Peroxiredoxins: a less studied component of hydrogen peroxide detoxification in photosynthetic organisms

Peroxiredoxins (Prx) are ubiquitous thiol-dependent peroxidases capable of reducing a broad range of toxic peroxides and peroxinitrites. A cysteinyl residue of peroxiredoxins reacts with the peroxides as primary catalytic center and oxidizes to sulfenic acid. The regeneration of the reduced form of Prx is required as a next step to allow its entry into next catalytic cycle. Several proteins, such as thioredoxin, glutaredoxin, cyclophilin, among others, are known to facilitate the regeneration of the reduced (catalytically active) form of Prx in plants. Based on the cysteine residues conserved in the deduced amino acid sequence and their catalytic mechanisms, four groups of peroxiredoxins have been distinguished in plants, namely, 1-Cys Prx, 2-Cys Prx, Type II Prx and Prx Q. Peroxiredoxins are known to play an important role in combating the reactive oxygen species generated at the level of electron transport activities in the plant exposed to different types of biotic and abiotic stresses. In addition to their role in antioxidant defense mechanisms in plants, they also modulate redox signaling during development and adaptation. Besides these general properties, peroxiredoxins have been shown to protect DNA from damage in vitro and in vivo. They also regulate metabolism in thylakoids and mitochondria. The present review summarizes the most updated information on the structure and catalysis of Prx and their functional importance in plant metabolism.     

Plant peroxiredoxins: alternative hydroperoxide scavenging enzymes

The role of plant peroxiredoxins in the detoxification systems is discussed in relation with the existence of many isoforms of this protein in distinct plant compartments. Phylogenetic analyses indicate that plant peroxiredoxins can be divided into four classes. Two of these classes correspond to chloroplastic enzymes. All isoforms contain at least one conserved catalytic cysteine. The enzymes belonging to the 1-Cys Prx class seem to be seed restricted and to play a role of detoxification during the germination process. At least one putative cytosolic isoform can use both thioredoxin and glutaredoxin as an electron donor, but the chloroplastic isoforms characterized depend on reduced thioredoxin. Mutagenesis and plant transformation studies support the proposal that the chloroplastic peroxiredoxins play an important role in combating the ROS species generated at the level of the chloroplastic electron transfer chain. This revised version was published online in June 2006 with corrections to the Cover Date.     

The function of peroxiredoxins in plant organelle redox metabolism

In 1996, cDNA sequences referred to as plant peroxiredoxins (Prx), i.e. a 1-Cys Prx and a 2-Cys Prx, were reported from barley. Ten years of research have advanced our understanding of plant Prx as thiol-based peroxide reductases with a broad substrate specificity, ranging from hydrogen peroxide to alkyl hydroperoxides and peroxinitrite. Prx have several features in common. (i) They are abundant proteins that are routinely detected in proteomics approaches. (ii) They interact with proteins such as glutaredoxins, thioredoxins, and cyclophilins as reductants, but also non-dithiol-disulphide exchange proteins. By work with transgenic plants, their activity was shown to (iii) affect metabolic integrity, (iv) protect DNA from damage in vitro and as shown here in vivo, and (v) modulate intracellular signalling related to reactive oxygen species and reactive nitrogen species. (vi) In all organisms Prx are encoded by small gene families that are of particular complexity in higher plants. A comparison of the Prx gene families in rice and Arabidopsis thaliana supports previous suggestions on Prx function in specific subcellular and metabolic context. (vii) Prx gene expression and activity are subjected to complex regulation realized by an integration of various signalling pathways. 2-Cys Prx expression depends on redox signals, abscisic acid, and protein kinase cascades. Besides these general properties, the chloroplast Prx have acquired specific roles in the context of photosynthesis. The thioredoxin-dependent peroxidase activity can be measured in crude plant extracts and contributes significantly to the overall H(2)O(2) detoxification capacity. Thus organellar Prx proteins enable an alternative water-water cycle for detoxification of photochemically produced H(2)O(2), which acts independently from the ascorbate-dependent Asada-Halliwell-Foyer cycle. 2-Cys Prx and Prx Q associate with thylakoid membrane components. The mitochondrial PrxII F is essential for root growth under stress. Following a more general introduction, the paper summarizes present knowledge on plant organellar Prx, addressing Prx in signalling, and also suggests some lines for future research.     

Hydroperoxide and peroxynitrite reductase activity of poplar thioredoxin-dependent glutathione peroxidase 5: kinetics, catalytic mechanism and oxidative inactivation

Gpxs (glutathione peroxidases) constitute a family of peroxidases, including selenocysteine- or cysteine-containing isoforms (SeCys-Gpx or Cys-Gpx), which are regenerated by glutathione or Trxs (thioredoxins) respectively. In the present paper we show new data concerning the substrates of poplar Gpx5 and the residues involved in its catalytic mechanism. The present study establishes the capacity of this Cys-Gpx to reduce peroxynitrite with a catalytic efficiency of 106 M-1·s-1. In PtGpx5 (poplar Gpx5; Pt is Populus trichocarpa), Glu79, which replaces the glutamine residue usually found in the Gpx catalytic tetrad, is likely to be involved in substrate selectivity. Although the redox midpoint potential of the Cys44-Cys92 disulfide bond and the pKa of Cys44 are not modified in the E79Q variant, it exhibited significantly improved kinetic parameters (Kperoxide and kcat) with tert-butyl hydroperoxide. The characterization of the monomeric Y151R variant demonstrated that PtGpx5 is not an obligate homodimer. Also, we show that the conserved Phe90 is important for Trx recognition and that Trx-mediated recycling of PtGpx5 occurs via the formation of a transient disulfide bond between the Trx catalytic cysteine residue and the Gpx5 resolving cysteine residue. Finally, we demonstrate that the conformational changes observed during the transition from the reduced to the oxidized form of PtGpx5 are primarily determined by the oxidation of the peroxidatic cysteine into sulfenic acid. Also, MS analysis of in-vitro-oxidized PtGpx5 demonstrated that the peroxidatic cysteine residue can be over-oxidized into sulfinic or sulfonic acids. This suggests that some isoforms could have dual functions potentially acting as hydrogen-peroxide- and peroxynitrite-scavenging systems and/or as mediators of peroxide signalling as proposed for 2-Cys peroxiredoxins.     

Peroxiredoxin 1 and its role in cell signaling

Peroxiredoxins (Prdxs) are a family of small (22-27kDa) non-seleno peroxidases currently known to possess six mammalian isoforms. Although their individual roles in cellular redox regulation and antioxidant protection are quite distinct, they all catalyze peroxide reduction of H2O2, organic hydroperoxides and peroxynitrite. They are found to be expressed ubiquitously and in high levels, suggesting that they are both an ancient and important enzyme family. Prdxs can be divided into three major subclasses: typical 2-cysteine (2-Cys) Prdxs (Prdx1-4), atypical 2-Cys Prdx (Prdx 5) and 1-Cys Prdx (Prdx 6). Recent evidence suggests that 2-Cys peroxiredoxins are more than “just simple peroxidases”. This hypothesis has been discussed elegantly in recent review articles, considering “over”-oxidation of the protonated thiolate peroxidatic cysteine and post-translational modification of Prdxs as processes initiating a mechanistic switch from peroxidase to chaperon function. The process of over-oxidation of the peroxidatic cysteine (CP) occurs during catalysis in the presence of thioredoxin (Trx), thus rendering the sulfenic moiety to sulfinic acid , which can be reduced by sulfiredoxin (Srx). However, further oxidation to sulfonic acid is believed to promote Prdx degradation or, as recently shown, the formation of oligomeric peroxidase-inactive chaperones10 with questionable H2O2-scavenging capacity. In the light of this and given that Prdx1 has recently been shown by us and by others to interact directly with signaling molecules, we will explore the possibility that H2O2 regulates signaling in the cell in a temporal and spatial fashion via oxidizing Prdx1. Therefore, this review will focus on H2O2 modulating cell signaling via Prdxs by discussing: a) the activity of Prdxs towards H2O2; b) sub cellular localization and availability of other peroxidases, such as catalase or glutathione peroxidases; c) the availability of Prdxs reducing systems such as thioredoxin and sulfiredoxin and lastly, d) Prdx1 interacting signaling molecules.     

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.     

FePer 1, a gene encoding an evolutionarily conserved 1-Cys peroxiredoxin in buckwheat (Fagopyrum esculentum Moench), is expressed in a seed-specific manner and induced during seed germination

A cDNA corresponding to 1-Cys peroxiredoxin, an evolutionarily conserved thiol-specific antioxidant enzyme, was isolated from buckwheat (Fagopyrum esculentum Moench), a dicotyledonous plant species belonging to the Polygonaceae family. The cDNA, which we have designated as FePer1, contains a major open reading frame capable of encoding a polypeptide of 219 residues with a predicted molecular mass of 24.3kDa. The deduced primary structure of FePer1 polypeptide shows a high level (about 70%) of sequence homology to other recently identified plant 1-Cys peroxiredoxins. FePer1 also exhibits a significant level of sequence similarity to non-plant 1-Cys peroxiredoxins, sharing 52 and 42% identities with mammalian and fungal 1-Cys peroxiredoxins, respectively. As for all 1-Cys peroxiredoxins identified from various organisms, the amino acid sequence proposed to constitute the active site of the enzyme is highly conserved in FePer1 polypeptide. The gene corresponding to FePer1 cDNA is a single-copy gene in the buckwheat genome. Its expression is regulated in a seed-specific and temporal manner during seed development. FePer1 gene is induced transiently for a short period immediately after seed imbibition.     

Bacterial defenses against oxidants: mechanistic features of cysteine-based peroxidases and their flavoprotein reductases

Antioxidant defenses include a group of ubiquitous, non-heme peroxidases, designated the peroxiredoxins, which rely on an activated cysteine residue at their active site to catalyze the reduction of hydrogen peroxide, organic hydroperoxides, and peroxynitrite. In the typical 2-Cys peroxiredoxins, a second cysteinyl residue, termed the resolving cysteine, is also involved in intersubunit disulfide bond formation during the course of catalysis by these enzymes. Many bacteria also express a flavoprotein, AhpF, which acts as a dedicated disulfide reductase to recycle the bacterial peroxiredoxin, AhpC, during catalysis. Mechanistic and structural studies of these bacterial proteins have shed light on the linkage between redox state, oligomeric state, and peroxidase activity for the peroxiredoxins, and on the conformational changes accompanying catalysis by both proteins. In addition, these studies have highlighted the dual roles that the oxidized cysteinyl species, cysteine sulfenic acid, can play in eukaryotic peroxiredoxins, acting as a catalytic intermediate in the peroxidase activity, and as a redox sensor in regulating hydrogen peroxide-mediated cell signaling.     

Peroxiredoxins from Trypanosoma cruzi: virulence factors and drug targets for treatment of Chagas disease?

Cytosolic and mitochondrial Trypanosoma cruzi tryparedoxin peroxidases belong to the family of 2-Cys peroxiredoxins. These enzymes play an essential role as antioxidants by their peroxidase and peroxynitrite reductase activities. TXNPx are key components of the trypanosomatid peroxide detoxification pathways. The aim of this work was to determine the role of TXNPx as virulence factors in the parasite, and whether these enzymes are good candidates for drug design. We observed that peroxiredoxins are not highly abundant proteins expressed at similar levels throughout the T. cruzi life cycle. In order to study the role of c-TXNPx and m-TXNPx in invasion and infectivity, parasites overexpressing TXNPx were produced, and infection experiments were carried out using phagocytic and non-phagocytic cells. Parasites overexpressing peroxiredoxins showed a significant increase in infectivity with respect to the control ones. The results presented in this work point out that the T. cruzi peroxiredoxins are important in survival, replication and differentiation of T. cruzi and could constitute virulence factors. Moreover, their expression in the infective forms of the life cycle and their low intracellular concentration make them good candidates to become targets for drug design.     

The significance of type II and PrxQ peroxiredoxins for antioxidative stress response in the purple bacterium Rhodobacter sphaeroides

Two peroxiredoxins, classified as Type II and PrxQ, were characterized in the purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides. Both recombinant proteins showed remarkable thioredoxin-dependent peroxidase activity with broad substrate specificity in vitro. Nevertheless, PrxQ of R. sphaeroides, unlike typical PrxQs studied to date, does not contain one of the two conserved catalytic Cys residues. We found that R. sphaeroides PrxQ and other PrxQ-like proteins from several organisms conserve a different second Cys residue, indicating that these proteins should be categorized into a novel PrxQ subfamily. Disruption of either the Type II or PrxQ gene in R. sphaeroides had a dramatic effect on cell viability when the cells were grown under aerobic light or oxidative stress conditions created by exogenous addition of reactive oxygen species to the medium. Growth rates of the mutants were significantly decreased compared with that of wild type under aerobic but not anaerobic conditions. These results indicate that the peroxiredoxins are crucial for antioxidative stress response in this bacterium. The gene disruptants also demonstrated reduced levels of photopigment synthesis, suggesting that the peroxiredoxins are directly or indirectly involved in regulated synthesis of the photosynthetic apparatus.     

A selenocysteine-containing peroxiredoxin from the strictly anaerobic organism Eubacterium acidaminophilum

A strongly 75Se-labeled 22 kDa protein detected previously showed in its N-terminal sequence the highest similarity to the family of thiol-dependent peroxidases, now called peroxiredoxins. The respective gene prxU was cloned and analyzed. prxU encodes a protein of 203 amino acids (22,470 Da) and contains an in-frame UGA codon (selenocysteine) at the position of the so far strictly conserved and catalytically active Cys47. The second conserved cysteine present in 2-Cys peroxiredoxins was replaced by alanine. Heterologous expression of the Eubacterium acid-aminophilum PrxU as a recombinant selenoprotein in Escherichia coli was not possible. A cysteine-encoding mutant gene, prxU47C, containing UGC instead of UGA was strongly expressed. This recombinant PrxU47C mutant protein was purified to homogeneity by its affinity tag, but was not active as a thiol-dependent peroxidase. The identification of prxU reveals that the limited class of natural selenoproteins may in certain organisms also include isoenzymes of peroxiredoxins, previously only known as non-selenoproteins containing catalytic cysteine residues.     

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.     

Biochemical characterization of 2-Cys peroxiredoxins from Schistosoma mansoni

Peroxiredoxins are a large family of peroxidases that have important antioxidant and cell signaling functions. Genes encoding two novel 2-cysteine peroxiredoxin proteins were identified in the expressed sequence tag data base of the helminth parasite Schistosoma mansoni, a causative agent of schistosomiasis. The recombinant proteins showed peroxidase activity in vitro with a variety of hydroperoxides and used both the thioredoxin and the glutathione systems as electron donors. Steady-state kinetic analysis indicated that the new peroxiredoxins had saturable kinetics, whereas a previously identified schistosome peroxiredoxin was found to function with more typical unsaturable (ping-pong) kinetics. The catalytic efficiencies S. mansoni peroxiredoxins were similar to those for other peroxiredoxins studied (10(4)-10(5) m(-1) s(-1)). Mutagenesis of S. mansoni peroxiredoxins indicated that glutathione dependence and kinetic differences were conferred by the C-terminal alpha-helix forming 22 amino acids. This is the first report of 2-cysteine peroxiredoxins efficiently utilizing reducing equivalents from both the thioredoxin and glutathione systems. Studies to determine the resistance to oxidative inactivation, important in regulating cell signaling pathways, showed that S. mansoni possess both bacterial-like resistant and mammalian-like sensitive peroxiredoxins. The susceptibility to oxidative inactivation was conferred by the C-terminal tail containing a tyrosine-phenylalanine motif. S. mansoni is the first organism shown to possess both robust and sensitive peroxiredoxins. The ability of schistosome peroxiredoxins to use alternative electron donors, and their variable resistance to overoxidation may reflect their presence in different cellular sites and emphasizes the significant differences in overall redox balance mechanisms between the parasite and its mammalian host.     

Isolation and characterization of a thioredoxin-dependent peroxidase from Chlamydomonas reinhardtii.

All living organisms contain redox systems involving thioredoxins (Trx), proteins featuring an extremely conserved and reactive active site that perform thiol-disulfide interchanges with disulfide bridges of target proteins. In photosynthetic organisms, numerous isoforms of Trx coexist, as revealed by sequencing of Arabidopsis genome. The specific functions of many of them are still unknown. In an attempt to find new molecular targets of Trx in Chlamydomonas reinhardtii, an affinity column carrying a cytosolic Trx h mutated at the less reactive cysteine of its active site was used to trap Chlamydomonas proteins that form mixed disulfides with Trx. The major protein bound to the column was identified by amino-acid sequencing and mass spectrometry as a thioredoxin-dependent 2Cys peroxidase. Isolation and sequencing of its gene revealed that this peroxidase is most likely a chloroplast protein with a high homology to plant 2Cys peroxiredoxins. It is shown that the Chlamydomonas peroxiredoxin (Ch-Prx1) is active with various thioredoxin isoforms, functions as an antioxidant toward reactive oxygen species (ROS), and protects DNA against ROS-induced degradation. Expression of the peroxidase gene in Chlamydomonas was found to be regulated by light, oxygen concentration, and redox state. The data suggest a role for the Chlamydomonas Prx in ROS detoxification in the chloroplast.     

Peroxiredoxins,a new family of antioxidant proteins

Current ideas are discussed about the structures and mechanisms of action of proteins that have been united at present into a family of thiol-specific antioxidants or peroxiredoxins, which protect the cells of different organisms from the action of hydrogen peroxide. Peroxiredoxins fulfill the same function as antioxidant enzymes such as catalases and glutathione-dependent peroxidases; however, their catalytic activity is lower than that of these enzymes. The level of expression of genes of peroxiredoxins is increased in many pathological states accompanied by oxidative stress, and today there is direct evidence for the important role of peroxiredoxins in the vital activity of cells.     

The function of the chloroplast 2-cysteine peroxiredoxin in peroxide detoxification and its regulation

The Arabidopsis genome contains nine open reading frames with homology to members of the peroxiredoxin (prx) family: one 1-Cys-prx, two 2-Cys-prx, five type II-prx, and one peroxiredoxin Q. The function of the peroxiredoxins in plant metabolism is only slowly emerging. They are assumed to reduce toxic peroxides to their corresponding alcohols with a rather broad substrate specificity. The 2-Cys peroxiredoxins (2-CP) were recently identified as members of the antioxidant defence system of chloroplasts. Knock-out mutants of Synechocystis and antisense mutants of Arabidopsis have provided insight into the function of 2-CPs in the photosynthetic antioxidant network. This review summarizes present knowledge on the enzymatic mechanism, the physiological context and the genetic regulation of the 2-CPs in plants and cyanobacteria. In addition, an extrapolation on the metabolic role of the chloroplast 2-CP is attempted based on the molecular features of 2-CPs from other organisms.     

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.     

Peroxiredoxins

Present knowledge on peroxiredoxins is reviewed with special emphasis on catalytic principles, specificities and biological function. Peroxiredoxins are low efficiency peroxidases using thiols as reductants. They appear to be fairly promiscuous with respect to the hydroperoxide substrate; the specificities for the donor substrate vary considerably between the subfamilies, comprising GSH, thioredoxin, tryparedoxin and the analogous CXXC motifs in bacterial AhpF proteins. Peroxiredoxins are definitely responsible for antioxidant defense in bacteria (AhpC), yeast (thioredoxin peroxidase) and trypanosomatids (tryparedoxin peroxidase). They are considered to determine virulence of mycobacteria and trypanosomatids. In higher plants they are involved in balancing hydroperoxide production during photosynthesis. In higher animals peroxiredoxins appear to be involved in the redox-regulation of cellular signaling and differentiation, displaying in part opposite effects.