Structural snapshots of yeast alkyl hydroperoxide reductase Ahp1 peroxiredoxin reveal a novel two-cysteine mechanism of electron transfer to eliminate reactive oxygen species

Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that protect cells against reactive oxygen species and are involved in cellular signaling pathways. Alkyl hydroperoxide reductase Ahp1 belongs to the Prx5 subfamily and is a two-cysteine (2-Cys) Prx that forms an intermolecular disulfide bond. Enzymatic assays and bioinformatics enabled us to re-assign the peroxidatic cysteine (C_P) to Cys-62 and the resolving cysteine (C_R) to Cys-31 but not the previously reported Cys-120. Thus Ahp1 represents the first 2-Cys Prx with a peroxidatic cysteine after the resolving cysteine in the primary sequence. We also found the positive cooperativity of the substrate t-butyl hydroperoxide binding to Ahp1 homodimer at a Hill coefficient of ∼2, which enabled Ahp1 to eliminate hydroperoxide at much higher efficiency. To gain the structural insights into the catalytic cycle of Ahp1, we determined the crystal structures of Ahp1 in the oxidized, reduced, and Trx2-complexed forms at 2.40, 2.91, and 2.10 Å resolution, respectively. Structural superposition of the oxidized to the reduced form revealed significant conformational changes at the segments containing C_P and C_R. An intermolecular C_P-C_R disulfide bond crossing the A-type dimer interface distinguishes Ahp1 from other typical 2-Cys Prxs. The structure of the Ahp1-Trx2 complex showed for the first time how the electron transfers from thioredoxin to a peroxidase with a thioredoxin-like fold. In addition, site-directed mutagenesis in combination with enzymatic assays suggested that the peroxidase activity of Ahp1 would be altered upon the urmylation (covalently conjugated to ubiquitin-related modifier Urm1) of Lys-32.     

Structural properties of the peroxiredoxin AhpC2 from the hyperthermophilic eubacterium Aquifex aeolicus

Peroxiredoxins (Prxs) are thiol peroxidases that scavenge various peroxide substrates such as hydrogen peroxide (H₂O₂), alkyl hydroperoxides and peroxinitrite. They also function as chaperones and are involved in signal transduction by H₂O₂ in eukaryotic cells. The genome of Aquifex aeolicus, a microaerophilic, hyperthermophilic eubacterium, encodes four Prxs, among them an alkyl hydroperoxide reductase AhpC2 which was found to be closely related to archaeal 1-Cys peroxiredoxins. We determined the crystal structure of AhpC2 at 1.8?Å resolution and investigated its oligomeric state in solution by electron microscopy. AhpC2 is arranged as a toroid-shaped dodecamer instead of the typically observed decamer. The basic folding topology and the active site structure are conserved and possess a high structural similarity to other 1-Cys Prxs. However, the C-terminal region adopts an opposite orientation. AhpC2 contains three cysteines, Cys⁴⁹, Cys²¹², and Cys²¹⁸. The peroxidatic cysteine C_P⁴⁹ was found to be hyperoxidized to the sulfonic acid (SO₃H) form, while Cys²¹² forms an intra-monomer disulfide bond with Cys²¹⁸. Mutagenesis experiments indicate that Cys²¹² and Cys²¹⁸ play important roles in the oligomerization of AhpC2. Based on these structural characteristics, we proposed the catalytic mechanism of AhpC2. This study provides novel insights into the structure and reaction mechanism of 1-Cys peroxiredoxins.     

Distinct Characteristics of Two 2-Cys Peroxiredoxins of Vibrio vulnificus Suggesting Differential Roles in Detoxifying Oxidative Stress

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes reducing toxic peroxides. Two distinct 2-Cys Prxs, Prx1 and Prx2, were identified in Vibrio vulnificus, a facultative aerobic pathogen. Both Prxs have two conserved catalytic cysteines, C_P and C_R, but Prx2 is more homologous in amino acid sequences to eukaryotic Prx than to Prx1. Prx2 utilized thioredoxin A as a reductant, whereas Prx1 required AhpF. Prx2 contained GGIG and FL motifs similar to the motifs conserved in sensitive Prxs and exhibited sensitivity to overoxidation. MS analysis and C_P-SO₃H specific immunoblotting demonstrated overoxidation of C_P to C_P-SO₂H (or C_P-SO₃H) in vitro and in vivo, respectively. In contrast, Prx1 was robust and C_P was not overoxidized. Discrete expression of the Prxs implied that Prx2 is induced by trace amounts of H₂O₂ and thereby residential in cells grown aerobically. In contrast, Prx1 was occasionally expressed only in cells exposed to high levels of H₂O₂. A mutagenesis study indicated that lack of Prx2 accumulated sufficient H₂O₂ to induce Prx1. Kinetic properties indicated that Prx2 effectively scavenges low levels of peroxides because of its high affinity to H₂O₂, whereas Prx1 quickly degrades higher levels of peroxides because of its high turnover rate and more efficient reactivation. This study revealed that the two Prxs are differentially optimized for detoxifying distinct ranges of H₂O₂, and proposed that Prx2 is a residential scavenger of peroxides endogenously generated, whereas Prx1 is an occasional scavenger of peroxides exogenously encountered. Furthermore, genome sequence database search predicted widespread coexistence of the two Prxs among bacteria.     

Crystal structure of Escherichia coli thiol peroxidase in the oxidized state: insights into intramolecular disulfide formation and substrate binding in atypical 2-Cys peroxiredoxins

Thioredoxin-dependent thiol peroxidase (Tpx) from Escherichia coli represents a group of antioxidant enzymes that are widely distributed in pathogenic bacterial species and which belong to the peroxiredoxin (Prx) family. Bacterial Tpxs are unique in that the location of the resolving cysteine (CR) is different from those of other Prxs. E. coli Tpx (EcTpx) shows substrate specificity toward alkyl hydroperoxides over H2O2 and is the most potent reductant of alkyl hydroperoxides surpassing AhpC and BCP, the other E. coli Prx members. Here, we present the crystal structure of EcTpx in the oxidized state determined at 2.2-A resolution. The structure revealed that Tpxs are the second type of atypical 2-Cys Prxs with an intramolecular disulfide bond formed between the peroxidatic (CP, Cys61) and resolving (Cys95) cysteine residues. The extraordinarily long N-terminal chain of EcTpx folds into a beta-hairpin making the overall structure very compact. Modeling suggests that, in atypical 2-Cys Prxs, the CR-loop as well as the CP-loop may alternately assume the fully folded or locally unfolded conformation depending on redox states, as does the CP-loop in typical 2-Cys Prxs. EcTpx exists as a dimer stabilized by hydrogen bonds. Its substrate binding site extends to the dimer interface. A modeled structure of the reduced EcTpx in complex with 15-hydroperoxyeicosatetraenoic acid suggests that the size and shape of the binding site are particularly suited for long fatty acid hydroperoxides consistent with its greater reactivity.     

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

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

Overview on Peroxiredoxin

Peroxiredoxins (Prxs) are a very large and highly conserved family of peroxidases that reduce peroxides, with a conserved cysteine residue, designated the “peroxidatic” Cys (C_P) serving as the site of oxidation by peroxides (Hall et al., 2011; Rhee et al., 2012). Peroxides oxidize the C_P-SH to cysteine sulfenic acid (C_P–SOH), which then reacts with another cysteine residue, named the “resolving” Cys (C_R) to form a disulfide that is subsequently reduced by an appropriate electron donor to complete a catalytic cycle. This overview summarizes the status of studies on Prxs and relates the following 10 minireviews.     

A 1-Cys Peroxiredoxin from a Thermophilic Archaeon Moonlights as a Molecular Chaperone to Protect Protein and DNA against Stress-Induced Damage

Peroxiredoxins (Prxs) act against hydrogen peroxide (H₂O₂), organic peroxides, and peroxynitrite. Thermococcus kodakaraensis KOD1, an anaerobic archaeon, contains many antioxidant proteins, including three Prxs (Tk0537, Tk0815, and Tk1055). Only Tk0537 has been found to be induced in response to heat, osmotic, and oxidative stress. Tk0537 was found to belong to a 1-Cys Prx6 subfamily based on sequence analysis and was named 1-Cys TkPrx. Using gel filtration chromatography, electron microscopy, and blue-native polyacrylamide gel electrophoresis, we observed that 1-Cys TkPrx exhibits oligomeric forms with reduced peroxide reductase activity as well as decameric and dodecameric forms that can act as molecular chaperones by protecting both proteins and DNA from oxidative stress. Mutational analysis showed that a cysteine residue at the N-terminus (Cys⁴⁶) was responsible for the peroxide reductase activity, and cysteine residues at the C-terminus (Cys²⁰⁵ and Cys²¹¹) were important for oligomerization. Based on our results, we propose that interconversion between different oligomers is important for regulating the different functions of 1-Cys TkPrx.     

Structure,mechanism and regulation of peroxiredoxins

Peroxiredoxins (Prxs) are a ubiquitous family of antioxidant enzymes that also control cytokine-induced peroxide levels which mediate signal transduction in mammalian cells. Prxs can be regulated by changes to phosphorylation, redox and possibly oligomerization states. Prxs are divided into three classes: typical 2-Cys Prxs; atypical 2-Cys Prxs; and 1-Cys Prxs. All Prxs share the same basic catalytic mechanism, in which an active-site cysteine (the peroxidatic cysteine) is oxidized to a sulfenic acid by the peroxide substrate. The recycling of the sulfenic acid back to a thiol is what distinguishes the three enzyme classes. Using crystal structures, a detailed catalytic cycle has been derived for typical 2-Cys Prxs, including a model for the redox-regulated oligomeric state proposed to control enzyme activity.     

A colorimetric assay for sulfiredoxin activity using inorganic phosphate measurement

2-Cys peroxiredoxin (Prx) is the major subgroup of a family of Prx enzymes that reduce peroxide molecules such as hydrogen peroxide (H₂O₂). 2-Cys Prxs are inactivated when their active site cysteine residue is hyperoxidized to sulfinic acid. Sulfiredoxin (Srx) is an enzyme that catalyzes reduction of hyperoxidized 2-Cys Prxs in the presence of ATP, Mg²⁺, and thiol equivalent. Therefore, Srx activity is crucial for cellular function of 2-Cys Prxs. The method currently available for the determination of Srx activity relies on immunoblot detection using antibodies to hyperoxidized enzymes. Here we introduce a simple quantitative assay for Srx activity based on the colorimetric determination of inorganic phosphate released in Srx-dependent reduction of hyperoxidized Prx using the malachite green. The colorimetric assay was used for high-throughput screening of 25,000 chemicals to find Srx inhibitors.     

Novel Protective Mechanism against Irreversible Hyperoxidation of Peroxiredoxin: N��-TERMINAL ACETYLATION OF HUMAN PEROXIREDOXIN II*

Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (C_P), cycles between thiol (C_P-SH) and disulfide (–S–S–) states via a sulfenic (C_P-SOH) intermediate. Hyperoxidation of the C_P thiol to its sulfinic (C_P-SO₂H) derivative has been shown to be reversible, but its sulfonic (C_P-SO₃H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with C_P-SO₃H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that N^α-terminal acetylation (N^α-Ac) occurred exclusively on Prx II after demethionylation. N^α-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-N^α-acetylated and N^α-terminal acetylated Prx II revealed that N^α-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of T_m from 59.6 to 70.9 °C. These findings suggest that the structural maintenance of Prx II by N^α-Ac may be responsible for preventing its hyperoxidation to form C_P-SO₃H.Peroxiredoxins (Prxs)⁴ are a family of peroxidases that possess a conserved cysteine residue at the catalytic site for the reduction of peroxide/peroxynitrite. Using thiol-based reducing equivalents, like thioredoxin, Prxs catalyze the reduction of hydrogen peroxide, alkylhydroperoxides, and peroxynitrite to water, corresponding alcohols, and nitrite, respectively (1–8). Based on the number and location of conserved cysteine residue(s) directly involved in peroxide reduction, the six isotypes of mammalian Prx can be grouped into three distinct subgroups as follows: 2-Cys Prx, atypical 2-Cys Prx, and 1-Cys Prx, (1–2, 5). Human Prx I (hPrx I) and Prx II (hPrx II) are members of the 2-Cys Prx subgroup and thus contain two conserved cysteine residues that are directly involved in peroxidase activity. Cys⁵² for hPrx I and Cys⁵¹ for hPrx II are designated the peroxidatic cysteines (C_P). These residues attack the O–O bond of the peroxide (ROOH) substrate to form the product (ROH) and the sulfenic derivative C_P-SOH. This sulfenic derivative then forms a disulfide bond with the other conserved cysteine residue, which is referred to as the resolving cysteine (C_R; Cys¹⁷³ in hPrx I and Cys¹⁷² in hPrx II). In the case of 2-Cys Prxs, the disulfide partners, C_P and C_R, reside within different subunits; therefore, the disulfide bond established between C_P and C_R (C_P-S–S-C_R) is intermolecular. The reduced thioredoxin molecule is responsible for reducing the C_P-S–S-C_R disulfide bond to generate sulfhydryls (1–3, 5, 9).The C_P of eukaryotic 2-Cys Prxs is vulnerable to hyperoxidation, which results in the loss of its peroxidase activity. This feature is referred to as the “floodgate” mechanism, by which Prxs function as a redox sensor for the regulation of cell signaling (10–11). Hyperoxidation of C_P does not occur when the disulfide bond (C_P-S–S-C_R) is formed. However, the thiol (C_P-SH) can be hyperoxidized via the sulfenic (C_P-SOH) derivative intermediate in the absence of C_P-S-S-C_R formation during catalysis (12). Two different hyperoxidation products of C_P, the reversible sulfinic (C_P-SO₂H) derivative and the irreversible sulfonic (C_P-SO₃H) derivative, have been identified. The irreversible C_P-SO₃H was reported in Tsa1p, a yeast 2-Cys Prx, based on in vivo and in vitro regeneration assay results, and a stronger reactivity to an anti-Tsa1p-SO₃H antibody, which exhibits high specificity toward Tsa1p-C_P-SO₃H relative to Tsa1p-C_P-SO₂H (13). Both forms of hyperoxidized Prxs, C_P-SO₂H and C_P-SO₃H, are superimposed on the acidic migrated spot instead of the Prx-SH spot on a two-dimensional polyacrylamide gel because of the introduction of one negative charge by hyperoxidation (12–16). The protein sulfinic acid reductase, sulfiredoxin, is responsible for reversing 2-Cys Prx-SO₂H to Prx-SH in the presence of ATP and thiol-reducing equivalents like thioredoxin or glutathione (17–24). Until now, an intracellular enzymatic regeneration system for Prx-SO₃H has not been reported.Because mammalian Prx I and Prx II have been studied independently in a number of different organisms and cultured cells, the comparative biochemical data supporting their distinctive functional identities is still very limited. Recombinant Prx I (rPrx I) showed a 2.6-fold higher specific activity as a peroxidase than the recombinant Prx II (rPrx II) without any obvious catalytic or mechanistic differences (25, 26). Recent competition kinetics studies of hPrx II revealed a rate constant of 1.3 × 10⁷ m^–1 s^–1, which is fast enough to favor an intracellular hydrogen peroxide target even in competition with catalase or glutathione peroxidase (27, 28). The kinetic parameters of the competition assay for hPrx I are still not available. Mammalian Prx I interacts with and regulates a broad spectrum of proteins, such as the Src homology domain 3 of c-Abl (29), the Myc box II (MBII) domain of c-Myc (30), the macrophage migration inhibitory factor (MIF, 31), the androgen receptor (32), and the apoptosis signal-regulating kinase-1 (ASK-1) (33). The suggested roles of Prx I in interactions with these molecules are those of a tumor repressor, a survival enhancer, and a growth regulator. Although these suggested functions are controversial (34), all of them can be attributed to the peroxide-scavenging capacity of Prx I (at least in part), except for the enhancement of androgen receptor transactivation (32). Prx II interacts with platelet-derived growth factor receptor and functions as a negative regulator for platelet-derived growth factor signaling (35). Prx II also binds to phospholipase D1 (PLD1) and functions as a hydrogen peroxide-stimulated PLD1 signal terminator (36). Both of these suggested Prx II roles are attributable to the peroxidase activity of Prx II. The major phenotypes of Prx I knock-out mice involve the development of a variety of age-related cancers, hemolytic anemia (37), and dramatic shifts in subcellular reactive oxygen species localization (38). Prx II knock-out mice exhibit splenomegaly and a lack of tumor development in any cell type or tissue (39). Until now, the protein molecule that interacts with Prx I and Prx II has not been characterized, and there is no indication of a heterodimer between Prx I and Prx II. Despite their similar peroxide-scavenging capacities, it is reasonable to conclude that the Prx I and Prx II are unable to compensate for each other in terms of physiological roles. There are several examples of tissue- or cell type-specific expression patterns, such as exclusive Prx I expression in astrocytes and Leydig cells and Prx II expression in neurons and Sertoli cells (40, 41); however, Prx I and Prx II are coexpressed in the majority of mammalian cells and tissues, suggesting distinguished biochemical characteristics of their cellular regulatory mechanisms. Recently, the unique presence of Cys⁸³ in hPrx I, which contributes to the stability of the dimer-dimer interface and suppresses local unfolding, has been claimed to be prone to overoxidation of Prx I (42). The contribution of the dimer-decamer interconversion to the regulation of Prx I activity has also been reported (43).In this study, we confirmed that hPrx II was more susceptible to hyperoxidation as well as more prone to regeneration than hPrx I in HeLa cells. We also found that the difficulty in regenerating hPrx I was caused by irreversible sulfonic (C_P-SO₃H) hyperoxidation. Using AspN (EC 3.4.24.33) peptide fingerprints, we identified the N^α-terminal acetylation exclusively on hPrx II. In addition, we provide evidence for the structural maintenance offered by N^α-terminal acetylation of hPrx II, which possibly contributes to preventing irreversible overoxidation of C_P-SO₃H.     

Overoxidation of 2-Cys Peroxiredoxin in Prokaryotes: CYANOBACTERIAL 2-CYS PEROXIREDOXINS SENSITIVE TO OXIDATIVE STRESS*?

In eukaryotic organisms, hydrogen peroxide has a dual effect; it is potentially toxic for the cell but also has an important signaling activity. According to the previously proposed floodgate hypothesis, the signaling activity of hydrogen peroxide in eukaryotes requires a transient increase in its concentration, which is due to the inactivation by overoxidation of 2-Cys peroxiredoxin (2-Cys Prx). Sensitivity to overoxidation depends on the structural GGLG and YF motifs present in eukaryotic 2-Cys Prxs and is believed to be absent from prokaryotic enzymes, thus representing a paradoxical gain of function exclusive to eukaryotic organisms. Here we show that 2-Cys Prxs from several prokaryotic organisms, including cyanobacteria, contain the GG(L/V/I)G and YF motifs characteristic of sensitive enzymes. In search of the existence of overoxidation-sensitive 2-Cys Prxs in prokaryotes, we have analyzed the sensitivity to overoxidation of 2-Cys Prxs from two cyanobacterial strains, Anabaena sp. PCC7120 and Synechocystis sp. PCC6803. In vitro analysis of wild type and mutant variants of the Anabaena 2-Cys Prx showed that this enzyme is overoxidized at the peroxidatic cysteine residue, thus constituting an exception among prokaryotes. Moreover, the 2-Cys Prx from Anabaena is readily and reversibly overoxidized in vivo in response to high light and hydrogen peroxide, showing higher sensitivity to overoxidation than the Synechocystis enzyme. These cyanobacterial strains have different strategies to cope with hydrogen peroxide. While Synechocystis has low content of less sensitive 2-Cys Prx and high catalase activity, Anabaena contains abundant and sensitive 2-Cys Prx, but low catalase activity, which is remarkably similar to the chloroplast system.     

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

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

OsPRX2 contributes to stomatal closure and improves potassium deficiency tolerance in rice

Peroxiredoxins (Prxs) which are thiol-based peroxidases have been implicated in the toxic reduction and intracellular concentration regulation of hydrogen peroxide. In Arabidopsis thaliana At2-CysPrxB (At5g06290) has been demonstrated to be essential in maintaining the water-water cycle for proper H₂O₂ scavenging. Although the mechanisms of 2-Cys Prxs have been extensively studied in Arabidopsis thaliana, the function of 2-Cys Prxs in rice is unclear. In this study, a rice homologue gene of At2-CysPrxB, OsPRX2 was investigated aiming to characterize the effect of 2-Cys Prxs on the K⁺-deficiency tolerance in rice. We found that OsPRX2 was localized in the chloroplast. Overexpressed OsPRX2 causes the stomatal closing and K⁺-deficiency tolerance increasing, while knockout of OsPRX2 lead to serious defects in leaves phenotype and the stomatal opening under the K⁺-deficiency tolerance. Detection of K⁺ accumulation, antioxidant activity of transgenic plants under the starvation of potassium, further confirmed that OsPRX2 is a potential target for engineering plants with improved potassium deficiency tolerance.     

Crystal Structure and Functional Analysis of the Glutaminyl Cyclase from Xanthomonas campestris

Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the formation of pyroglutamate (pGlu) at the N-terminus of many proteins and peptides, a critical step for the maturation of these bioactive molecules. Proteins having QC activity have been identified in animals and plants, but not in bacteria. Here, we report the first bacterial QC from the plant pathogen Xanthomonas campestris (Xc). The crystal structure of the enzyme was solved and refined to 1.44-Å resolution. The structure shows a five-bladed β-propeller and exhibits a scaffold similar to that of papaya QC (pQC), but with some sequence deletions and conformational changes. In contrast to the pQC structure, the active site of XcQC has a wider substrate-binding pocket, but its accessibility is modulated by a protruding loop acting as a flap. Enzyme activity analyses showed that the wild-type XcQC possesses only 3% QC activity compared to that of pQC. Superposition of those two structures revealed that an active-site glutamine residue in pQC is substituted by a glutamate (Glu⁴⁵) in XcQC, although position 45 is a glutamine in most bacterial QC sequences. The E45Q mutation increased the QC activity by an order of magnitude, but the mutation E45A led to a drop in the enzyme activity, indicating the critical catalytic role of this residue. Further mutagenesis studies support the catalytic role of Glu⁸⁹ as proposed previously and confirm the importance of several conserved amino acids around the substrate-binding pocket. XcQC was shown to be weakly resistant to guanidine hydrochloride, extreme pH, and heat denaturations, in contrast to the extremely high stability of pQC, despite their similar scaffold. On the basis of structure comparison, the low stability of XcQC may be attributed to the absence of both a disulfide linkage and some hydrogen bonds in the closure of β-propeller structure. These results significantly improve our understanding of the catalytic mechanism and extreme stability of type I QCs, which will be useful in further applications of QC enzymes.     

Molecular recognition in the interaction of chloroplast 2-Cys peroxiredoxin with NADPH-thioredoxin reductase C (NTRC) and thioredoxin x

In addition to the standard NADPH thioredoxin reductases (NTRs), plants hold a plastidic NTR (NTRC), with a thioredoxin module fused at the C-terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs). The interaction of NTRC and chloroplastic thioredoxin x with 2-Cys Prxs has been confirmed in vivo, by bimolecular fluorescence complementation (BiFC) assays, and in vitro, by isothermal titration calorimetry (ITC) experiments. In comparison with thioredoxin x, NTRC interacts with 2-Cys Prx with higher affinity, both the thioredoxin and NTR domains of NTRC contributing significantly to this interaction, as demonstrated by using the NTR and thioredoxin modules of the enzyme expressed separately. The presence of the thioredoxin domain seems to prevent the interaction of NTRC with thioredoxin x.     

Backbone chemical shift assignments for <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> peroxiredoxin Q in the reduced and oxidized states: a dramatic change in backbone dynamics

Peroxiredoxins (Prx) are ubiquitous enzymes that reduce peroxides as part of antioxidant defenses and redox signaling. While Prx catalytic activity and sensitivity to hyperoxidative inactivation depend on their dynamic properties, there are few examples where their dynamics has been characterized by NMR spectroscopy. Here, we provide a foundation for studies of the solution properties of peroxiredoxin Q from the plant pathogen Xanthomonas campestris (XcPrxQ) by assigning the observable ¹H^N, ¹⁵N, ¹³C^α, ¹³C^β, and ¹³C′ chemical shifts for both the reduced (dithiol) and oxidized (disulfide) states. In the reduced state, most of the backbone amide resonances (149/152, 98 %) can be assigned in the XcPrxQ ¹H–¹⁵N HSQC spectrum. In contrast, a remarkable 51 % (77) of these amide resonances are not visible in the ¹H–¹⁵N HSQC spectrum of the disulfide state of the enzyme, indicating a substantial change in backbone dynamics associated with the formation of an intramolecular C48–C84 disulfide bond.     

Catalytic Mechanism of Sulfiredoxin from Saccharomyces cerevisiae Passes through an Oxidized Disulfide Sulfiredoxin Intermediate That Is Reduced by Thioredoxin

Sulfiredoxin catalyzes the ATP-dependent reduction of overoxidized eukaryotic 2-Cys peroxiredoxin PrxSO₂ into sulfenic PrxSOH. Recent mechanistic studies on sulfiredoxins have validated a catalytic mechanism that includes formation of a phosphoryl intermediate on the sulfinyl moiety of PrxSO₂, followed by an attack of the catalytic cysteine of sulfiredoxin on the phosphoryl intermediate that leads to formation of a thiosulfinate intermediate PrxSO-S-sulfiredoxin. Formation of this intermediate implies the recycling of sulfiredoxin into the reduced form. In this study, we have investigated how the reductase activity of the Saccharomyces cerevisiae sulfiredoxin is regenerated. The results show that an oxidized sulfiredoxin under disulfide state is formed between the catalytic Cys⁸⁴ and Cys⁴⁸. This oxidized sulfiredoxin species is shown to be catalytically competent along the sulfiredoxin-recycling process and is reduced selectively by thioredoxin. The lack of Cys⁴⁸ in the mammalian sulfiredoxins and the low efficiency of reduction of the thiosulfinate intermediate by thioredoxin suggest a recycling mechanism in mammals different from that of sulfiredoxin from Saccharomyces cerevisiae.     

Crystal structure of an archaeal peroxiredoxin from the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix K1

Peroxiredoxins (Prxs) are thiol-dependent peroxidases that catalyze the detoxification of various peroxide substrates such as H2O2, peroxinitrite, and hydroperoxides, and control some signal transduction in eukaryotic cells. Prxs are found in all cellular organisms and represent an enormous superfamily. Recent genome sequencing projects and biochemical studies have identified a novel subfamily, the archaeal Prxs. Their primary sequences are similar to those of the 1-Cys Prxs, which use only one cysteine residue in catalysis, while their catalytic properties resemble those of the typical 2-Cys Prxs, which utilize two cysteine residues from adjacent monomers within a dimer in catalysis. We present here the X-ray crystal structure of an archaeal Prx from the aerobic hyperthermophilic crenarchaeon, Aeropyrum pernix K1, determined at 2.3 A resolution (Rwork of 17.8% and Rfree of 23.0%). The overall subunit arrangement of the A.pernix archaeal Prx is a toroid-shaped pentamer of homodimers, or an (alpha2)5 decamer, as observed in the previously reported crystal structures of decameric Prxs. The basic folding topology and the peroxidatic active site structure are essentially the same as those of the 1-Cys Prx, hORF6, except that the C-terminal extension of the A.pernix archaeal Prx forms a unique helix with its flanking loops. The thiol group of the peroxidatic cysteine C50 is overoxidized to sulfonic acid. Notably, the resolving cysteine C213 forms the intra-monomer disulfide bond with the third cysteine, C207, which should be a unique structural characteristic in the many archaeal Prxs that retain two conserved cysteine residues in the C-terminal region. The conformational flexibility near the intra-monomer disulfide linkage might be necessary for the dramatic structural rearrangements that occur in the catalytic cycle.