Cysteine pK_a Values for the Bacterial Peroxiredoxin AhpC

Salmonella typhimurium AhpC is a founding member of the peroxiredoxin family, a ubiquitous group of cysteine-based peroxidases with high reactivity toward hydrogen peroxide, organic hydroperoxides, and peroxynitrite. For all of the peroxiredoxins, the catalytic cysteine, referred to as the peroxidatic cysteine (C_P), acts as a nucleophile in attacking the peroxide substrate, forming a cysteine sulfenic acid at the active site. Because thiolates are far stronger nucleophiles than thiol groups, it is generally accepted that cysteine-based peroxidases should exhibit pK_a values lower than an unperturbed value of 8.3−8.5. In this investigation, several independent approaches were used to assess the pK_a of the two cysteinyl residues of AhpC. Methods using two different iodoacetamide derivatives yielded unperturbed pK_a values (7.9−8.7) for both cysteines, apparently due to reactivity with the wrong conformation of C_P (i.e., locally unfolded and flipped out of the active site), as supported by X-ray crystallographic analyses. A functional pK_a of 5.94 ± 0.10 presumably reflecting the titration of C_P within the fully folded active site was obtained by measuring AhpC competition with horseradish peroxidase for hydrogen peroxide; this value is quite similar to that obtained by analyzing the pH dependence of the ε₂₄₀ of wild-type AhpC (5.84 ± 0.02) and similar to those obtained for two typical 2-cysteine peroxiredoxins from Saccharomyces cerevisiae (5.4 and 6.0). Thus, the pK_a value of AhpC balances the need for a deprotonated thiol (at pH 7, ∼90% of the C_P would be deprotonated) with the fact that thiolates with higher pK_a values are stronger nucleophiles.