Model for the Exceptional Reactivity of Peroxiredoxins 2 and 3 with Hydrogen Peroxide

Peroxiredoxins (Prx) are thiol peroxidases that exhibit excep- tionally high reactivity toward peroxides, but the chemical basis for this is not well understood. We present strong experimental evidence that two highly conserved arginine residues play a vital role in this activity of human Prx2 and Prx3. Point mutation of either ArgI or ArgII (in Prx3 Arg-123 and Arg-146, which are 3- 4 Aor 6- 7 Aaway from the active site peroxidative cys- teine (Cp), respectively) in each case resulted in a 5 orders of magnitude loss in reactivity. A further 2 orders of magnitude decrease in the second-order rate constant was observed for the double arginine mutants of both isoforms, suggesting a cooper- ative function for these residues. Detailed ab initio theoretical calculations carried out with the high level G4 procedure sug- gest strong catalytic effects of H-bond-donating functional groups to the Cp sulfur and the reactive and leaving oxygens of the peroxide in a cooperative manner. Using a guanidinium cat- ion in the calculations to mimic the functional group of arginine, we were able to locate two transition structures that indicate rate enhancements consistent with our experimentally observed rate constants. Our results provide strong evidence for a vital role of ArgI in activating the peroxide that also involves H-bond- ing to ArgII. This mechanism could explain the exceptional reactivity of peroxiredoxins toward H2O2 and may have wider implications for protein thiol reactivity toward peroxides.