Reactive oxygen species (ROS), such as the superoxide anion and hydrogen peroxide, are generated by the photosystems because photoexcited electrons are often generated in excess of requirements for CO2 fixation and used for reducing molecular oxygen, even under normal environmental conditions. Moreover, ROS generation is increased in chloroplasts if plants are subjected to stresses, such as drought, high salinity and chilling. Chloroplast-localized isoforms of ascorbate peroxidase and possibly peroxiredoxins assume the principal role of scavenging hydrogen peroxide. However, in vitro studies revealed that both types of peroxidases are easily damaged by hydrogen peroxide and lose their catalytic activities. This is one contributing factor for cellular damage that occurs under severe oxidative stress. In this review, I describe mechanisms of hydrogen peroxide-mediated inactivation of these two enzymes and discuss a reason why they became susceptible to damage by hydrogen peroxide.
In chloroplasts of plants, water molecules at the lumenal side of the thylakoid are split by the light energy to molecular oxygen, protons and electrons. The excited electrons are transferred through photosystems II and cytocrome b6/f complex to photosystem I, where they reduce NADP+ to form NADPH, which is then used for CO2 fixation in the stroma. Even under normal environmental conditions, the number of excited electrons is in excess of NADP+ reduction, and a part of surplus electrons are transferred to molecular oxygen at photosystem I to form superoxide anions, O2−. Although such reduction of molecular oxygen also occurs in the electron transport system of mitochondria, it is very much faster in chloroplasts because the photosystems are continuously excited by sun light and the chloroplast contains larger amounts of molecular oxygen derived from water at over 250 μm, three orders of magnitude higher than that in the liver cell of animal (1). The generated O2− is disproportionated to molecular oxygen and hydrogen peroxide (H2O2) in a reaction that is catalyzed by the chloroplastic superoxide dismutase. H2O2 is then reduced to water by ascorbate peroxidase (APX) and possibly 2-cysteine peroxiredoxin (2-Cys Prx). The electron donor, ascorbate for APX and generally thioredoxin and NTRC, a novel type of NADPH thioredoxin reductase, for 2-Cys Prx, are regenerated using reducing equivalents supplied by the photosystems. The whole system thus dissipates excess light energy of photosystems by splitting and regenerating water (water–water cycle, 1–3). Depending on the environmental conditions, electron flux through the water–water cycle accounts for up to 40% of the total electron flux of photosystems (1,4). Electron flux through the water–water cycle is increased under low CO2 concentrations and temperatures (5), low temperature (6,7) and salt stress (8). In addition, upon illumination of dark-adapted leaves, the Calvin cycle enzymes have not been photoactivated and thus much of the electrons are transferred to the water–water cycle during the induction period of CO2 fixation (9).
If H2O2 could not be scavenged efficiently, it would accumulate in the chloroplast and damage chloroplastic proteins; for example, thiol enzymes of the Calvin cycle and heme proteins. Furthermore, H2O2 reacts with Fe2+, derived from damaged heme proteins, leading to the formation of a more reactive hydroxyl radical, •OH (Fenton reaction).
Therefore, it is very surprising that both APX and 2-Cys Prx are easily inactivated by excess amounts of H2O2. In vitro analysis showed that chloroplastic APX lost its activity within 1 min in the absence of ascorbate when 20 mol equivalent of H2O2 was added (10,11). This inactivation is very much faster than those reported for other peroxidases, such as the cytosol-localized APX isoform of higher plants (12) and red algae (10), and the cytochrome c peroxidase of yeast (13,14), even though all of them share the sequence and structural similarity to chloroplastic APXs. Eukaryotic 2-Cys Prxs, including plant 2-Cys Prx (15), are more sensitive to H2O2 than bacterial homologs. For example, human PrxII is 100 times more susceptible than the Salmonella homolog AhpC (16).
In this review, I describe mechanisms of H2O2-mediated inactivation of these two enzymes, and then propose a reason why they became susceptible to H2O2.
Mechanism of H2O2-Mediated Inactivation of Chloroplastic APX
Ascorbate peroxidase, which catalyzes the reaction monodehydroascorbate radical, is found in eukaryotic photosynthetic organisms from eukaryotic algae to higher plants, and is a member of the class I heme peroxidase gene family, which includes yeast cytochrome c peroxidase and bacterial bifunctional catalase-peroxidase (17). The molecular mass ranges between 28 and 38 kDa. APX isoforms are localized in cytosol, peroxisomes, chloroplasts and mitochondria. Chloroplasts of higher plants have two APX isoforms, one localized to the stroma and the other bound to the stromal side of the thylakoid membrane because of the presence of a C-terminal hydrophobic tail. In many higher plants, both chloroplastic APXs are produced from a common gene by alternative splicing (18). While both of the homodimer (19,20) and monomer (21–23) were found in cytosolic APX isoform, only monomer was reported in chloroplastic APX isoforms (22,24–26).
Local concentrations of thylakoid-bound APX and thylakoid-attached superoxide dismutase around photosystem I are estimated to be about 1 mm (1,2). In cooperation with the ferredoxin-dependent regeneration system of ascorbate, they form the first line of defense against reactive oxygen species (ROS). Stromal APX at about 40 μm (given that it uniformly distributes in stroma, 1) and stromal superoxide dismutase form the second line of defense.
When APX reacts with one molecule of H2O2, the ferric (FeIII) atom of heme is oxidized into the oxyferryl (FeIV = O) species, and a porphyrin-based cation radical intermediate is formed (compound I). The porphyrin-based radical of compound I is then reduced by one ascorbate molecule to form another intermediate (compound II), the oxyferryl species. The compound II is then reduced by a second ascorbate molecule to the ferric resting state. If compound I is not subsequently reduced because of the absence of ascorbate, the porphyrin-based radical abstracts electron from an amino acid residue of apoprotein. Inactivation of APX is thought to be due to an attack on the reaction intermediates by H2O2 (27).
The molecular mechanism of H2O2-mediated inactivation was first reported by Kitajima et al. (11). They demonstrated that, in the stromal APX, irreversible cross-linking of heme to a tryptophan residue (Trp35, Fig. 1) facing the distal cavity formed by the heme and N-terminal half of the apoprotein occurred in response to H2O2-mediated inactivation. The chemical structure of the Trp-heme complex is not known but mass spectrometric analysis suggested that an oxygen was incorporated in it. Mutation of this Trp to Phe abolished the H2O2-mediated cross-linking and increased the half-time for inactivation from <10 to 62 s. Because the indole ring of Trp35 is 3.18 Å from the porphyrin ring in the stromal APX (the distance between N of the indole ring and C6 of the porphyrin ring ), the heme must move toward Trp35 to form the covalent bond. In active cytosolic APX, ascorbate binds to the γ-meso edge of heme (a propionated side of heme) (29). The loss of ascorbate oxidizing activity in the cross-linked form of chloroplastic APX may therefore be due to the repositioning of heme, preventing it from interacting with ascorbate. Cross-linking of the Trp strongly suggests that this residue reduced the porphyrin-based cation radical of compound I and formed a Trp cation radical.
Cys26 (Fig. 1), which is located near the propionate side chain of heme and forms an ascorbate-binding site, also becomes a radical in an H2O2-dependent manner. This was demonstrated by using the radical scavenger 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO•) and spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) in conjunction with mass spectrometry (30). Cys126, located apart from the catalytic site (Fig. 1), may also be a radical site (30). Other residues may also form radicals to enable the electron transfer among heme, Trp35, Cys26 and Cys126. In certain situations, radical formation of Cys26 may lead to inactivation of APX; TEMPO• not only binds to the thiyl radical of Cys residue but also irreversibly oxidizes it to form sulfinylated (SO2H) and sulfonylated (SO3H) cysteine (30,31). APX with hyperoxidized Cys26 showed decreased APX activity (30). There have been no in vivo studies demonstrating the hyperoxidation of Cys26 of APX, but it has been reported that oxidative stress hyperoxidizes many proteins in vivo (32). Although Cys26 may be protected by glutathione by forming a reversible mixed disulfide bond under moderate oxidative stress (30), glutathione may no longer protect Cys26 if severe oxidative stress decreases the reduced form of glutathione.
In cytosolic APX, the porphyrin cation radical also abstracts electron from the distal Trp, resulting in formation of the cross-link (33), as well as from the Cys near the propionate side chain of heme (30). However, it retains 50% of APX activity after treatment of 20 mol equivalent of H2O2 for 10 min in the absence of ascorbate (10). In the case of the cytochrome c peroxidase of yeast, when ferrocytochrome c is absent, the radical is transferred to and disrupts at least eight tryptophan and tyrosine residues distant from the heme (34), in addition to the proximal Trp which is a site for interaction with ferrocytochrome c. Also, in fungal lignin peroxidase (35), when the reducing substrate is absent the porphyrin radical is thought to transfer to tryptophans far from heme, resulting in their hydroxylation. Such redox-active residues may have a role in protection of the active site of the peroxidases by donating electrons directly or indirectly to porphyrin when reduction of heme by substrate electron donor could not proceed. It is thus possible that one of the reasons for rapid inactivation of chloroplastic APX is that the distal Trp preferentially functions as an electron donor to porphyrin-based cation radicals, but electron donation by other residues (such as Cys26) is relatively slower.
The structure of the active site in chloroplastic APX is quite similar to that of cytosolic APX and the cytochrome c peroxidase, but the loop structure in the vicinity of propionate side chains of the heme is quite different because of 16 amino acid residues of inserted sequence (Fig. 1). In contrast to cytosolic APX, whose loop binds noncovalently to a propionate side chain of porphyrin, the corresponding loop of chloroplastic APX does not bind to the propionate side chain. When the 16-residue inserted sequence of chloroplastic APX is inserted into cytosolic APX, it becomes susceptible to H2O2 (10). In a theoretical study, the propionate side chain is shown to be involved in electron transfer from amino acid residues to the porphyrin (36). Thus, because of the different interaction of the loop with the propionate side chain, the heme of chloroplastic APX cannot accept electrons rapidly from amino acid residues near the propionate side chain to prevent cross-linking of Trp35 with heme.
The distal Trp is conserved in the class I peroxidase family, namely APX, cytochrome c peroxidase and bacterial catalase-peroxidase. The distal Trp is also a radical site in catalase-peroxidase (37) and is necessary for its catalase activity but not for its peroxidase activity (38,39). This residue is also unnecessary for peroxidase activity in APX (11) and cytochrome c peroxidase (40). The conservation of the distal Trp and its radical formation in APX may be a relic of an ancestral enzyme which had catalase activity. In contrast, the corresponding site is Phe in class II and III peroxidases, namely lignin peroxidase (41), manganese peroxidase (42) and horseradish peroxidase (17).
Why is Chloroplastic APX Susceptible to H2O2?
Chloroplastic APX loses its activity by H2O2 in the absence of ascorbate. The resulting accumulation of H2O2 may initiate ROS-related signaling to induce expression of defense-related genes. The acquisition of H2O2 susceptibility by APX could thus be viewed in terms of its evolution as an efficient H2O2 sensor. Alternatively, H2O2 susceptibility may simply be a defect of chloroplastic APX, and regarded as an evolutionary tradeoff that has allowed development by APX of a far higher turnover rate for catalysis; the value of chloroplastic APX is several times to one order of magnitude higher than that of cytosolic APX (references cited in 11). This may have been achieved by forcing the cation radical of the reaction intermediate to remain near the catalytic site by preventing it from distributing to amino acid residues further away. This strategy is effective through the high levels (up to 25 mm) of ascorbate present in chloroplasts (43), which protects the H2O2-sensitive APX.
A search of the peroxidase database PeroxiBase (44, http://peroxibase.isb-sib.ch/) found APXs which are likely to be stromal isoforms not only in Chlorophyta but also in Rhodophyta (CmeAPX02 of Cyanidioschyzon merolae) and in diatoms (PtrAPX01 of Phaeodactylum tricornutum, TpsAPX02 of Thalassiosira pseudonana). However, no APXs likely to be thylakoid-bound isoforms could be found in aquatic plants, though this may be because the peroxidase database currently has only a limited amount of registered data relating to lower plants. Thylakoid-bound isoforms could be found only in land plants: cryptogams (PpaAPX01 of Physcomitrella patens) and higher plants. This suggests an evolutionary adaptation whereby aquatic plants, moving to a terrestrial environment where larger amounts of ROS are generated in photosystems because of higher light intensity and water limitation, may require an APX with a high turnover number targeted to the thylakoid (the site of H2O2 generation), rather than increasing the tolerance of stromal APX.
It is noteworthy that, in Arabidopsis, gene products of water–water cycle enzymes including stroma-localized APX (45,46) are dual targeted into mitochondria as well as chloroplasts. Therefore, the susceptibility of APX to H2O2 is also important for understanding the mitochondrial ROS-scavenging system.
Mechanism of H2O2-Mediated Inactivation of PRX
Prx is a nonheme and thiol-based peroxidase that reduces H2O2, alkyl hydroperoxides and peroxinitrite with electrons provided by a cellular thiol, generally thioredoxin. The molecular mass ranges between 17 and 22 kDa. In recent years, animal Prxs have been thought to have a critical role in scavenging H2O2. Depending on their reaction mechanism, Prx are divided into three classes: typical 2-Cys Prx, atypical 2-Cys Prx and 1-Cys Prx (47). Based on the amino acid sequence, atypical 2-Cys Prxs of plants are subdivided into PrxQ and Prx II (3,48). In chloroplasts of Arabidopsis and rice, only three isoforms, i.e. typical 2-Cys, PrxQ and PrxII are found (3). Both of the 2-Cys Prxs share the same catalytic mechanism, in which an active site Cys (the peroxidatic Cys) is oxidized to a sulfenic acid (Cys-SOH) by H2O2. The sulfenic acid then forms a disulfide bond with the second Cys residue (resolving Cys) to release water. However, there are exceptions to this mechanism, e.g. in some cyanobacterial PrxQ that lack the resolving Cys (49). In the case of typical 2-Cys Prx, the disulfide bond is formed between two subunits of head-to-tail homodimers, but formed within a subunit in atypical 2-Cys. The disulfide bond is reduced back to Cys residues by an electron donor such as thioredoxin.
Typical 2-Cys Prx is most abundant among chloroplastic Prxs, and accounts for 0.6% of chloroplast protein (60 μm, 3, 15). The active form of 2-Cys Prx forms a decamer and is bound to the thylakoid membrane (15,50). PrxQ, which is also bound to the thylakoid membrane, accounts for 0.3% (51). In contrast, PrxII is localized in stroma (3).
The peroxidase acitivity of chloroplastic Prxs, which varies depending on the reducing agent (dithiothreitol, thioredoxin or glutaredoxin), is generally very low: 1–60 mol H2O2 mol enzyme−1 min−1 with the exception of poplar Prx Q (176) (3). This value is three–four orders of magnitude lower than that of chloroplastic APX, for which, for example, 42,000 mol H2O2 mol enzyme−1 min−1 at 0.5 mm ascorbate and 0.1 mm H2O2 (values can be calculated from Km and kcat values reported in 30. Note that one molecule of H2O2 is scavenged per oxidation of two molecules of ascorbate). Pérez-Ruiz et al. (52) reported that novel chloroplastic protein NTRC, which has NADPH-dependent thioredoxin reductase domain at N-terminus and thioredoxin domain at C-terminus, could more efficiently reduce 2-Cys Prx using NADPH as the electron donor than thioredoxin. Currently, the relative contribution of Prx to the overall water–water cycle activity remains unclear (3).
If the reaction intermediate, the sulfinylated form of the peroxidatic Cys residue, does not form a disulfide bond rapidly, it is oxidized by another H2O2 to form sulfinylated Cys. Protein sulfinic acid cannot be reduced by cellular reductants such as glutathione or thioredoxin, and therefore once peroxiredoxin has been sulfinylated, it no longer has peroxidase activity. In eukaryotic typical 2-Cys Prx, including plant homologs (15), the peroxidatic Cys are very easily sulfinylated by H2O2 compared to bacterial homolog. Crystallographic studies revealed that the distance between the peroxidatic and resolving Cys residues are approximately 10 (53) or 14 Å (16), which are too far to form the disulfide bond (Fig. 2). To allow the sulfinylated peroxidatic Cys to react with the resolving Cys, the protein adopts a locally unfolded conformation. Eukaryotic typical 2-Cys Prxs, including those of plants, have a unique loop (GGLG motif) between α4 and β5, and a helix (YF motif) at the C-terminal end, both of which are not present in the bacterial homolog (16). In the fully folded state, they pack next to each other and bury the active site helix containing the peroxidatic Cys (16). As a result of the presence of the unique motifs, the fully folded conformation is stabilized and the disulfide bond is less efficiently formed (16). This is one reason for the higher susceptibility of eukaryotic typical 2-Cys Prx to H2O2.
Restoration of Peroxidase Activity of Hyperoxidized Prx by Sulfiredoxin
Sulfiredoxin, which exists only in eukaryotes, specifically reduces sulfinylated Cys of Prx and restores peroxidase activity. It requires ATP hydrolysis, Mg2+and a thiol-reductant such as glutathione, thioredoxin or DTT. GTP or dATP can substitute for ATP in the reaction (54). The Km values for glutathione and thioredoxin are 1.8 mm and 1.2 μm, respectively, suggesting they are potential physiological electron donors for the sulfiredoxin reaction (54). The reaction is very slow: kcat = 0.18 min−1 (43). The Arabidopsis genome has one copy of the sulfiredoxin gene and its 13.9 kDa product is targeted to chloroplasts.
Sulfiredoxin access to the sulfinylated Cys is restricted because of the YF and GGLC motifs, and the sulfinic acid moiety is stabilized by a salt bridge to a neighboring Arg. It was thus unclear how sulfiredoxin can access and reduce sulfinylated Cys. Recently, the crystal structure of the human sulfinylated Prx–sulfiredoxin complex was resolved (55) and it was found that sulfiredoxin is able to locally unfold the active site of Prx and move aside the YF motif to allow the sulfinylated Cys to be exposed to the Prx–sulfiredoxin interface. Based on the structure and location of the ATP binding site, the catalytic process of sulfiredoxin was proposed; the sulfinylated Cys is first phosphorylated to form a sulphinic phosphoryl ester (Prx-Cys-SO2PO32−), which is then converted to a thiosulphinated bond (Prx-S(O)-S-sulfiredoxin, where active site Cys of sulfiredoxin is included). A thiol-reductant such as glutathione finally converts it to free sulfiredoxin and Prx Cys-SOH (55).
Why Typical 2-Cys Prx is Susceptible to H2O2
In view of its lower peroxidase activity compared with other peroxidases, and higher susceptibility compared with bacterial homolog of 2-Cys Prx, it has been questioned whether the sole function of 2-Cys Prx protein is to act as a peroxidase. Active 2-Cys Prx is suggested to scavenge H2O2 and other hydroperoxides under normal conditions. But once inactivated by excess H2O2, accumulated H2O2 would initiate ROS-mediated signaling to induce expression of defense-related genes. The peroxidase activity of 2-Cys Prx thus functions as “flood gate” of the signaling (47). For example, when animal Prxs are inactivated by the accumulation of H2O2 caused by injury, inflammation and activation of NADPH oxidase, H2O2 accumulates in the cells and initiates ROS-mediated signaling. The cell then rapidly synthesizes more peroxiredoxin, and reactivates the inactive form, so that the extra H2O2 can be removed after it has done its job. In fission yeast, hyperoxidation of 2-Cys Prx activates Sty1, stress-activated protein kinase, which leads to expression of sulfiredoxin (56,57).
In plants, a change in H2O2 levels in the chloroplast also acts as signal for nuclear gene expression via redox regulation and phosphorylation of transcription factors. A study by Rey et al. (58) suggests that inactivation of Prx may function in a similar manner. Contrary to expectation, Arabidopsis plants lacking a sulfiredoxin gene showed decreased rather than increased oxidative damage relative to wild-type plants in terms of lipid peroxidation and Fv/Fm under moderate stresses (which means the plants can adapt to it), although accumulation of hyperoxidized 2-Cys Prx was enhanced in the chloroplast. This suggests that H2O2 accumulated by 2-Cys Prx hyperoxidation or hyperoxidized 2-Cys Prx itself initiate signaling for defense-related gene expression. On the other hand, unique functions have been found for hyperoxidized Prx. The protein structure of 2-Cys Prx from yeast (59) and human (60) changes from low–molecular weight to high–molecular weight complexes (up to 1,000 kDa) in response to H2O2-mediated hyperoxidation of peroxidatic Cys. The high–molecular weight complex showed no peroxidase activity but acts as a molecular chaperone (61). It is not known whether plant 2-Cys Prx also acquires chaperone activity in response to hyperoxidation.
In summary, considering that the chloroplast is a major source of H2O2 generation, it is interesting to know why the H2O2-scavenging enzymes of chloroplasts, namely APX and 2-Cys Prx, are so susceptible to H2O2. In the case of 2-Cys Prx, it is thought that loss of peroxidase activity caused by hyperoxidation of the active site Cys may act as ROS-signal sensor. It may also be that hyperoxidized Prx of plants acquires chaperone activity, as shown for yeast and animals. In the case of APX, its susceptibility to H2O2 may be an evolutionary tradeoff that has allowed development of higher turnover rate for catalysis by locating cation radicals of reaction intermediates at the active site.