Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed the realtime assessment of glutaredoxin structure-function relationships inside living cells. Finally, we employed this assay to rapidly screen multiple glutaredoxin mutants, ultimately enabling us to convert enzymatically active and inactive glutaredoxins into each other. In summary, we have gained a comprehensive understanding of the mechanistic underpinnings of glutaredoxin catalysis and have elucidated the determinant structural differences between the two main classes of glutaredoxins.
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.
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