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. 2008 Dec 2;47(48):12860-8.
doi: 10.1021/bi801718d.

Cysteine pK(a) values for the bacterial peroxiredoxin AhpC

Kimberly J Nelson  1 , Derek Parsonage, Andrea Hall, P Andrew Karplus, Leslie B Poole
Affiliations

Cysteine pK(a) values for the bacterial peroxiredoxin AhpC

Kimberly J Nelson et al. Biochemistry. .

Abstract

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 epsilon(240) 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, approximately 90% of the C(P) would be deprotonated) with the fact that thiolates with higher pK(a) values are stronger nucleophiles.

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Figures

FIGURE 1
FIGURE 1. Monitoring cysteine thiolate absorption of wild type AhpC at 240 nm over a pH range yields a single pKa value of 5.84 ± 0.02
(a) The A240 and A280 values for oxidized (o) and reduced (●) AhpC (3-10 μM) were measured over a range of pH values and converted to ε240 assuming an ε280 value of 24,300 M-1 cm-1. The pKa was determined from the ε240 versus pH plot by direct fit to Equation 1 as described in Methods. (b) As described for panel a, the pH-dependent change in absorbance was measured for C46S (o), C165S (●) and the C46S, C165S double mutant of AhpC (Δ). These data were not able to be fit to equation 1.
FIGURE 2
FIGURE 2
pKa determination using two different iodoacetamide-based compounds and single cysteine mutants of AhpC provide pKa values for C46 and C165 close to 8.5. (a) Reduced C165S (—●—) or C46S (- -○- -) AhpC (24 μM) in various pH buffers was incubated with 180 μM 5-iodoacetamidofluorescein for various amounts of time, quenched with excess 2-mercaptoethanol, then analyzed on a 12% SDS-polyacrylamide gel to determine the % fluorescence in the protein fraction; the data were fit to a single exponential equation to determine kobs at each pH. Data plotted as kobs versus pH were fit to equation 1 as described in Methods. (b) Reduced wild-type AhpC (40 μM) in various pH buffers was incubated with 400 μM d0-IAAn over a time course, and aliquots were quenched with excess 2-mercaptoethanol. A standard amount of AhpC labeled with d5-IAAn was added to each sample, and the protein was digested with trypsin overnight as described in Experimental Procedures. The extent of alkylation of C46 (—●—) and C165 (- -○- -) was determined by measuring the ratio of the peak intensities at 3836/3841 or 1745/1750 Da, respectively. The ratios were fit to a single exponential equation to determine the kobs and used to calculate the pKa of each residue as in panel (a).
FIGURE 3
FIGURE 3
Crystal structure of C165S AhpC adduct with iodoacetanilide shows that the protein is in the locally unfolded conformation. In each panel, the Cα chain of residues 38 through 50 from the fully folded (FF) model (magenta) and the locally unfolded (LU) model (green) are shown along with 2Fo-Fc electron density contoured at 0.7·ρrms. (a and c) Electron density calculated using the FF or LU model, respectively; (b and d) Electron density calculated using the FF or LU model, respectively, with residues 41 through 48 omitted. Figures were prepared using Pymol.
FIGURE 4
FIGURE 4. pKa determination for wild type AhpC using a competition assay with horseradish peroxidase (HRP) yields a pKa of 5.94 ± 0.10
Three μM H2O2 was added to HRP (7.5 μM) and reduced AhpC (0, 2, 4, 8, 12, or 16 μM) in various pH buffers. The extent of HRP oxidation by H2O2 was monitored using A403 (to measure complex I formation in HRP) before peroxide addition and again within 90 s after initiation of the reaction. The percentage of inhibition of HRP oxidation (F/1–F) versus [AhpC] was used to calculate the second-order rate constant for AhpC (kAhpC) using equation 2 (see Methods and Supplemental Figures S2 a and b). Data plotted as either kAhpC or relative rate versus pH were fit to equation 1 as described in Experimental Procedures.
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