Prxs (peroxiredoxins) are a family of proteins that are extremely effective at scavenging peroxides. The Prxs exhibit a number of intriguing properties that distinguish them from conventional antioxidants, including a susceptibility to inactivation by hyperoxidation in the presence of excess peroxide and the ability to form complex oligomeric structures. These properties, combined with a high cellular abundance and reactivity with hydrogen peroxide, have led to speculation that the Prxs function as redox sensors that transmit signals as part of the cellular response to oxidative stress. Multicellular organisms express several different Prxs that can be categorized by their subcellular distribution. In mammals, Prx 3 and Prx 5 are targeted to the mitochondrial matrix. Mitochondria are a major source of hydrogen peroxide, and this oxidant is implicated in the damage associated with aging and a number of pathologies. Hydrogen peroxide can also act as a second messenger, and is linked with signalling events in mitochondria, including the induction of apoptosis. A simple kinetic competition analysis estimates that Prx 3 will be the target for up to 90% of hydrogen peroxide generated in the matrix. Therefore, mitochondrial Prxs have the potential to play a major role in mitochondrial redox signalling, but the extent of this role and the mechanisms involved are currently unclear.
- hydrogen peroxide
- redox signalling
Prxs (peroxiredoxins) are a family of thiol peroxidases that scavenge peroxides in cells. The first Prx was discovered in Saccharomyces cerevisiae, where it protected the enzyme glutamine synthase from oxidation in a system that, serendipitously, included reduced thiols to generate hydrogen peroxide . The unknown protein was dependent on these thiols for its protective activity, and was named thiol-specific antioxidant. This was changed to thioredoxin peroxidase when the protein was discovered to be oxidized by hydrogen peroxide and reduced back to its active state by Trx (thioredoxin) [2,3]. Concurrent studies identified a homologous protein in Salmonella typhimurium known as alkylhydroperoxide reductase, which was important for conferring resistance to oxidative stress [4–6]. Once it became clear that many other homologous proteins shared this peroxidase activity, the term peroxiredoxin was introduced [2,7], and the nomenclature has been widely adopted since then.
Prxs are unique among the peroxidases in their utilization of cysteine at their active site. Catalysis is initiated by the reaction of this peroxidatic cysteine residue with hydrogen peroxide to a sulfenic acid. Although free cysteine reacts very slowly with hydrogen peroxide, the reactivity of the peroxidatic cysteine residue is increased by up to seven orders of magnitude [8–11]. The structural features responsible for this dramatic increase in reactivity have not been determined. The Prxs are also highly abundant proteins, comprising up to 1% of soluble cellular protein [12,13], which accounts for their appearance in several proteomic studies. This high reactivity and abundance has led to the realization that Prxs will have a central role in the response of cells to oxidative stress.
Structural analysis of the Prx family has stimulated speculation that they do not function solely as antioxidants. The Prx catalytic cycle differs depending on the class of Prx, but a common feature is that the sulfenic acid generated at the active site reacts with a neighbouring thiol to produce a disulfide. Eukaryotic Prxs were recognized to have evolved a C-terminal domain that interferes with disulfide bond formation . This results in a ‘kinetic pause’ in which the sulfenic acid can react with another molecule of hydrogen peroxide to be hyperoxidized and inactivated. This is a major impediment to antioxidant activity, and suggests an alternative function linked to the increased sensitivity to hyperoxidation. Prominent hypotheses include conversion of Prxs into molecular chaperones upon hyperoxidation [15–17], and the adaptation of Prxs to function as redox sensors that transmit signals upon increased environmental or endogenous peroxide flux [18,19]. Both possibilities are supported by the complex quaternary structures of Prxs, consisting of ring-shaped decamers and dodecamers that have the capacity to stack and intercalate . These structures are considerably more complex than would be required for decomposition of peroxides, and are regulated by post-translational modifications including phosphorylation and oxidation .
The Prxs of multicellular organisms can be categorized by their subcellular localization. Mammals have six Prxs, with Prx 1, 2 and 6 found in the cytoplasm, Prx 4 in the endoplasmic reticulum, Prx 3 in the mitochondria, and Prx 5 found in various compartments in the cell, including peroxisomes and mitochondria. Mitochondria are a major site of hydrogen peroxide generation in cells . There is considerable interest in the factors that control mitochondrial oxidant production and removal, and the role of disturbances in mitochondrial redox homoeostasis in fundamental processes such as apoptosis and metabolic signalling . This review focuses on the biochemical properties of the mammalian mitochondrial Prxs and their ability to protect mitochondria from oxidative stress, and discusses emerging evidence that these proteins play a central role in regulating redox-mediated signalling events.
BIOCHEMICAL PROPERTIES OF THE MITOCHONDRIAL PRXS
Research into the mitochondrial Prxs began in 1989 with the cloning of the mer5 gene, which was preferentially expressed in murine erythroleukaemia . Mer5, subsequently named murine Prx 3, was thought to play a significant role in the differentiation of erythrocytes . Subsequent studies revealed that Mer5 shared significant sequence homology with the bacterial Prx alkyl hydroperoxide reductase (AhpC) . During this period, Watabe et al. were investigating substrates of a mitochondrial ATP-dependent protease isolated from bovine adrenal cortex , later identified as the Lon protease . They discovered a 22 kDa substrate of Lon protease, which they named SP-22. In 1994, SP-22 was purified to homogeneity and characterized as a highly abundant mitochondrial matrix protein (1.6% of soluble mitochondrial protein) that shared 90% homology with Mer5 and significant homology with AhpC , and is now recognized to be bovine Prx 3. Later studies revealed that SP-22 activity was enhanced in the presence of an additional factor, identified as bovine mitochondrial Trx . In the period during these initial studies, Prx 3 was also known as antioxidant protein 1 (Aop1). The human prx 3 gene is located on chromosome 10 (q25–q26) and is transcribed from seven exons. Prx 3 is a 256 amino acid-containing protein with an N-terminal mitochondrial-targeting sequence (61 amino acids) that is cleaved to give a 21.5 kDa protein localized to the mitochondrial matrix. The peroxidatic cysteine residue is at residue 47 of the mature protein, in a highly conserved region (Figure 1A).
At much the same time as the mer5 gene was cloned, a novel gene was identified in the yeast Candida boidinii known as PMP20 . PMP20 encodes a 20 kDa peroxisomal membrane-associated protein that was abundantly expressed in Candida supplied with methanol as the sole carbon source. Human and murine homologues of PMP20 were identified 10 years later and characterized as peroxisomal proteins with thiol-specific antioxidant activity . That same year, parallel studies investigating the composition of human bronchoalveolar lavage fluid identified a novel protein, termed AEB166, which shared 65% sequence homology with the bacterial Prx AhpC [32,33]. This protein has subsequently been designated as Prx 5. The human prx 5 gene is located on chromosome 11 (q13) and is transcribed from six exons. The Prx 5 sequence is more divergent from the other human Prxs, sharing 28–30% overall sequence identity with Prx 1, Prx 2 and Prx 3. Prx 5 is targeted to the mitochondria by a 52 amino acid leader sequence that is subsequently cleaved off the mature enzyme yielding a 17 kDa protein (Figure 1A). The protein also has a C-terminal SQL peroxisomal-targeting sequence that enables the protein to be sorted to both mitochondria and peroxisomes [33,34]. The N-terminus contains an alternative translational start codon that produces a form of Prx 5 that is localized to the nucleus . The peroxidatic cysteine is residue 48 of the mature protein, and the active site has several residues in common with other family members.
The Prx family of proteins can be divided into three classes (typical 2-Cys, atypical 2-Cys and 1-Cys) with respect to the mechanism by which they decompose hydrogen peroxide . Mitochondrial Prx 3 belongs to the typical 2-Cys class of Prx, meaning it is a homodimer orientated in a head-to-tail fashion and utilizes peroxidatic (CysP) and resolving (CysR) cysteine residues on opposite subunits for catalysis. In the first step, hydrogen peroxide oxidizes the peroxidatic cysteine residue (Cys47) to a sulfenic acid (CysP-SOH), which condenses with the resolving cysteine (Cys168) of the second subunit to form an intermolecular disulfide bond (Figure 2A). In contrast, Prx 5 is an atypical 2-Cys Prx that becomes oxidized at the peroxidatic cysteine (Cys48) to a sulfenic acid, which condenses with a resolving cysteine (Cys152) within the same polypeptide to form an intramolecular disulfide bond  (Figure 2B). The oxidized forms of both mitochondrial Prxs are reduced back to the native state by Trx 2, which is itself reduced by TrxR 2 (Trx reductase 2). Trx 2 and TrxR 2 reside in the mitochondrial matrix and operate independently from the cytosolic Trx network.
Two complementary approaches have been used to determine kinetic parameters for the Prxs, namely steady-state analysis (Michaelis–Menton approach) and pre-steady-state analysis of a specific step in the catalytic cycle. Initial steady-state studies in the presence of reductants led to the conclusion that Prxs are relatively inefficient catalytic peroxidases that would compete poorly for hydrogen peroxide [36–39]. Indeed, the catalytic efficiency of mitochondrial Prx 3 (kcat=1–5 s−1) is low when compared to haem peroxidases [12,40]. Competitive kinetic approaches have revealed that the bimolecular reaction rate for Prx with hydrogen peroxide is in many cases comparable to catalase (k~107 M−1·s−1) [8–11,41,42]. Using this approach, we found that mitochondrial Prx 3 reacts with hydrogen peroxide with a rate constant of 2×107 M−1·s−1 . In contrast, Prx 5 reacts more slowly with hydrogen peroxide with a rate constant of 3×105 M−1·s−1 . In steady-state conditions, the mitochondrial Prxs appear to have a low KM for hydrogen peroxide (<20 μM) [12,34], that in all likelihood will be similar to that of the bacterial Prx AhpC (KM=1.4 μM) . These studies suggest that the mitochondrial Prxs will compete strongly for hydrogen peroxide in vivo at low levels of hydrogen peroxide but are likely to become inefficient at higher levels as the recycling rate becomes a more important factor.
The kinetic constants for the mitochondrial Prxs reacting with their physiological electron donor Trx 2 in steady-state conditions have not been determined. However, both Prxs exhibit a high affinity for Escherichia coli Trx (KM=1–5 μM) . Prx 3 is reduced by E. coli Trx with an apparent rate constant of 106 M−1·s−1 , whereas Prx 5 is reduced by Trx 2 with a rate constant of 2×106 M−1·s−1 . The reduction potential of mitochondrial Trx 2 (E0=−292 mV) , is more than 50 mV more negative (making it more reducing) than cytosolic Trx 1 (E0=−230 mV)  and GSH (E0=−240 mV) . We have determined the midpoint reduction potential of mitochondrial Prx 3 to be −290 mV . With its low reduction potential, Prx 3 requires an equally strong reductant to recycle its oxidized form. This could be achieved by Trx 2, but reduction by alternative electron donors such as Trx 1 or GSH will be thermodynamically unfavourable.
The active sites of the mitochondrial Prxs share many similarities despite exhibiting different mechanisms of disulfide formation (Figure 1B). Both consist of a solvent-exposed positively charged pocket surrounding a highly reactive peroxidatic cysteine residue in the thiolate form. The peroxidatic cysteine residue have a low pKa due to the presence of a conserved positively charged arginine group (Arg123 in Prx 3, Arg128 in Prx 5) in the active site [38,47]. The peroxidatic cysteine residue of Prx 5 has a calculated pKa of 5.2 , whereas that of Prx 3 is below 6 . The thiolate anion is also hydrogen bonded to a conserved threonine residue (Thr44 in Prx 3, Thr45 in Prx 5) that stabilizes the catalytic triad (cysteine, arginine and threonine) . Deprotonation of the peroxidatic cysteine residue facilitates the nucleophilic attack on the peroxyl bond of hydroperoxides. The active site also harbours an invariant proline residue (Pro40 in Prx 3 and Pro41 in Prx 5) that is believed to limit solvent accessibility and thereby shield the sulfenic acid intermediate from reaction with other thiols . Although the low pKa of the peroxidatic cysteine residue enhances its reactivity, it could only account for a maximum of two to three orders of magnitude difference in reactivity compared with free cysteine, not the six to seven that is observed. Furthermore, Prx 3 reacts with hydrogen peroxide six to nine orders of magnitude faster than with thiol alkylating agents such as N-ethylmaleimide or iodoacetamide [9,11]. This is in stark contrast with other thiol proteins such as Trx and glyceraldehyde-3-phosphate dehydrogenase that have greater reactivity with alkylating agents, and display considerably less difference between alkylation and peroxide reactivity [49,50]. These findings suggest that other features within the active site provide selectivity for reaction with peroxides.
One of the most remarkable properties of the typical 2-Cys class of Prxs, which includes mitochondrial Prx 3, is their propensity to form complex quaternary structures including doughnut-shaped toroids [13,51,52] (Figure 3A). The mitochondrial Prx 3 toroid is made up of 12 monomers (dodecamer), which contrasts with the decameric structure of Prx 1 and Prx 2 . Prx toroids can interact to form higher-order structures such as filaments (stacked toroids), concatenanes (interlinked toroids) and dodecahedrons (12 toroids associated as 12 faces of a polygon) . In vitro studies by Lindsay and co-workers have determined that the Prx 3 toroid can stack laterally to form filamentous structures [53,55]. Resolution of the crystal structure has provided further insight, revealing that Prx 3 crystallizes as a dodecameric toroid (Figure 3A), that can form higher-order catenane structures . The interlocked toroids are positioned at a 55 ° angle and therefore have numerous points of contact. As with other 2-Cys Prxs, the monomeric subunit of Prx 3 is a compact globular structure consisting of a seven-stranded twisted β sheet surrounded by four α-helices (Figure 3B). The peroxidatic cysteine is situated in the first turn of the N-terminal section of helix α2.
Oligomerization of the 2-Cys Prxs is dependent on a number of factors including protein concentration, with Prxs existing as dimers at low concentrations (<2 μM) and favouring co-operative assembly into toroids at higher concentrations (>2 μM) . The physiological significance of Prx oligomerization is not known, but given the high abundance of Prxs in cells, it appears that the formation of toroids in vivo will be favourable. Oligomerization of the 2-Cys Prxs is redox-dependent, with toroids favoured in the reduced form of the enzyme, whereas discrete dimers favoured in the reversibly oxidized form [40,52]. In addition to protein concentration and redox status, there are several other factors known to affect the oligomeric state of Prxs, including salt concentration, pH and phosphorylation. An elegant study by Parsonage et al.  showed that the oligomeric state of the bacterial Prx AhpC has a major effect on the catalytic activity of the enzyme. The authors found that AhpC mutants that could not form toroids exhibited a 100-fold decrease in catalytic efficiency, whereas mutants that stabilized the toroid structure had enhanced reactivity.
A great deal is known about the structural properties of Prx 5 as high-resolution crystal structures have been solved for both the reduced and oxidized forms of the enzyme [57,58]. Unlike the typical 2-Cys Prxs, Prx 5 does not oligomerize into higher-order structures. Prx 5 shares many of the structural elements present in other mammalian Prxs, but lacks the C-terminal domain of the typical 2-Cys Prxs (Figure 1B). The peroxidatic cysteine residue is present in a small cavity within the N-terminal region of kinked helix α2, whereas the resolving cysteine residue is located in a loop region between β7 and helix α6 (Figure 3C) . These cysteine residues are 13.8 Å (1 Å=0.1 nm) apart in the reduced form of the enzyme, necessitating a dramatic conformational shift in Prx 5 following oxidation. Indeed, the recent crystal structure of oxidized Prx 5 reveals a local unfolding of the loops containing both the peroxidatic (displaced by 9.5 Å) and resolving (displaced by 4 Å) cysteine residues, enabling the formation of an intramolecular disulfide bond .
Structural studies investigating requirements for the reduction of Prx 5 by Trx 2 have shown that oxidation of the Prx induces a conformational change that exposes the intramolecular disulfide bond to the surface of the molecule, enabling it to be reduced by Trx 2 . The crystal structures of Trx 2 in both the reduced and oxidized forms have also provided insight into the reduction mechanism . Molecular simulations of the interaction of the two proteins suggest that the protruding positively charged Lys49 of Prx 5 may interact with three negatively charged aspartate residues (Asp58, Asp60 and Asp61) within Trx 2.
In the presence of excess hydrogen peroxide, typical 2-Cys Prxs are highly sensitive to oxidative inactivation. This process, known as hyperoxidation, occurs when the sulfenic acid intermediate is oxidized by a second molecule of hydrogen peroxide, forming a sulfinic acid derivative that is enzymatically inactive (Figure 2A). The sulfenic acid intermediate that forms on the peroxidatic cysteine residue of typical 2-Cys Prxs during catalysis is 14 Å from the resolving cysteine residue of an adjacent subunit and therefore local unfolding is required for disulfide formation. Eukaryotic typical 2-Cys Prxs, such as Prx 3, are more susceptible to hyperoxidation than their prokaryotic counterparts due to a C-terminal extension that further delays disulfide formation . The additional α-helix in the C-terminal extension (α7) contains a ‘YF’ motif that interacts with a conserved ‘GGLG’ motif present in a loop neighbouring the peroxidatic cysteine residue (Figure 1B). These two features, unique to the eukaryotic Prxs, provide a kinetic pause during catalysis that provides an opportunity for reaction with an additional molecule of hydrogen peroxide. Consequently, truncation of the C-terminus makes susceptible Prxs more resistant to hyperoxidation . In the presence of excess levels of hydrogen peroxide, the typical 2-Cys Prxs can become irreversibly oxidized as hydrogen peroxide oxidizes the sulfinic acid to a sulfonic acid. The process of irreversible hyperoxidation can be prevented in human Prx 2 by acetylation of the N-terminus . The study emphasized that structural features remote from the C-terminal extension can also alter protein sensitivity to hyperoxidation.
Kinetic studies investigating the hyperoxidation of human Prx 1 in low steady-state concentrations of hydrogen peroxide have shown that the peroxidatic cysteine residue becomes hyperoxidized to the sulfinic acid at a rate of 0.072% per turnover (1 in 1300 turnovers) . Hyperoxidation of Prx 3 has been observed in cultured cells following prolonged exposure to high levels of hydrogen peroxide or drugs that generate hydrogen peroxide [63–66], and in the liver of aged rats . However, mitochondrial Prx 3 is more resistant to hyperoxidation than the cytosolic Prxs (Prx 1 and Prx 2) both in cells exposed to hydrogen peroxide and in vitro . There are several differences in the C-terminus of Prx 3 that may contribute to the resilience to hyperoxidation . For as yet unknown reasons, Prx 5 is insensitive to hyperoxidation.
The reduction of hyperoxidized 2-Cys Prxs is catalysed by Srx (sulfiredoxin), a 13 kDa enzyme that uses ATP to reduce sulfinylated Prxs [69,70]. The mechanism involves major structural rearrangements of the Prxs , and operates very slowly (kcat=0.18 min−1) [70,72]. Although Srx is cytosolic, it is reported to slowly traffic to the mitochondria, by an as yet uncharacterized mechanism, and reduce hyperoxidized Prx 3 following oxidative stress . Hyperoxidized Prx 3 is retroreduced more slowly than hyperoxidized Prxs 1 and 2 in the cytoplasm [64,66]. Therefore, although Prx 3 is more resistant to hyperoxidation, the slow reduction will enable the hyperoxidized form of Prx 3 to accumulate under certain conditions.
The hyperoxidation of typical 2-Cys Prxs is associated with the formation of stabilized toroids that are structurally distinct from Prx toroids in the reduced form . The hyperoxidized Prx toroids are reported to exhibit a chaperone-like function [15–17]. They can protect yeast cells from heat shock by preventing thermal aggregation  and have been proposed to protect mammalian cells from hydrogen peroxide-induced apoptosis . Consequently, the hyperoxidation of Prxs has been described as a molecular switch that shuts down peroxidase activity and activates the chaperone function of the enzyme. Human Prx 1 has been shown to function more effectively as a molecular chaperone than Prx 2 . This may reflect a greater propensity of the peroxidatic cysteine residue to be hyperoxidized to the sulfinic acid or to the sulfonic acid , which has an even greater chaperone activity . To date, there has been no investigation of the chaperone activity of the mitochondrial Prxs.
Although initially recognized for their ability to consume peroxides, Prxs can scavenge other oxidants. Bryk et al.  were the first to show that the bacterial Prx AhpC reduces peroxynitrite to nitrite with a rate constant of 106 M−1·s−1. Since then, other Prxs have been reported to have extremely high reactivity towards peroxynitrite [42,43,77–79]. Of the mammalian Prxs, Prx 5 appears to react the fastest (k=7×107 M−1·s−1) , and on this basis it has been estimated to be a major target for peroxynitrite in mitochondria . The rate of reaction between Prx 3 and peroxynitrite has not been tested. However, studies with Prx 2 have demonstrated a rate constant that is ~10-fold less than that for hydrogen peroxide .
The mitochondrial Prxs also have the capacity to scavenge organic hydroperoxides that might form during oxidative stress. Trujillo et al.  found that Prx 5 decomposes the model organic hydroperoxides tButOOH (t-butyl hydroperoxide) (k=5×106 M−1·s−1) and CumOOH (cumene hydroperoxide) (k=8×107 M−1·s−1) even more effectively than hydrogen peroxide (k=3×105 M−1·s−1). Cao et al.  demonstrated that Prx 3 decomposes tButOOH and CumOOH in a coupled enzymatic assay with Trx 2 and TrxR 2 with ~80% and ~40% respectively of the activity exhibited with hydrogen peroxide as a substrate. Although the rate constants for the reaction between Prx 3 and tButOOH or CumOOH are not known, values for the bacterial 2-Cys Prx, AhpC, reacting with tButOOH (k=2×105 M−1·s−1) and CumOOH (k=5×105 M−1·s−1) have been measured .
To date, there have been no in vitro studies investigating reactions between mitochondrial Prxs and organic hydroperoxides of physiological relevance such as the lipid hydroperoxides. However, Cordray et al. have shown that fatty acid hydroperoxides produced by lipoxygenase and cyclo-oxygenase activity in cells lead to the hyperoxidation of typical 2-Cys Prxs such as Prx 3 , supporting the hypothesis that Prxs contribute to lipid hydroperoxide decomposition. Our laboratory has recently found that Prx 3 and Prx 2 scavenge amino acid and peptide hydroperoxides with rate constants of up to 105 M−1·s−1 (A. V. Peskin, A. G. Cox, P. Nagy, P. E. Morgan, M. B. Hampton, M. J. Davies and C. C. Winterbourn, unpublished work). In addition, both Prx 3 and Prx 2 scavenge hydroperoxides formed on model proteins by photolysis or γ-irradiation. No other antioxidant enzymes so far examined have the capacity to decompose protein hydroperoxides.
Together, these studies suggest that the mitochondrial Prxs can accommodate a broad range of peroxide substrates. Furthermore, the mitochondrial Prxs may exhibit complementary antioxidant activities in the matrix with Prx 3 scavenging hydrogen peroxide and Prx 5 behaving more effectively as a scavenger of peroxynitrite. Both Prxs have the capacity to decompose organic hydroperoxides. However, it remains to be seen whether the scavenging of organic peroxides by mitochondrial Prxs is an integral aspect of their antioxidant function in vivo.
ANTIOXIDANT FUNCTION OF MITOCHONDRIAL PRXS
Although the in vivo generation rate is unknown, electrons can transfer from the respiratory chain to molecular oxygen to generate superoxide within the mitochondrial matrix . Superoxide is rapidly converted into hydrogen peroxide by Mn-SOD (manganese superoxide dismutase). The high reactivity (k=2×109 M−1·s−1) and concentration (10 μM) of the dismutase ensures that the steady-state concentration of superoxide remains very low [82,83]. Mitochondrial hydrogen peroxide can be decomposed by GPxs (glutathione peroxidases) 1 and 4, and Prxs 3 and 5. GPx 4 also has a specialized function in breaking down phospholipid hydroperoxides . Catalase has been detected in mitochondria purified from rat heart and liver [85,86], but this is at low nanomolar concentrations and therefore is generally not regarded to play a significant role in mitochondrial hydrogen peroxide detoxification. The mitochondrial genome encodes for a disproportionately high number of methionine residues in the respiratory chain and these have been proposed to protect other sensitive sites within the respiratory complexes by acting as oxidant scavengers . However, methionine only reacts slowly with hydrogen peroxide, and only a small percentage of mitochondrial proteins are actually encoded in the mitochondrial genome.
Mitochondrial peroxidase activity is dependent on the thiol reductants GSH for the GPxs, and Trx 2 for the mitochondrial Prxs. Therefore, both of these species are an integral component of mitochondrial antioxidant defence. The oxidized forms of glutathione and Trx 2 are reduced by GR (glutathione reductase) and TrxR 2 respectively in an NADPH-dependent manner [88,89]. Oxidized methionine residues in mitochondrial proteins are reduced by methionine sulfoxide reductase A and B2, which also use NADPH for reducing equivalents [90–92]. As such, the entire mitochondrial antioxidant firewall is dependent on an adequate supply of NADPH, which is maintained by a combination of mechanisms that include the enzymatic reduction of NADP+ by transhydrogenase, malic enzyme and isocitrate dehydrogenase . The mitochondrial NADPH/NADP+, GSH/GSSG and Trx 2red/Trx 2ox redox couples function independently from the cytosolic redox couples and operate under stable non-equilibrium conditions [94–96]. The essential role of the mitochondrial Trx network is highlighted by studies revealing that mice deficient in Trx 2 or TrxR 2 die during embryogenesis [97,98].
Kinetic hierarchy of mitochondrial proteins that react with hydrogen peroxide
Before recognition of the Prxs, GPx 1 was viewed as the primary scavenger of mitochondrial hydrogen peroxide. The likelihood of mitochondrial Prxs reacting directly with hydrogen peroxide can be assessed by comparing their reactivity with other potential mitochondrial targets using published rate constants and abundance data (Figure 4). If it is assumed that all targets are equally accessible, this competitive kinetic analysis estimates that approx. 90% of mitochondrial hydrogen peroxide should react with Prx 3. Although GPx 1 has a higher rate constant than Prx 3, its lower abundance limits its ability to compete directly with Prx 3. This analysis is consistent with estimates that only 15% of mitochondrial hydrogen peroxide can be accounted for by GPx 1 scavenging . The accuracy of the analysis model will obviously vary depending on abundance of the key peroxidases in different cell types and tissues. It is also important to emphasize that the continuing ability of Prx 3 to function as an antioxidant will depend on recycling by the mitochondrial Trx network. Trx 2 is present at considerably lower concentrations than Prx 3 [29,100], and is likely to become a limiting factor under sustained levels of hydrogen peroxide generation.
Prx 5 is likely to react with a small but significant fraction of mitochondrial hydrogen peroxide. GPx 4 will compete for only a small percentage of the hydrogen peroxide, which is consistent with this enzyme preferring phospholipid hydroperoxides as substrates . Other targets of hydrogen peroxide such as the phosphatases PTP-1B (protein tyrosine phosphatase 1B) and SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) will not be directly competitive with the peroxidases. The kinetic considerations are based on the assumption that the mitochondrial proteins are competing for hydrogen peroxide in a homogenous system. In vivo these proteins reside in different niches within the mitochondria, which may enable the oxidation of less reactive targets, although there is currently no evidence to support this hypothesis.
Oxidation of mitochondrial Prxs in cells and protection from oxidative stress
Changes in the oxidation state of Prx 3 have been seen in oxidant-treated or stressed cells. We have demonstrated that Prx 3 becomes oxidized following treatment with the cytotoxic agents phenethyl isothiocyanate and auranofin [101,102]. The oxidation was specific for mitochondrial Prx 3 as no changes were seen in Prx 1 or Prx 2. The mechanisms are not yet clear, but it is likely to involve an increase in oxidant generation in combination with disruption of the Trx reduction pathway. Exposure of cells to the lipid peroxidation product acrolein or the industrial toxin hexavalent chromium also results in the oxidation of both Prx 3 and its physiological reductant Trx 2 [103,104]. Perfusion of isolated rat hearts with hydrogen peroxide causes both Prx 3 and Prx 5 to become oxidized , and oxidation of Prx 3 occurs during ischaemia in a mouse heart Lagendorff perfusion system . Accumulation of oxidized Prx 3 should increase steady-state levels of mitochondrial hydrogen peroxide, but this has not been demonstrated in any of these models.
Studies investigating the effects of modulating mitochondrial Prx expression in cells have helped to clarify the function of these proteins in vivo. The overexpression of Prx 3 decreases ROS (reactive oxygen species) production and lipid peroxidation. It also renders cells resistant to numerous inducers of apoptosis including hydrogen peroxide, hypoxia, staurosporine, TNFα (tumour necrosis factor α), the plant toxin abrin, and drugs that generate ROS [107–111]. In contrast, knockdown of Prx 3 expression by RNA interference increases mitochondrial ROS production, enhances protein carbonylation, alters mitochondrial morphology and sensitizes cells to apoptosis [108,110,112,113]. Consistent with a regulatory role in apoptosis, the overexpression of Prx 3 enhances transformation and tumour growth in fibroblasts overexpressing the oncogene c-Myc . The modulation of Prx 3 expression also affects the differentiation of erythroleukaemic cells [24,114]. Overexpression of mitochondrial Prx 5 prevents peroxide or drug-induced DNA damage and p53-induced apoptosis [115–118]. Cells deficient in Prx 5 have increased levels of protein carbonylation and DNA damage, and are hypersensitive to hydrogen peroxide, irradiation, adriamycin, etoposide, and the mitochondrial toxin 1-methyl-4-phenylpyridium [113,116]. The different outcomes following Prx 3 or Prx 5 manipulation suggest that these enzymes are non-redundant.
In vivo effects of altering mitochondrial Prx expression
Prx 3 knockout mice develop with no overt phenotype, but exhibit a hypersensitivity to lipopolysaccharide-induced lung inflammation . Higher levels of ROS are detectable in macrophages derived from these mice and they release increased amounts of of TNFα . They also show oxidative damage to lung tissue . In addition, Prx 3-deficient maternal mice display a much higher frequency of stillbirths , which has been attributed to Prx 3 being important in placental antioxidant defence. The overexpression of Prx 3 in mice has been shown to exert a neuroprotective effect, inhibiting nitrotyrosine formation and preventing excitotoxic injury in hippocampal neurons . Transgenic mice overexpressing Prx 3 are also protected from left ventricular remodelling and failure after myocardial infarction . Prx 3 overexpression has also been shown to improve glucose homoeostasis, with transgenic mice displaying resistance to diet-induced elevations in blood glucose and increased glucose clearance . These mice display modulation in the activity of the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway, suggesting that Prx 3 may be involved in the regulation of cell signalling . To date, Prx 5 knockout mice have not been described. However, in Drosophila, the overexpression of Prx 5 extends lifespan by ~30%, whereas Prx 5 deficiency produces signs of oxidative stress, increased apoptosis and shortened lifespan .
MITOCHONDRIAL PRXS AND REDOX SIGNALLING
Cells are capable of sensing and adapting to a disruption in redox homoeostasis. In unicellular organisms, exogenous hydrogen peroxide induces the upregulation of antioxidant defences . Cells can also be exposed to endogenous oxidative stress from superoxide and hydrogen peroxide derived from the mitochondrial respiratory chain. Though not well characterized, there is clear evidence that mitochondrial oxidative stress causes the upregulation of antioxidants such as Mn-SOD, GPx 1 and Prx 3 [126–130]. This adaptive response has been termed mitohormesis [131–133]. Given that the majority of mitochondrial proteins are encoded within the nuclear genome, it is apparent that retrograde signalling has to occur. Although the mechanism is unclear, disruption of mitochondrial redox homoeostasis causes activation of nuclear transcription factors such as PGC-1α (peroxisome-proliferator-activated receptor γ co-activator 1α) and FoxO3a (forkhead box O3a), which appear to be responsible for the upregulation of Prx 3 and other mitochondrial antioxidants [126,129,130].
In addition to the adaptive responses that maintain homoeostasis, cells also appear capable of generating oxidants as second messengers during cell signalling [134,135]. The best-studied source is the membrane-associated NADPH oxidases that are specifically activated in response to a variety of extracellular ligands . The primary product of the NADPH oxidases is superoxide, which may act directly or through its dismutation product hydrogen peroxide. Hydrogen peroxide is well-suited as a signalling molecule because of its membrane permeability and selective reactivity . Mitochondrial-derived hydrogen peroxide is also proposed to have a signalling role, and levels could be elevated by increasing the constitutive generation rate from the respiratory chain or impairment of mitochondrial antioxidant defences. However, insight into the mechanisms of production and stimulation is necessary before mitochondrial hydrogen peroxide can be classified as a second messenger as opposed to a metabolic byproduct that provokes an adaptive response.
A detailed description of the cellular processes in which mitochondrial redox signalling is invoked is beyond the scope of this review, but they range from growth factor and hypoxia signalling through to autophagy and the induction of apoptosis. EGF (epidermal growth factor) signalling is attenuated by mitochondrial uncouplers and the overexpression of GPx1 or mitochondrial-targeted catalase [138,139], implicating mitochondrial-derived oxidants. These oxidants have also been proposed to play an essential role in hydrogen peroxide-induced transactivation of growth factor receptors . There is increasing evidence that the activation of HIF-1α (hypoxia-inducible factor 1α) in response to hypoxia is mediated by mitochondrial oxidants generated by complex III of the respiratory chain [141,142]. Recently, several studies have indicated that mitochondrial oxidants play a role in the induction of the lysosomal degradation pathway known as autophagy [143–145]. Furthermore, it has emerged that the cysteine protease Atg4, which is essential for autophagosome formation, is a target for hydrogen peroxide .
One of the most extensively studied, but still unresolved, relationships is that between mitochondrial hydrogen peroxide and the induction of apoptosis. Hydrogen peroxide itself can trigger apoptosis, though often over a limited concentration range [147,148]; however, endogenous generation is proposed to contribute to the initiation process. Oxidation of Prx 3 has been observed during receptor-mediated apoptosis , indicative of selective disruption of mitochondrial redox homoeostasis. There are several points in the apoptosis process susceptible to redox modulation, including the ANT (adenine nucleotide translocator) [150,151] and cyclophilin D  that interact to regulate mitochondrial membrane permeability during cell death. There is evidence to suggest that cytochrome c uses hydrogen peroxide to catalyse the peroxidation of cardiolipin during the initiation of apoptosis, and that this enables the mobilization of cytochrome c into the intermembrane space [153,154]. The enzyme p66Shc traffics to the mitochondria in response to oxidative stress and oxidizes cytochrome c to generate hydrogen peroxide, which is proposed to be necessary to trigger apoptosis [155,156], and p66Shc activation itself requires oxidation of critical cysteine residues in the redox-active domain . Similarly, ASK1 (apoptosis signalling kinase-1) is a pro-apoptotic kinase that traffics to the mitochondria, is activated by hydrogen peroxide and can be inhibited by Trx 2 [158–160]. In contrast with the oxidation-sensitive pro-apoptotic proteins, DJ-1 is anti-apoptotic protein associated with Parkinson's disease , which has recently been described as a Prx-like sensor that in its oxidized form protects mitochondria from oxidative stress and apoptosis [162,163].
Involvement of mitochondrial Prxs in redox signalling
There are indications, albeit still speculative, that Prx 3, and by extrapolation Prx 5, may play a role in mitochondrial redox signalling. In Figure 5 we have illustrated three major mechanisms that the Prxs could utilize for redox signalling. First, our kinetic analysis implies that these proteins will be a major target of mitochondrial hydrogen peroxide, and oxidation is likely to influence steady-state levels of hydrogen peroxide available for oxidation of redox-sensitive signalling proteins. The evolution of structural motifs that impair antioxidant capability and promote hyperoxidation and inactivation of eukaryotic Prxs led Wood et al.  to postulate the floodgate hypothesis. In this signalling model, Prxs keep levels of hydrogen peroxide low under normal conditions, but when a signal increases hydrogen peroxide generation then Prx hyperoxidation occurs, permitting accumulation of higher levels of hydrogen peroxide and the oxidation of less reactive thiol proteins. Although the rate of hyperoxidation during catalysis is low in vitro (1 in 1300 turnovers for Prx 1) , the cytoplasmic Prxs appear more susceptible in a cellular environment, thereby supporting the floodgate model . However, significant Prx hyperoxidation has not been reported during cell signalling [149,164,165]. It is possible that the floodgate model may operate in small cellular microenvironments within the location of a specific oxidant source, meaning that hyperoxidized Prxs would not be detected on a global scale . However, such a scenario has yet to be demonstrated, and the diffusability of hydrogen peroxide would still limit the extent to which other thiols could be selectively oxidized. Mitochondria provide an environment that would aid operation of a floodgate model, with Prxs present at high concentrations and an internal source of hydrogen peroxide, all within a double membrane system. Also, hydrogen peroxide transport from the mitochondria is potentially regulated by membrane aquaporin activity [167,168]. However, Prx 3 is more resilient to hyperoxidation than the cytoplasmic Prxs, both in vivo and in the purified protein , which impacts on the feasibility of this model. Accumulation of the oxidized disulfide Prx through impaired reduction would have a similar net effect to hyperoxidation and allow increased levels of mitochondrial hydrogen peroxide. It is worth noting, however, that the kinetic model indicates more than 90% of the Prx 3 would have to be oxidized before the other peroxidases become more competitive than Prx 3.
The second major mechanism is based on the possibility that specific protein–protein interactions of Prxs vary depending on the redox status of the protein. As discussed earlier, the toroid quaternary structure is favoured by reduced Prxs, whereas oxidation promotes disassembly into dimers. There is limited knowledge of Prx 3-binding proteins, although there are reports of association with cyclophilin A , Fanconi anaemia-associated protein FancG , the adapter protein RPK118 . It is unclear if the redox status of Prx 3 alters these interactions. Little is known about physiological binding partners of Prx 5.
Many of the target proteins reported to be oxidized during redox signalling display relatively weak reactivity and are unlikely to be directly oxidized, regardless of the redox status of the Prxs . These include the protein tyrosine phosphatases PTP-1B and SHP-2 that can traffic to mitochondria . They are categorized as redox-sensitive on the basis of low pKa catalytic cysteine residues , but are considerably less-reactive than Prxs [173,174]. This leads to the third putative mechanism, that Prxs can also act to facilitate the oxidation of less reactive thiol proteins. The concept has a precedent in the seminal yeast studies, where oxidation of the transcription factors Yap1 in S. cerevisiae or Pap1 in Schizosaccharomyces pombe was shown to be essential for their adaptive response to oxidative stress . In their oxidized state, Yap1 and Pap1 traffic to the nucleus and activate the transcription of antioxidant genes such as catalase, GPx, Prxs, GR, TrxR and Trx . The sulfenic acid formed on reaction of a Prx with hydrogen peroxide was shown to react with a cysteine residue on the transcription factors to generate a transient intermolecular disulfide [176–179]. A second cysteine residue on the target proteins then reduces the disulfide to regenerate the reduced Prx and the oxidized transcription factor. Although appealing, as yet there are no reports of this mechanism operating with mammalian Prxs. Further investigation is warranted to determine whether facilitated oxidation pathways involving Prxs operate in multicellular organisms.
In a variation on the binding and facilitated oxidation mechanisms, mitochondrial Trx 2 is likely to become transiently oxidized as a consequence of Prx 3 oxidation by hydrogen peroxide, thereby altering its interaction partners or triggering the oxidation of mitochondrial signalling proteins dependent on Trx remaining reduced. There is evidence that Trx 2 and mitochondrial GSH are involved in cell signalling [94,95], with both reported to be oxidized during early apoptosis [180–182]. The redox status of Trx influences its interaction and modulation of the signalling kinase Ask-1 [158–160]. This provides a mechanism by which hydrogen peroxide could selectively influence a kinase pathway through the action of a Prx.
CONCLUSIONS AND FUTURE CHALLENGES
Prxs have unique biochemical and physical attributes that allow interplay between their antioxidant activity and an ability to function as redox sensors. There is considerable interest in mitochondrial redox signalling, both in maintenance of homoeostasis and the integration of metabolic signals. The mitochondrial Prxs clearly operate independently of their cytoplasmic family members, and there are reports of Prx 3 oxidation during cell signalling. However, a direct role of mitochondrial Prxs in signalling remains speculative.
There are several key questions that need to be resolved to increase our understanding of the antioxidant and signalling properties of mitochondrial Prxs. The physical features of the Prxs that promote the dramatic reactivity and selectivity for hydroperoxides are unclear. Within the mitochondrion, we need to know the major biological substrates of the Prxs, what the rate of Prx turnover is during normal metabolism, and what the maximal capacity of the system is under conditions of oxidative stress. No major defects in mitochondrial integrity or signalling have been revealed in Prx 3-deficient mice, but more extensive studies are required. Double knockouts of Prx 3 and Prx 5 and transient knockdowns may be necessary to limit compensatory effects.
It is also unclear what the biological role is, if any, of the complex quaternary structures of Prxs. One obvious role is in the regulation of protein–protein interactions, which would provide a key layer of selectivity in a Prx-mediated signalling pathway; however, this remains hypothetical. As physiologically relevant binding partners are discovered, it will be valuable to use molecular techniques to engineer proteins in which these interactions are disrupted, yet maintain the antioxidant capacity of the Prxs. The significance of Prx hyperoxidation is also unclear. The discovery of this phenomenon in eukaryotic Prxs was one of the major stimuli for discussion of Prxs as signalling proteins. However, neither Prx 3 nor Prx 5 appear sensitive to hyperoxidation relative to the major cytoplasmic Prxs. Indeed, it is not clear if hyperoxidation of the cytoplasmic Prxs is directly involved in cell signalling or if it is a side-effect associated with other Prx properties, for example, stabilization of the sulfenic acid intermediate for facilitated oxidation. Even so, the report of hyperoxidized Prx 3 accumulation associated with aging is intriguing and warrants further investigation.
Finally, improved experimental tools are required. In particular, there are no specific antibodies to the oxidized and hyperoxidized forms of individual Prxs, which would enable visualization of redox interconversions at an individual cell level. This information will be important in obtaining further insight into the timing and significance of Prx 3 oxidation during apoptotic signalling, and other physiological processes with which this intriguing family of proteins has been associated.
The authors acknowledge funding from the Health Research Council of New Zealand and the Royal Society Marsden Fund. A.G.C. is a recipient of a Top Achiever Doctoral Scholarship from the Tertiary Education Commission.
Abbreviations: CumOOH, cumene hydroperoxide; CysP, peroxidatic cysteine residue; GPx, glutathione peroxidase; GR, glutathione reductase; Mn-SOD, manganese superoxide dismutase; Prx, peroxiredoxin; PTP-1B, protein tyrosine phosphatase 1B; ROS, reactive oxygen species; SHP-2, Src homology 2 domain-containing protein tyrosine phosphatase 2; Srx, sulfiredoxin; tButOOH, t-butyl hydroperoxide; TNFα, tumour necrosis factor α; Trx, thioredoxin; TrxR, Trx reductase
- © The Authors Journal compilation © 2010 Biochemical Society