Factors Affecting Protein Thiol Reactivity and Specificity in Peroxide Reduction

Abstract

Protein thiol reactivity generally involves the nucleophilic attack of the thiolate on an electrophile. A low pK(a) means higher availability of the thiolate at neutral pH but often a lower nucleophilicity. Protein structural factors contribute to increasing the reactivity of the thiol in very specific reactions, but these factors do not provide an indiscriminate augmentation in general reactivity. Notably, reduction of hydroperoxides by the catalytic cysteine of peroxiredoxins can achieve extraordinary reaction rates relative to free cysteine. The discussion of this catalytic efficiency has centered in the stabilization of the thiolate as a way to increase nucleophilicity. Such stabilization originates from electrostatic and polar interactions of the catalytic cysteine with the protein environment. We propose that the set of interactions is better described as a means of stabilizing the anionic transition state of the reaction. The enhanced acidity of the critical cysteine is concurrent but not the cause of catalytic efficiency. Protein stabilization of the transition state is achieved by (a) a relatively static charge distribution around the cysteine that includes a conserved arginine and the N-terminus of an α-helix providing a cationic environment that stabilizes the reacting thiolate, the transition state, and also the anionic leaving group; (b) a dynamic set of polar interactions that stabilize the thiolate in the resting enzyme and contribute to restraining its reactivity in the absence of substrate; but upon peroxide binding these active/binding site groups switch interactions from thiolate to peroxide oxygens, simultaneously increasing the nucleophilicity of the attacking sulfur and facilitating the correct positioning of the substrate. The switching of polar interaction provides further acceleration and, importantly, confers specificity to the thiol reactivity. The extraordinary thiol reactivity and specificity toward H(2)O(2) combined with their ubiquity and abundance place peroxiredoxins, along with glutathione peroxidases, as obligate hydroperoxide cellular sensors.

Peroxynitrite, a Stealthy Biological Oxidant

“…The remarkable velocity for peroxiredoxin reactions with peroxynitrite (Table 1) extended earlier observations for H 2 O 2 (30); the molecular determinants of such reactivity are under scrutiny but seem to depend of the stabilization of the enzyme-activated complex (30). The complete catalytic cycle to restore peroxiredoxin to the resting state is analyzed elsewhere (30). Due to the high concentration of peroxiredoxins, the fast rate constant, and its thorough distribution across various cellular compartments, it constitutes a prime endogenous antioxidant mechanism for the catalytic detoxification of peroxynitrite.…”

“…This reaction was later shown in mammalian peroxiredoxin systems (13). The remarkable velocity for peroxiredoxin reactions with peroxynitrite (Table 1) extended earlier observations for H 2 O 2 (30); the molecular determinants of such reactivity are under scrutiny but seem to depend of the stabilization of the enzyme-activated complex (30). The complete catalytic cycle to restore peroxiredoxin to the resting state is analyzed elsewhere (30).…”

“…The reactivity of peroxynitrite with thiols is intermediary between that of H 2 O 2 and that of hypohalous acids, resulting in a moderately strong and selective biological oxidant in a similar way to what has been proposed for thiol oxidation mediated by chloramines (k ϳ 100 -200 M Ϫ1 s Ϫ1 ) (28). Importantly, peroxynitrite can oxidize at even more remarkable rates some "fast reacting thiols," such as those present in mammalian and microbial peroxiredoxins (30). Indeed, peroxiredoxins react with peroxynitrite with constants on the order of 10 6 -10 7 M Ϫ1 s Ϫ1 (Table 1) and represent a first line of enzymatic antioxidant defense against peroxynitrite.…”

“…; however, the actual (pH-independent) rate constant of the reaction is on the order of 10 5 M Ϫ1 s 1 (27,30). c The product of the second-order rate constant times the concentration of the reactant provides a pseudo-first-order rate constant in s Ϫ1 that allows ready comparison of the kinetic biological relevance among different peroxynitrite targets.…”

How does the protein environment optimize the thermodynamics of thiol sulfenylation? Insights from model systems to QM/MM calculations on human 2-Cys peroxiredoxin

“…Rate constants depend on the thiol and on the oxidant, for example, with H 2 O 2 as an oxidant, the rate constant varies between 1 M -1 s -1 (for gluthation, GSH) and 10 8 M -1 s -1 (for Prx) at pH 7.4-7.6 and 37 °C [6] ; with GSH as thiol, the rate constants range between 10 2 M -1 s -1 (for peroxynitrite) and 10 10 M -1 s -1 (for hydroxyl radicals) at pH 7.4 [4] .…”