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

Abstract

Peroxiredoxins (Prx) are thiol peroxidases that exhibit exceptionally 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 ϳ3-4 Å or ϳ6 -7 Å away from the active site peroxidative cysteine (C p ), 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 cooperative function for these residues. Detailed ab initio theoretical calculations carried out with the high level G4 procedure suggest strong catalytic effects of H-bond-donating functional groups to the C p sulfur and the reactive and leaving oxygens of the peroxide in a cooperative manner. Using a guanidinium cation 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-bonding to ArgII. This mechanism could explain the exceptional reactivity of peroxiredoxins toward H 2 O 2 and may have wider implications for protein thiol reactivity toward peroxides.

Molecular Basis for the Resistance of Human Mitochondrial 2-Cys Peroxiredoxin 3 to Hyperoxidation

“…hyperoxidation of the catalytic Cys-S P H residue to Cys sulfinic acid (Cys-S P O 2 H) (24). To evaluate this difference, a panel of human Prx2 and Prx3 variants was expressed and purified from E. coli.…”

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

“…Kinetic experiments with Arg127 mutated to Lys show that the rate constant drops with a factor 5 [12] . Perhaps here, when Lys is present in the position of Arg, it cannot fix the cysteine thiolate side chain in a position optimal for the reaction with H 2 O 2 in the same way as the guanidinium group of Arg does.…”

ATP and Mg2+ Promote the Reversible Oligomerization and Aggregation of Chloroplast 2-Cys Peroxiredoxin

“…When compared with the effects on the peroxidase and chaperone activities, it is evident that 2-Cys Prx performs autonomous and unrelated activities without partitioning into different domains of the protein. As expected (44,47), the replacement of Arg 129 and Arg 152 by alanine strongly reduced the peroxidase activity, but unexpectedly, the former enhanced whereas the latter almost abrogated the chaperone activity. In consequence, these conserved arginines play important roles not only in the peroxidase activity but also in the chaperone activity and the ATP/ Mg-mediated self-assembly of 2-Cys Prx, revealing that determinants responsible for these disparate functions overlap.…”

“…Earlier studies have revealed the necessary participation of two conserved arginines, Arg 129 and Arg 152 , in the peroxidase activity, but detailed information on the chaperone activity and the ATP/Mg-dependent oligomerization/aggregation was missing (43)(44)(45)(46)(47). Therefore, we set out to find whether mutants whose positively charged residues were replaced by the neutral alanine were functional in the latter functions.…”