomposition and Possible Deactivation Mechanisms by Thiols

Kinetics of formation and decomposition of peroxynitrite was studied in acidic and buffer solutions. Peroxynitrous acid was formed in acidic media by the reaction of H2O2 with HNO2 and NO +. Bimolecular rate constant for reaction of H2O2 with HNO2 at 25°C was determined to be 3.7 × 10 3 M-1 s-1. Peroxynitrous acid formed is highly unstable with half life of approximately 1 second. Decomposition of peroxynitrite is highly pH-dependent. In acidic media, peroxynitrous acid decomposes to nitrate while at neutral to slightly basic media, products of decomposition is nitrite, oxygen and acidic protons. In strongly basic pH, the products of peroxynitrite decomposition were nitrite and oxygen in a 2:1 ratio. Decomposition dynamics showed first order dependence with respect to pH. Rate constant of decomposition of peroxynitrite in absence of a substrate at pH 3.0 was determined to be 0.98 s-1 and 0.10 s-1 at pH 8.0. Data generated from EPR studies indicates generation of ·OH radical as a decomposition product of peroxynitrite. Kinetics and mechanism of oxidation of cysteine (CYSH) and glutathione (GSH) by peroxynitrite, as radical deactivation agents were also studied. Peroxynitrite reacts with CYSH/GSH at acidic pH to produce corresponding S-nitrosothiol and disulfide. The reaction is first order in peroxynitrite, acid and thiol. Mass spectrometer technique confirms formation of nitrosothiol as product of oxidation. Mechanism of formation can be rationalized as occurring through acid catalyzed decomposition of PN through intermediacy of HNO2/ +N=O, which is capable of nitrosating thiol function. In basic pH, peroxynitrite oxidizes CYSH/GSH through a two electron oxidation mechanism via sulfinic acid to corresponding disulfide. Mass spectrometer technique suggests that stable product of CYSH/GSH oxidation by peroxynitrite at pH 7.4 is disulfide of corresponding initial thiol. EPR studies on oxidation of CYSH/GSH by peroxynitrite at pH 7.4 suggest pathway of oxidation goes via formation of thiyl radicals. Bimolecular rate constants for oxidation of GSH by peroxynitrite at 25°C (pH 7.0) was determined to be 587 M-1 s-1, while the bimolecular rate constants for oxidation of CYSH by peroxynitrite at 25°C (pH 7.4) was determined to be 2.0 × 103 M-1 s-1.