Peroxiredoxins efficiently remove hydroperoxides and peroxynitrite in pro- and eukaryotes. However, isoforms of one subfamily of peroxiredoxins, the so-called Prx6-type enzymes, usually have very low activities in standard peroxidase assays in vitro. In contrast to other peroxiredoxins, Prx6 homologues share a conserved histidyl residue at the bottom of the active site. Here we addressed the role of this histidyl residue for redox catalysis using the Plasmodium falciparum homologue PfPrx6 as a model enzyme. Steady-state kinetics with tert-butyl hydroperoxide (tBuOOH) revealed that the histidyl residue is nonessential for Prx6 catalysis and that a replacement with tyrosine can even increase the enzyme activity four- to six-fold in vitro. Stopped-flow kinetics with reduced PfPrx6 , PfPrx6 , and PfPrx6 revealed a preference for H O as an oxidant with second order rate constants for H O and tBuOOH around 2.5 × 10 M s and 3 × 10 M s , respectively. Differences between the oxidation kinetics of PfPrx6 , PfPrx6 , and PfPrx6 were observed during a slower second-reaction phase. Our kinetic data support the interpretation that the reductive half-reaction is the rate-limiting step for PfPrx6 catalysis in steady-state measurements. Whether the increased activity of PfPrx6 is caused by a facilitated enzyme reduction because of a destabilization of the fully folded enzyme conformation remains to be analyzed. In summary, the conserved histidyl residue of Prx6-type enzymes is non-essential for catalysis, PfPrx6 is rapidly oxidized by hydroperoxides, and the gain-of-function mutant PfPrx6 might provide a valuable tool to address the influence of conformational changes on the reactivity of Prx6 homologues.

So-called 1-Cys peroxiredoxins (Prx) employ only one cysteine residue for the reduction of hydroperoxides and require an external thiol for the reduction of a reactive sulfenic acid during the catalytic cycle. Hence, 1-Cys Prx, which often belong to the structural Prx5-or the Prx6-type subfamily, are potentially promiscuous enzymes that could react with a variety of thiols. Furthermore, the dependence on an external thiol could affect the susceptibility of 1-Cys Prx to hyperoxidation, i.e., the formation of a sulfinic or sulfonic acid. Here, we compared the reaction mechanisms and kinetics of the Prx5-and Prx6-type enzymes PfAOP and PfPrx6 from the malaria parasite Plasmodium falciparum to address the hyperoxidation susceptibility and potential substrate promiscuity of 1-Cys Prx. While PfAOP did not react with common thiol-disulfide oxidoreductases, the enzyme turned out to be promiscuous regarding the reduction by synthesized glutathione analogues and other low-molecular-weight thiols. Furthermore, we established a complete single turnover experiment for PfAOP with glutathione and the glutaredoxin PfGrx and identified the rapid H 2 O 2 -dependent hyperoxidation of PfAOP as the cause for the apparent preference of this Prx5-type enzyme for alkylhydroperoxides in vitro. Unlike promiscuous PfAOP, PfPrx6 was inactive with ascorbate, the physiological low-molecular-weight thiols glutathione, cysteine, cysteamine, coenzyme A, and dihydrolipoamide, as well as physiological protein thiols, including PfTrx1, PfGrx, and the resolving cysteine of the Prx1-type enzyme PfPrx1a in potential hetero-oligomers. Reduction of PfPrx6 was only observed with dithiothreitol and required the presence of a histidine residue, which protects the enzyme from hyperoxidation and is the major structural difference between the active sites of Prx5-and Prx6-type enzymes. We propose two alternative evolutionary adaptations of the 1-Cys Prx mechanism to hyperoxidation and the formation of alternative mixed disulfides that could explain the co-existence of promiscuous Prx5-and protected Prx6-type enzymes in a variety of organisms and subcellular compartments.