The reduction of oxidized glutathione GSSG by hydrated electrons and hydrogen atoms to form GSSG•− is quantitative. The radical anion dissociates into GS• and GS−, and the S-centered radical subsequently abstracts a hydrogen intramolecularly. We observe sequential development of UV absorbance signatures that indicate the formation of both α- and β-carbon-centered radicals. From experiments performed at pH 2 and pH 11.8, we determined forward and reverse rate constants for the overall equilibrium between sulfur-centered and carbon-centered radicals: kforward = 3·105 s−1, kreverse = 7·105 s−1, and K = 0.4. Furthermore, on the basis of the differences between the kinetics traces at 240 and 280 nm, we estimate that α- and β-carbon-centered radicals are formed at a surprising ratio of 1:3. The ratios found at pH 2 also apply to pH 7, with the conclusion that the equilibrium ratio of S-centered:β-centered:α-centered radicals is, very approximately, 8:3:1. The formation of carbon-centered radicals could lead to irreversible damage in proteins via the formation of carbon−carbon bonds or backbone fragmentation.

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Cysteine, cysteinyl-glycine, glutathione, phenylalanyl-cysteinyl-glycine, and histidyl-cysteinyl-glycine were dissolved in acidic and neutral D(2)O in the presence of the radical generator 2,2'-azobis(2-methylpropionamidine) dihydrochloride and radical mediator compounds (benzyl alcohol and 2-propanol). An exchange of H-atoms by D-atoms took place in these peptides due to intramolecular H-abstraction equilibria. NMR measurements allow one to follow the extent of H-D exchanges and to identify the sites where these exchanges take place. Significant exchanges occur in acidic media in GSH at positions Glu-β and Glu-γ, in Phe-Cys-Gly at positions Phe ortho, Phe-β, Cys-α, Cys-β, and Gly-α, and in His-Cys-Gly at positions His H1, His H2, His β, Cys β, and Gly α. In neutral media, exchanges occur in Cys-Gly at position Cys β and in GSH at position Cys α. Phe-Cys-Gly and His-Cys-Gly were not examined in neutral media. Sites participating in the radical exchange equilibria are highly dependent on structure and pH; the availability of electron density in the form of lone pairs appears to increase the extent of exchange. Interestingly, and unexpectedly, 2D NMR experiments show that GSH rearranges itself in acidic solution: the signals shift, but their patterns do not change. The formation of a thiolactone from Gly and Cys residues matches the changes observed.

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