Negatively Charged Lipids Are Essential for Functional and Structural Switch of Human 2-Cys Peroxiredoxin II

The function of ubiquitous 2-Cys peroxiredoxins (Prxs) can be converted alternatively from peroxidases to molecular chaperones. This conversion has been reported to occur by the formation of high-molecular-weight (HMW) complexes upon overoxidation of or ATP/ADP binding to 2-Cys Prxs, but its mechanism is not well understood. Here, we show that upon binding to phosphatidylserine or phosphatidylglycerol dimeric human 2-Cys PrxII (hPrxII) is assembled to trefoil-shaped small oligomers (possibly hexamers) with full chaperone and null peroxidase activities. Spherical HMW complexes are formed, only when phosphatidylserine or phosphatidylglycerol is bound to overoxidized or ATP/ADP-bound hPrxII. The spherical HMW complexes are lipid vesicles covered with trefoil-shaped oligomers arranged in a hexagonal lattice pattern. Thus, these lipids with a net negative charge, which can be supplied by increased membrane trafficking under oxidative stress, are essential for the structural and functional switch of hPrxII and possibly most 2-Cys Prxs.     

Oligomerization and chaperone activity of a plant 2-Cys peroxiredoxin in response to oxidative stress

Plant 2-Cys peroxiredoxins (2-Cys Prxs) have been reported to localize to chloroplasts and perform antioxidative roles during plant development and photosynthesis. In this study, we identified that, in addition to the well-known function of thioredoxin (Trx)-dependent peroxidase, the plant 2-Cys Prx in Chinese cabbage 2-Cys Prx1, designated C2C-Prx1, also behaves as a molecular chaperone under oxidative stress conditions, like the yeast and mammalian 2-Cys Prxs. By the chaperone function of C2C-Prx1, the protein efficiently prevented the denaturation of citrate synthase and insulin from heat shock and dithiothreitol (DTT)-induced chemical stresses. Also, the protein structure of C2C-Prx1 was shown to have discretely sized multiple structures, whose molecular sizes were in the diverse ranges of low molecular weight (LMW) proteins to high molecular weight (HMW) protein complexes. The dual functions of C2C-Prx1 acting as a peroxidase and as a molecular chaperone are alternatively switched by heat shock and oxidative stresses, accompanying with its structural changes. The peroxidase function predominates in the lower MW forms, but the chaperone function predominates in the higher MW complexes. The precise regulation of C2C-Prx1 structures and functions may play a pivotal role in the protection of plant chloroplasts from photo-oxidative stress.     

New antioxidant with dual functions as a peroxidase and chaperone in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Thiol-based peroxiredoxins (Prxs) are conserved throughout all kingdoms. We have found that a conserved typical 2-Cys Prx-like protein (PaPrx) from Pseudomonas aeruginosa bacteria displays diversity in its structure and apparent molecular weight (MW), and can act alternatively as a peroxidase and molecular chaperone. We have also identified a regulatory factor involved in this structural and functional switching. Exposure of P. aeruginosa to hydrogen peroxide (H₂O₂) causes PaPrx to convert from a high MW (HMW) complex to a low MW (LMW) form, which triggers a chaperone to peroxidase functional switch. This structural switching is primarily guided by either the thioredoxin (Trx) or glutathione (GSH) systems. Furthermore, comparison of our structural data [native and non-reducing polyacrylamide gel electrophoresis (PAGE) analysis, size exclusion chromatography (SEC) analysis, and electron microscopy (EM) observations] and enzymatic analyses (peroxidase and chaperone assay) revealed that the formation of oligomeric HMW complex structures increased chaperone activity of PaPrx. These results suggest that multimerization of PaPrx complexes promotes chaperone activity, and dissociation of the complexes into LMW species enhances peroxidase activity. Thus, the dual functions of PaPrx are clearly associated with their ability to form distinct protein structures.     

The C-terminal extension of chloroplast 2-Cys peroxiredoxin is critical for interaction with ATP

2-Cys peroxiredoxins (2-Cys Prxs) are ubiquitous enzymes that have been implicated in peroxide-mediated signaling of markedly different processes, such as cancer and photosynthesis. A highly conserved C-terminal extension of eukaryotic homologues modulates both the overoxidation of cysteines and the formation of oligomers. Here, we reveal that the plant counterpart regulates the self-polymerization of 2-Cys Prx triggered by ATP and Mg(2+). This feature is of particular importance under oxidative stress because the interaction of ATP with 2-Cys Prx rapidly integrates nonredox chemistry of signaling pathways into a network hub governed by multiple redox transformations at cysteine residues.     

Microbial 2-Cys Peroxiredoxins: Insights into Their Complex Physiological Roles

The peroxiredoxins (Prxs) constitute a very large and highly conserved family of thiol-based peroxidases that has been discovered only very recently. We consider here these enzymes through the angle of their discovery, and of some features of their molecular and physiological functions, focusing on complex phenotypes of the gene mutations of the 2-Cys Prxs subtype in yeast. As scavengers of the low levels of H₂O₂ and as H₂O₂ receptors and transducers, 2-Cys Prxs have been highly instrumental to understand the biological impact of H₂O₂, and in particular its signaling function. 2-Cys Prxs can also become potent chaperone holdases, and unveiling the in vivo relevance of this function, which is still not established, should further increase our knowledge of the biological impact and toxicity of H₂O₂. The diverse molecular functions of 2-Cys Prx explain the often-hard task of relating them to peroxiredoxin genes phenotypes, which underscores the pleiotropic physiological role of these enzymes and complex biologic impact of H₂O₂.     

How pH Modulates the Dimer-Decamer Interconversion of 2-Cys Peroxiredoxins from the Prx1 Subfamily

2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H₂O₂ and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His¹¹³) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp⁷⁶ from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H₂O₂ can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H₂O₂ and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress.     

ATP and Mg2+ promote the reversible oligomerization and aggregation of chloroplast 2-Cys peroxiredoxin

2-Cys peroxiredoxins (2-Cys Prxs) are ubiquitous peroxidases with important roles in cellular antioxidant defense and hydrogen peroxide-mediated signaling. Post-translational modifications of conserved cysteines cause the transition from low to high molecular weight oligomers, triggering the functional change from peroxidase to molecular chaperone. However, it remains unclear how non-covalent interactions of 2-Cys Prx with metabolites modulate the quaternary structure. Here, we disclose that ATP and Mg(2+) (ATP/Mg) promote the self-polymerization of chloroplast 2-Cys Prx (polypeptide 23.5 kDa) into soluble higher order assemblies (>2 MDa) that proceed to insoluble aggregates beyond 5 mM ATP. Remarkably, the withdrawal of ATP or Mg(2+) brings soluble oligomers and insoluble aggregates back to the native conformation without compromising the associated functions. As confirmed by transmission electron microscopy, ATP/Mg drive the toroid-like decamers (diameter 13 nm) to the formation of large sphere-like particles (diameter ～30 nm). Circular dichroism studies on ATP-labeled 2-Cys Prx reveal that ATP/Mg enhance the proportion of β-sheets with the concurrent decrease in the content of α-helices. In line with this observation, the formation of insoluble aggregates is strongly prevented by 2,2,2-trifluoroethanol, a cosolvent employed to induce α-helical conformations. We further find that the response of self-polymerization to ATP/Mg departs abruptly from that of the associated peroxidase and chaperone activities when two highly conserved residues, Arg(129) and Arg(152), are mutated. Collectively, our data uncover that non-covalent interactions of ATP/Mg with 2-Cys Prx modulate dynamically the quaternary structure, thereby coupling the non-redox chemistry of cell energy with redox transformations at cysteine residues.     

The 1-Cys peroxiredoxin, a regulator of seed dormancy, functions as a molecular chaperone under oxidative stress conditions

Peroxiredoxins are antioxidative enzymes that catalyze the reduction of alkyl hydroperoxides to alcohols and hydrogen peroxide to water. 1-Cys peroxiredoxins (1-Cys Prxs) perform important roles during late seed development in plants. To characterize their biochemical functions in plants, a 1Cys-Prx gene was cloned from a Chinese cabbage cDNA library and designated as “C1C-Prx”. Glutamine synthetase (GS) protection and hydrogen peroxide reduction assays indicated that C1C-Prx was functionally active as a peroxidase. Also C1C-Prx prevented the thermal- or chemical-induced aggregation of malate dehydrogenase and insulin. Hydrogen peroxide treatment changed the mobility of C1C-Prx on a two-dimensional gel, which implies overoxidation of the conserved Cys residue. Furthermore, after overoxidation, the chaperone activity of C1C-Prx increased approximately two-fold, but its peroxidase activity decreased to the basal level of the reaction mixture without enzyme. However, according to the structural analysis using far-UV circular dichroism spectra, intrinsic tryptophan fluorescence spectra, and native-PAGE, overoxidation did not lead to a conformational change in C1C-Prx. Therefore, our results suggest that 1-Cys Prxs function not only to relieve mild oxidative stresses but also as molecular chaperones under severe conditions during seed germination and plant development, and that overoxidation controls the switch in function of 1-Cys-Prxs from peroxidases to molecular chaperones.     

The C-type Arabidopsis thioredoxin reductase ANTR-C acts as an electron donor to 2-Cys peroxiredoxins in chloroplasts

2-Cys peroxiredoxins (Prxs) play important roles in the antioxidative defense systems of plant chloroplasts. In order to determine the interaction partner for these proteins in Arabidopsis, we used a yeast two-hybrid screening procedure with a C175S-mutant of Arabidopsis 2-Cys Prx-A as bait. A cDNA encoding an NADPH-dependent thioredoxin reductase (NTR) isotype C was identified and designated ANTR-C. We demonstrated that this protein effected efficient transfer of electrons from NADPH to the 2-Cys Prxs of chloroplasts. Interaction between 2-Cys Prx-A and ANTR-C was confirmed by a pull-down experiment. ANTR-C contained N-terminal TR and C-terminal Trx domains. It exhibited both TR and Trx activities and co-localized with 2-Cys Prx-A in chloroplasts. These results suggest that ANTR-C functions as an electron donor for plastidial 2-Cys Prxs and represents the NADPH-dependent TR/Trx system in chloroplasts.     

Repeated Superovulation via PMSG/hCG Administration Induces 2-Cys Peroxiredoxins Expression and Overoxidation in the Reproductive Tracts of Female Mice

Superovulation induced by exogenous gonadotropin treatment (PMSG/hCG) increases the number of available oocytes in humans and animals. However, Superovulatory PMSG/hCG treatment is known to affect maternal environment, and these effects may result from PMSG/hCG treatment-induced oxidative stress. 2-Cys peroxiredoxins (2-Cys Prxs) act as antioxidant enzymes that protect cells from oxidative stress induced by various exogenous stimuli. Therefore, the objective of this study was to test the hypothesis that repeated PMSG/hCG treatment induces 2-Cys Prx expression and overoxidation in the reproductive tracts of female mice. Immunohistochemistry and western blotting analyses further demonstrated that, after PMSG/hCG treatment, the protein expression levels of 2-Cys Prxs increased most significantly in the ovaries, while that of Prx1 was most affected by PMSG/hCG stimulation in all tissues of the female reproductive tract. Repeated PMSG/hCG treatment eventually leads to 2-Cys Prxs overoxidation in all reproductive organs of female mice, and the abundance of the 2-Cys Prxs-SO_2/3 proteins reported here supports the hypothesis that repeated superovulation induces strong oxidative stress and damage to the female reproductive tract. Our data suggest that excessive oxidative stress caused by repeated PMSG/hCG stimulation increases 2-Cys Prxs expression and overoxidation in the female reproductive organs. Intracellular 2-Cys Prx therefore plays an important role in maintaining the reproductive organ environment of female mice upon exogenous gonadotropin treatment.     

A eukaryotic-like sulfiredoxin involved in oxidative stress responses and in the reduction of the sulfinic form of 2-Cys peroxiredoxin in the cyanobacterium Anabaena PCC 7120

The overoxidation of 2-Cys peroxiredoxins (Prxs) into a sulfinic form was thought to be an irreversible protein inactivation process until sulfiredoxins (Srxs) were discovered. These are enzymes occurring among eukaryotes, which are able to reduce sulfinylated Prxs. Although Prxs are present in the three domains of life, their reduction by Srxs has been described only in eukaryotes so far. Here it was established that the cyanobacterium Anabaena PCC 7120 has a Srx homologue (SrxA), which is able to specifically reduce the sulfinic form of the 2-Cys Prx (PrxA) both in vivo and in vitro. A mutant lacking the srxA gene was found to be more sensitive than the wild type to oxidative stress. Sulfiredoxin homologues are restricted to the cyanobacterial and eukaryotic genomes sequenced so far. The present phylogenetic analysis of Srx and 2-Cys Prx sequences showed a pattern of coevolution of the enzyme and its substrate that must have involved an ancient gene transfer between ancestors of Cyanobacteria and Eukaryotes, followed by a more recent transfer from Cyanobacteria to Plantae through the chloroplastic endosymbiosis. This is the first functional characterization of a Srx enzyme in a prokaryotic organism.     

Functional switching of a novel prokaryotic 2-Cys peroxiredoxin (PpPrx) under oxidative stress

Many proteins have been isolated from eukaryotes as redox-sensitive proteins, but whether these proteins are present in prokaryotes is not clear. Redox-sensitive proteins contain disulfide bonds, and their enzymatic activity is modulated by redox in vivo. In the present study, we used thiol affinity purification and mass spectrometry to isolate and identify 19 disulfide-bond-containing proteins in Pseudomonas putida exposed to potential oxidative damages. Among these proteins, we found that a typical 2-Cys Prx-like protein (designated PpPrx) displays diversity in structure and apparent molecular weight (MW) and can act as both a peroxidase and a molecular chaperone. We also identified a regulatory factor involved in this structural and functional switching. Exposure of pseudomonads to hydrogen peroxide (H₂O₂) caused the protein structures of PpPrx to convert from high MW complexes to low MW forms, triggering a chaperone-to-peroxidase functional switch. This structural switching was primarily guided by the thioredoxin system. Thus, the peroxidase efficiency of PpPrx is clearly associated with its ability to form distinct protein structures in response to stress.     

Engineering of 2-Cys peroxiredoxin for enhanced stress-tolerance

A typical 2-cysteine peroxiredoxin (2-Cys Prx)-like protein (PpPrx) that alternatively acts as a peroxidase or a molecular chaperone in Pseudomonas putida KT2440 was previously characterized. The dual functions of PpPrx are regulated by the existence of an additional Cys(112) between the active Cys(51) and Cys(171) residues. In the present study, additional Cys residues (Cys(31), Cys(112), and Cys(192)) were added to PpPrx variants to improve their enzymatic function. The optimal position of the additional Cys residues for the dual functionality was assessed. The peroxidase activities of the S31C and Y192C mutants were increased 3- to 4-fold compared to the wild-type, while the chaperone activity was maintained at > 66% of PpPrx. To investigate whether optimization of the dual functions could enhance stress-tolerance in vivo, a complementation study was performed. The S31C and Y192C mutants showed a much greater tolerance than other variants under a complex condition of heat and oxidative stresses. The optimized dual functions of PpPrx could be adapted for use in bioengineering systems and industries, such as to develop organisms that are more resistant to extreme environments.     

Switching between the Alternative Structures and Functions of a 2-Cys Peroxiredoxin,by Site-Directed Mutagenesis

Members of the typical 2-Cys peroxiredoxin (Prx) subfamily represent an intriguing example of protein moonlighting behavior since this enzyme shifts function: indeed, upon chemical stimuli, such as oxidative stress, Prx undergoes a switch from peroxidase to molecular chaperone, associated to a change in quaternary structure from dimers/decamers to higher-molecular-weight (HMW) species. In order to detail the structural mechanism of this switch at molecular level, we have designed and expressed mutants of peroxiredoxin I from Schistosoma mansoni (SmPrxI) with constitutive HMW assembly and molecular chaperone activity. By a combination of X-ray crystallography, transmission electron microscopy and functional experiments, we defined the structural events responsible for the moonlighting behavior of 2-Cys Prx and we demonstrated that acidification is coupled to local structural variations localized at the active site and a change in oligomerization to HMW forms, similar to those induced by oxidative stress. Moreover, we suggest that the binding site of the unfolded polypeptide is at least in part contributed by the hydrophobic surface exposed by the unfolding of the active site. We also find an inverse correlation between the extent of ring stacking and molecular chaperone activity that is explained assuming that the binding occurs at the extremities of the nanotube, and the longer the nanotube is, the lesser the ratio binding sites/molecular mass is.     

Molecular cloning and expression of a 2-Cys peroxiredoxin gene in the crustacean Eurypanopeus depressus induced by acute hypo-osmotic stress

Peroxiredoxins (Prxs) are a family of ubiquitous proteins that help minimize the harmful effects of oxidative stress by catalyzing the reduction of hydrogen peroxide (H₂O₂) and organic hydroperoxides to less harmful forms. A full-length cDNA corresponding to a 2-Cys Prx gene was isolated from the flatback mud crab Eurypanopeus depressus and designated as EdPrx-1 (GenBank accession no. EU684547). EdPrx-1 has a major open-reading frame of 594 bp and is capable of encoding a polypeptide of 198 amino acid residues. Like other 2-Cys Prxs, EdPrx-1 protein possesses two conserved cysteine residues that play an essential role for the antioxidant activity of the proteins. The EdPrx-1 protein, as deduced from the cDNA sequence, shows a high level (74–93%) of sequence similarity to the 2-Cys Prxs from other crustaceans as well as those from many arthropod species (73–76% similarity). It shares about 70% sequence similarity with homologs from mammalian species. EdPrx-1 gene is expressed at low level in the gill, hypodermis, and hepatopancreas tissues of the crab under non-stressed condition; however, its expression is elevated about three-fold in the gills under hypo-osmotic stress. This suggests a possible role in protecting against oxidative stress caused by the increased metabolic activities associated with hyperosmoregulation.     

Chloroplast NADPH-Dependent Thioredoxin Reductase from Chlorella vulgaris Alleviates Environmental Stresses in Yeast Together with 2-Cys Peroxiredoxin

Chloroplast NADPH-dependent thioredoxin reductase (NTRC) catalyzes the reduction of 2-Cys peroxiredoxin (2-Cys Prx) and, thus, probably functions as an antioxidant system. The functions of the enzyme in oxidative and salt stresses have been reported previously. We have previously identified and characterized NTRC in Chlorella vulgaris. In the present study, we isolated a full-length cDNA clone encoding 2-Cys Prx from C. vulgaris and investigated the involvement of Chlorella NTRC/2-Cys Prx system in several environmental stress tolerances by using yeast as a eukaryotic model. Deduced Chlorella 2-Cys Prx was homologous to those of chloroplast 2-Cys Prxs from plants, and two conserved cysteine residues were found in the deduced sequence. Enzyme assay showed that recombinant mature C. vulgaris NTRC (mCvNTRC) transferred electrons from NADPH to recombinant mature C. vulgaris 2-Cys Prx (mCvPrx), and mCvPrx decomposed hydrogen peroxide, tert-butyl hydroperoxide, and peroxynitrite by cooperating with mCvNTRC. Based on the results, the mCvNTRC/mCvPrx antioxidant system was identified in Chlorella. The antioxidant system genes were expressed in yeast separately or coordinately. Stress tolerances of yeast against freezing, heat, and menadione-induced oxidative stresses were significantly improved by expression of mCvNTRC, and the elevated tolerances were more significant when both mCvNTRC and mCvPrx were co-expressed. Our results reveal a novel feature of NTRC: it functions as an antioxidant system with 2-Cys Prx in freezing and heat stress tolerances.     

Mitochondria of Saccharomyces cerevisiae contain one-conserved cysteine type peroxiredoxin with thioredoxin peroxidase activity

Peroxiredoxins are ubiquitously expressed proteins that reduce hydroperoxides using disulfur-reducing compounds as electron donors. Peroxiredoxins (Prxs) have been classified in two groups dependent on the presence of either one (1-Cys Prx) or two (2-Cys Prx) conserved cysteine residues. Moreover, 2-Cys Prxs, also named thioredoxin peroxidases, have peroxide reductase activity with the use of thioredoxin as biological electron donor. However, the biological reducing agent for the 1-Cys Prx has not yet been identified. We report here the characterization of a 1-Cys Prx from yeast Saccharomyces cerevisiae that we have named Prx1p. Prx1p is located in mitochondria, and it is overexpressed when cells use the respiratory pathway, as well as in response to oxidative stress conditions. We show also that Prx1p has peroxide reductase activity in vitro using the yeast mitochondrial thioredoxin system as electron donor. In addition, a mutated form of Prx1p containing the absolutely conserved cysteine as the only cysteine residue also shows thioredoxin-dependent peroxide reductase activity. This is the first example of 1-Cys Prx that has thioredoxin peroxidase activity. Finally, exposure of null Prx1p mutant cells to oxidant conditions reveals an important role of the mitochondrial 1-Cys Prx in protection against oxidative stress.     

The yeast Tsa1 peroxiredoxin is a ribosome-associated antioxidant

The yeast Tsa1 peroxiredoxin, like other 2-Cys peroxiredoxins, has dual activities as a peroxidase and as a molecular chaperone. Its peroxidase function predominates in lower-molecular-mass forms, whereas a super-chaperone form predominates in high-molecular-mass complexes. Loss of TSA1 results in aggregation of ribosomal proteins, indicating that Tsa1 functions to maintain the integrity of the translation apparatus. In the present study we report that Tsa1 functions as an antioxidant on actively translating ribosomes. Its peroxidase activity is required for ribosomal function, since mutation of the peroxidatic cysteine residue, which inactivates peroxidase but not chaperone activity, results in sensitivity to translation inhibitors. The peroxidatic cysteine residue is also required for a shift from ribosomes to its high-molecular-mass form in response to peroxide stress. Thus Tsa1 appears to function predominantly as an antioxidant in protecting both the cytosol and actively translating ribosomes against endogenous ROS (reactive oxygen species), but shifts towards its chaperone function in response to oxidative stress conditions. Analysis of the distribution of Tsa1 in thioredoxin system mutants revealed that the ribosome-associated form of Tsa1 is increased in mutants lacking thioredoxin reductase (trr1) and thioredoxins (trx1 trx2) in parallel with the general increase in total Tsa1 levels which is observed in these mutants. In the present study we show that deregulation of Tsa1 in the trr1 mutant specifically promotes translation defects including hypersensitivity to translation inhibitors, increased translational error-rates and ribosomal protein aggregation. These results have important implications for the role of peroxiredoxins in stress and growth control, since peroxiredoxins are likely to be deregulated in a similar manner during many different disease states.     

Structural and functional analysis of native peroxiredoxin 2 in human red blood cells

Peroxiredoxin 2, a typical 2-Cys peroxiredoxin, is the third most abundant protein in erythrocytes. It is understood that the physiologically functional state of peroxiredoxin 2 is the monomer, and that its role in scavenging low levels of H(2)O(2) results in the formation of disulfide-linked dimers, which are reversibly reduced to monomers by the thioredoxin-thioredoxin reductase system. Additionally, peroxiredoxins are highly susceptible to sulfinic acid formation through reactions with various peroxides. This overoxidized form, which is thought to convert peroxiredoxins into molecular chaperones and to be accompanied by a transition to polymeric forms, can be reversed by sulfiredoxins. However, physiological conformational changes and the antioxidant role of erythrocyte peroxiredoxin 2 are still unclear because there is low sulfiredoxin and thioredoxin-thioredoxin reductase activity in erythrocytes. In this study, we examined the structural and redox states of peroxiredoxin 2 in fresh hemolysates and estimated the activities of native and overoxidized peroxiredoxin 2 purified from red blood cells to clear the physiological roles of peroxiredoxin 2 in erythrocyte. Our findings demonstrate that native peroxiredoxin 2 exists as high molecular weight (>160 kDa) oligomers and that decamers or higher order molecular weight oligomers (260-460 kDa) have peroxidase activity. We further showed that peroxiredoxin 2 oligomers, which were predominantly composed of monomers in the reduced form, exert a chaperone activity equal to that of overoxidized peroxiredoxin 2 polymers. These results provide the novel insight that redox-active peroxiredoxin 2 functions in human red blood cells as high molecular weight oligomers that possess peroxidase and chaperone activities.