Cysteine pK(a) values for the bacterial peroxiredoxin AhpC

Area of Science:

• Biochemistry
• Enzymology
• Oxidative Stress

Background:

• Salmonella typhimurium AhpC is a key peroxiredoxin involved in antioxidant defense.
• Peroxiredoxins utilize a catalytic cysteine (C(P)) to neutralize reactive oxygen species.
• The nucleophilicity of the C(P) thiolate is critical for enzyme function, but its pKa is debated.

Purpose of the Study:

• To determine the pKa of the cysteinyl residues in Salmonella typhimurium AhpC.
• To understand the relationship between pKa, active site conformation, and enzymatic activity.

Main Methods:

• Utilized iodoacetamide derivatives to probe cysteinyl residue reactivity.
• Performed X-ray crystallography to analyze active site conformation.
• Measured enzyme kinetics by assessing AhpC competition with horseradish peroxidase for hydrogen peroxide.
• Analyzed the pH dependence of AhpC's UV absorbance at 240 nm.

Main Results:

• Iodoacetamide methods yielded unperturbed pKa values (7.9-8.7), suggesting reaction with non-native conformations.
• X-ray crystallography supported the presence of alternative conformations.
• Functional pKa of C(P) determined to be 5.94 +/- 0.10, reflecting the active conformation.
• This functional pKa is consistent with values from other 2-cysteine peroxiredoxins and UV-Vis spectroscopy.

Conclusions:

• The functional pKa of AhpC's catalytic cysteine is significantly lower than previously assumed.
• This lower pKa ensures a high proportion of the reactive thiolate form at physiological pH.
• The pKa value optimizes the balance between thiolate nucleophilicity and enzyme stability/function.