Dimer-oligomer interconversion of wild-type and mutant rat 2-Cys peroxiredoxin: disulfide formation at dimer-dimer interfaces is not essential for decamerization

Rat heme-binding protein 23 (HBP23)/peroxiredoxin (Prx I) belongs to the 2-Cys peroxiredoxin type I family and exhibits peroxidase activity coupled with reduced thioredoxin (Trx) as an electron donor. We analyzed the dimer-oligomer interconversion of wild-type and mutant HBP23/Prx I by gel filtration and found that the C52S and C173S mutants existed mostly as decamers, whereas the wild type was a mixture of various forms, favoring the decamer at higher protein concentration and lower ionic salt concentration and in the presence of dithiothreitol. The C83S mutant was predominantly dimeric, in agreement with a previous crystallographic analysis (Hirotsu, S., Abe, Y., Okada, K., Nagahara, N., Hori, H., Nishino, T., and Hakoshima, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12333-12338). X-ray diffraction analysis of the decameric C52S mutant revealed a toroidal structure (diameter, approximately 130A; inside diameter, approximately 55A; thickness, approximately 45A). In contrast to human Prx I, which was recently reported to exist predominantly as the decamer with Cys(83)-Cys(83) disulfide bonds at all dimer-dimer interfaces, rat HBP23/Prx I has a Cys(83)-Cys(83) disulfide bond at only one dimer-dimer interface (S-S separation of approximately 2.1A), whereas the interactions at the other interfaces (mean S-S separation of 3.6A) appear to involve hydrophobic and van der Waals forces. This finding is consistent with gel filtration analyses showing that the protein readily interconverts between dimer and oligomeric forms. The C83S mutant exhibited similar peroxidase activity to the wild type, which is exclusively dimeric, in the Trx/Trx reductase system. At higher concentrations, where the protein was mostly decameric, less efficient attack of reduced Trx was observed in a [(14)C]iodoacetamide incorporation experiment. We suggest that the dimerdecamer interconversion may have a regulatory role.     

Human peroxiredoxin 1 and 2 are not duplicate proteins: the unique presence of CYS83 in Prx1 underscores the structural and functional differences between Prx1 and Prx2

Human peroxiredoxins 1 and 2, also known as Prx1 and Prx2, are more than 90% homologous in their amino acid sequences. Prx1 and Prx2 are elevated in various cancers and are shown to influence diverse cellular processes. Although their growth regulatory role has traditionally been attributed to the peroxidase activity, the physiological significance of this function is unclear because the proteins are highly susceptible to inactivation by H(2)O(2). A chaperone activity appears to emerge when their peroxidase activity is lost. Structural studies suggest that they may form a homodimer or doughnut-shaped homodecamer. However, little information is available whether human Prx1 and Prx2 are duplicative in structure and function. We noted that Prx1 contains a cysteine (Cys(83)) at the putative dimer-dimer interface, which is absent in Prx2. We studied the role of Cys(83) in regulating the peroxidase and chaperone activities of Prx1, because the redox status of Cys(83) might influence the oligomeric structure and consequently the functions of Prx1. We show that Prx1 is more efficient as a molecular chaperone, whereas Prx2 is better suited as a peroxidase enzyme. Substituting Cys(83) with Ser(83) (Prx1C83S) results in dramatic changes in the structural and functional characteristics of Prx1 in a direction similar to those of Prx2. Here we also report the first crystal structure of human Prx1 and the presence of the Cys(83)-Cys(83) bond at the dimer-dimer interface of decameric Prx1. These findings are consistent with the hypothesis that human Prx1 and Prx2 possess unique functions and regulatory mechanisms and that Cys(83) bestows a distinctive identity to Prx1.     

Dimers to doughnuts: redox-sensitive oligomerization of 2-cysteine peroxiredoxins

2-Cys peroxiredoxins (Prxs) are a large and diverse family of peroxidases which, in addition to their antioxidant functions, regulate cell signaling pathways, apoptosis, and differentiation. These enzymes are obligate homodimers (alpha(2)), utilizing a unique intermolecular redox-active disulfide center for the reduction of peroxides, and are known to form two oligomeric states: individual alpha(2) dimers or doughnut-shaped (alpha(2))(5) decamers. Here we characterize both the oligomerization properties and crystal structure of a bacterial 2-Cys Prx, Salmonella typhimurium AhpC. Analytical ultracentrifugation and dynamic light scattering show that AhpC's oligomeric state is redox linked, with oxidization favoring the dimeric state. The 2.5 A resolution crystal structure (R = 18.5%, R(free) = 23.9%) of oxidized, decameric AhpC reveals a metastable oligomerization intermediate, allowing us to identify a loop that adopts distinct conformations associated with decameric and dimeric states, with disulfide bond formation favoring the latter. This molecular switch contains the peroxidatic cysteine and acts to buttress the oligomerization interface in the reduced, decameric enzyme. A structurally detailed catalytic cycle incorporating these ideas and linking activity to oligomeric state is presented. Finally, on the basis of sequence comparisons, we suggest that the enzymatic and signaling activities of all 2-Cys Prxs are regulated by a redox-sensitive dimer to decamer transition.     

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.     

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

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

Structural basis for the retroreduction of inactivated peroxiredoxins by human sulfiredoxin

Sufiredoxins (Srx) repair the inactivated forms of typical two-Cys peroxiredoxins (Prx) implicated in hydrogen peroxide-mediated cell signaling. The reduction of the cysteine sulfinic acid moiety within the active site of the Prx by Srx involves novel sulfur chemistry and the use of ATP and Mg(2+). The 1.65 A crystal structure of human Srx (hSrx) exhibits a new protein fold and a unique nucleotide binding motif containing the Gly98-Cys99-His100-Arg101 sequence at the N-terminus of an alpha-helix. HPLC analysis of the reaction products has confirmed that the site of ATP cleavage is between the beta- and gamma-phosphate groups. Cys99 and the gamma-phosphate of ATP, modeled within the active site of the 2.0 A ADP product complex structure, are adjacent to large surface depressions containing additional conserved residues. These features and the necessity for significant remodeling of the Prx structure suggest that the interactions between hSrx and typical two-Cys Prxs are specific. Moreover, the concave shape of the hSrx active site surface appears to be ideally suited to interacting with the convex surface of the toroidal Prx decamer.     

Reaction mechanism of plant 2-Cys peroxiredoxin. Role of the C terminus and the quaternary structure

Barley 2-cysteine peroxiredoxin (2-Cys Prx) was analyzed for peroxide reduction, quaternary structure, thylakoid attachment, and function as well as in vivo occurrence of the inactivated form, with emphasis on the role of specific amino acid residues. Data presented show the following. 1) 2-Cys Prx has a broad substrate specificity and reduces even complex lipid peroxides such as phosphatidylcholine dilineoyl hydroperoxide, although at low rates. 2) 2-Cys Prx partly becomes irreversibly oxidized by peroxide substrates during the catalytic cycle in a concentration-dependent manner, particularly by bulky hydroperoxides. 3) Using dithiothreitol and thioredoxin (Trx) as reductants, amino acids were identified that are important for peroxide reduction (Cys64, Arg140, and Arg163), regeneration by Trx (Cys185), and conformation changes from dimer to oligomer (Thr66, Trp99, and Trp189). 4) Oligomerization decreased the rate of Trx-dependent peroxide detoxification. 5) Comparison of PrxWT, W99L, and W189L using static and time-resolved LIF techniques demonstrated the contributions of the tryptophan residues and yielded information about their local environment. Data indicated protein dynamics in the catalytic site and the carboxyl terminus during the reduction-oxidation cycle. 6) Reduced and inactivated barley 2-Cys Prx oligomerized and attached to the thylakoid membrane in isolated chloroplasts. The in vivo relevance of inactivation was shown in leaves subjected to cold and wilting stress and during senescence. Based on these results, it is hypothesized that in addition to its function in peroxide detoxification, 2-Cys Prx may play a role as a structural redox sensor in chloroplasts.     

ATP-dependent modulation and autophosphorylation of rapeseed 2-Cys peroxiredoxin

2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous thiol-containing peroxidases that have been implicated in antioxidant defense and signal transduction. Although their biochemical features have been extensively studied, little is known about the mechanisms that link the redox activity and non-redox processes. Here we report that the concerted action of a nucleoside triphosphate and Mg(2+) on rapeseed 2-Cys Prx reversibly impairs the peroxidase activity and promotes the formation of high molecular mass species. Using protein intrinsic fluorescence in the analysis of site-directed mutants, we demonstrate that ATP quenches the emission intensity of Trp179, a residue close to the conserved Cys175. More importantly, we found that ATP facilitates the autophosphorylation of 2-Cys Prx when the protein is successively reduced with thiol-bearing compounds and oxidized with hydroperoxides or quinones. MS analyses reveal that 2-Cys Prx incorporates the phosphoryl group into the Cys175 residue yielding the sulfinic-phosphoryl [Prx-(Cys175)-SO(2)PO(3)(2-)] and the sulfonic-phosphoryl [Prx-(Cys175)-SO(3)PO(3)(2-)] anhydrides. Hence, the functional coupling between ATP and 2-Cys Prx gives novel insights into not only the removal of reactive oxygen species, but also mechanisms that link the energy status of the cell and the oxidation of cysteine residues.     

Direct evidence for the formation of a complex between 1-cysteine peroxiredoxin and glutathione S-transferase pi with activity changes in both enzymes

Glutathione S-transferase pi (GST pi) has been shown to reactivate oxidized 1-cysteine peroxiredoxin (1-Cys Prx, Prx VI, Prdx6, and AOP2). We now demonstrate that a heterodimer complex is formed between 1-Cys Prx with a C-terminal His6 tag and GST pi upon incubation of the two proteins at pH 8.0 in buffer containing 20% 1,6-hexanediol to dissociate the homodimers, followed by dialysis against buffer containing 2.5 mM glutathione (GSH) but lacking 1,6-hexanediol. The heterodimer can be purified by chromatography on nickel-nitriloacetic acid agarose in the presence of GSH. N-Terminal sequencing showed that equimolar amounts of the two proteins are present in the isolated complex. In the heterodimer, 1-Cys Prx is fully active toward either H2O2 or phospholipid hydroperoxide, while the GST pi activity is approximately 25% of that of the GST pi homodimer. In contrast, the 1-Cys Prx homodimer lacks peroxidase activity even in the presence of free GSH. The heterodimer is also formed in the presence of S-methylglutathione, but no 1-Cys Prx activity is found under these conditions. The yield of heterodimer is decreased in the absence of 1,6-hexanediol or GSH. Rapid glutathionylation of 1-Cys Prx in the heterodimer is detected by immunoblotting. Subsequently, a disulfide-linked dimer is observed on SDS-PAGE, and the free cysteine content is decreased by 2 per heterodimer. The involvement of particular binding sites in heterodimer formation was tested by site-directed mutagenesis of the two proteins. For 1-Cys Prx, neither Cys47 nor Ser32 is required for heterodimer formation but Cys47 is essential for 1-Cys Prx activation. For GST pi, Cys47 and Tyr7 (at or near the GSH-binding site) are needed for heterodimer formation but three other cysteines are not. We conclude that reactivation of oxidized 1-Cys Prx by GST pi occurs by heterodimerization of 1-Cys Prx and GST pi harboring bound GSH, followed by glutathionylation of 1-Cys Prx and then formation of an intersubunit disulfide. Finally, the GSH-mediated reduction of the disulfide regenerates the reduced active-site sulfhydryl of 1-Cys Prx.     

Engineering of 2-Cys peroxiredoxin for enhanced stress-tolerance

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

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.     

Assignment of the complete disulphide bridge pattern in the human recombinant follitropin beta-chain

The chemical assessment of the complete disulphide bridge pattern in the beta-chain of human recombinant follicotropin (betaFSH) was accomplished by integrating classical biochemical methodologies with mass spectrometric procedures. A proteolytic strategy consisting of a double digestion of native betaFSH using the broad-specificity protease subtilisin first, followed by trypsin, was employed. The resulting peptide mixture was directly analysed by FAB-MS, leading to the assignment of the first three disulphide bridges. The remaining S-S bridges were determined by HPLC fractionation of the proteolytic digest followed by ESMS analysis of the individual fractions. The pattern of cysteine couplings in betaFSH was determined as: Cys3-Cys5l, Cys17-Cys66, Cys20-Cys104, Cys28-Cys82, Cys32-Cys84 and Cys87-Cys94, confirming the arrangement inferred from the crystal structure of the homologous betaCG. A subset of the S-S bridge pattern comprising Cys3-Cys51, Cys28-Cys82 and Cys32-Cys84 constitutes a cysteine knot motif similar to that found in the growth factor superfamily.     

Crystal structure of the tryparedoxin peroxidase from the human parasite Trypanosoma cruzi

Tryparedoxin peroxidase from Trypanosoma cruzi (TcTXNPx) belongs to the family of typical 2-Cys peroxiredoxins. These enzymes function as antioxidants through their peroxidase and peroxynitrite reductase activities. In T. cruzi, as in all trypanosomatids, this enzyme is the final electron acceptor of a unique system for detoxifying hydroperoxides, constituting a relevant target for drug design. We have determined the crystal structure of TcTXPNx in the reduced active state. The structure comprises 10 subunits in the asymmetric unit, associated to form a decamer of toroidal shape obeying 52 (D5) point group symmetry. We have analyzed the structure of TcTXNPx by comparing it with other structures of typical 2-Cys peroxiredoxins in both redox states, and have identified key residues in the structural rearrangement taking place in the enzymatic cycle. This is the first report of the structure of an active peroxiredoxin that has peroxidase and peroxynitrite reductase activity, and it is noteworthy that it is from a human parasite. This knowledge is of interest for further understanding peroxide metabolism in these parasites, and in the design of new trypanosomatidal drugs against Chagas disease.     

Structural insights into the peroxidase activity and inactivation of human peroxiredoxin 4

Prx4 (peroxiredoxin 4) is the only peroxiredoxin located in the ER (endoplasmic reticulum) and a proposed scavenger for H2O2. In the present study, we solved crystal structures of human Prx4 in three different redox forms and characterized the reaction features of Prx4 with H2O2. Prx4 exhibits a toroid-shaped decamer constructed of five catalytic dimers. Structural analysis revealed conformational changes around helix α2 and the C-terminal reigon with a YF (Tyr-Phe) motif from the partner subunit, which are required for interchain disulfide formation between Cys87 and Cys208, a critical step of the catalysis. The structural explanation for the restricting role of the YF motif on the active site dynamics is provided in detail. Prx4 has a high reactivity with H2O2, but is susceptible to overoxidation and consequent inactivation by H2O2. Either deletion of the YF motif or dissociation into dimers decreased the susceptibility of Prx4 to overoxidation by increasing the flexibility of Cys87.     

Study of the quaternary structure of rat 1-Cys peroxiredoxin

Peroxiredoxins (Prx) are a family of antioxidant proteins with peroxidase activity. The ability of 1-Cys Prx to self-associate was studied with the use of native PAGE and Western blotting. Two protein bands corresponding to monomeric and dimeric forms were detected in the preparation of the recombinant 1-Cys Prx subjected to native PAGE, with dimers being more abundant. The third band corresponding to the oligomeric form was detected after incubation of the recombinant 1-Cys Prx with DTT, although monomers and dimers were also observed. These results indicate that monomeric, dimeric, and oligomeric states of the protein are likely to be interchangeable. Native PAGE in combination with Western blot analysis revealed that self-association of 1-Cys Prx also occurred at physiologically relevant concentrations in vivo. The native 1-Cys Prx existed in the monomeric and dimeric forms in rat olfactory epithelium, with monomers being more common. The structural sensitivity of the recombinant 1-Cys Prx to imidazole was shown.     

Functional structure of the somatomedin B domain of vitronectin

The N-terminal somatomedin B domain (SMB) of vitronectin binds PAI-1 and the urokinase receptor with high affinity and regulates tumor cell adhesion and migration. We have shown previously in the crystal structure of the PAI-1/SMB complex that SMB, a peptide of 51 residues, is folded as a compact cysteine knot of four pairs of crossed disulfide bonds. However, the physiological significance of this structure was questioned by other groups, who disputed the disulfide bonding shown in the crystal structure (Cys5-Cys21, Cys9-Cys39, Cys19-Cys32, Cys25-Cys31), notably claiming that the first disulfide is Cys5-Cys9 rather than the Cys5-Cys21 bonding shown in the structure. To test if the claimed Cys5-Cys9 bond does exist in the SMB domain of plasma vitronectin, we purified mouse and rat plasma vitronectin that have a Met (hence cleavable by cyanogen bromide) at residue 14, and also prepared recombinant human SMB variants from insect cells with residues Asn14 or Leu24 mutated to Met. HPLC and mass spectrometry analysis showed that, after cyanogen bromide digestion, all the fragments of the SMB derived from mouse or rat vitronectin or the recombinant SMB mutants are still linked together by disulfides, and the N-terminal peptide (residue 1-14 or 1-24) can only be released when the disulfide bonds are broken. This clearly demonstrates that Cys5 and Cys9 of SMB do not form a disulfide bond in vivo, and together with other structural evidence confirms that the only functional structure of the SMB domain of plasma vitronectin is that seen in its crystallographic complex with PAI-1.     

Disassembly of the ring‐type decameric structure of peroxiredoxin from Aeropyrum pernix K1 by amino acid mutation

The quaternary structure of peroxiredoxin from Aeropyrum pernix K1 (ApPrx) is a decamer, in which five homodimers are assembled in a pentagonal ring through hydrophobic interactions. In this study, we determined the amino acid (AA) residues of ApPrx crucial for forming the decamer using AA mutations. The ApPrx0Cys mutant, wherein all cysteine residues were mutated to serine, was prepared as a model protein to remove the influence of the redox states of the cysteines on its assembling behavior. The boundary between each homodimer of ApPrx0Cys contains characteristic aromatic AA residues forming hydrophobic interactions: F46, F80, W88, W210, and W211. We found that a single mutation of F46, F80, or W210 to alanine completely disassembled the ApPrx0Cys decamer to homodimers, which was clarified by gel‐filtration chromatography and dynamic light scattering measurements. F46A, F80A, and W210A mutants lacked only one aromatic ring compared with ApPrx0Cys, indicating that the assembly is very sensitive to the surface structure of the protein. X‐ray structures revealed two mechanisms of disassembly of the ApPrx decamer: loss of hydrophobicity between homodimers and flip of the arm domain. The AA residues targeted in this study are well conserved in ring‐type Prx proteins, suggesting the importance of these residues in the assembly. This study demonstrates the sensitivity and modifiability of peroxiredoxin assembly by a simple AA mutation.     

Oxidative stress-dependent structural and functional switching of a human 2-Cys peroxiredoxin isotype II that enhances HeLa cell resistance to H2O2-induced cell death

Although biochemical properties of 2-Cys peroxiredoxins (Prxs) have been extensively studied, their real physiological functions in higher eukaryotic cells remain obscure and certainly warrant further study. Here we demonstrated that human (h) PrxII, a cytosolic isotype of human 2-Cys Prx, has dual functions as a peroxidase and a molecular chaperone, and that these different functions are closely associated with its adoption of distinct protein structures. Upon exposure to oxidative stress, hPrxII assumes a high molecular weight complex structure that has a highly efficient chaperone function. However, the subsequent removal of stressors induces the dissociation of this protein structure into low molecular weight proteins and triggers a chaperone-to-peroxidase functional switch. The formation of a high molecular weight hPrxII complex depends on the hyperoxidation of its N-terminal peroxidatic Cys residue as well as on its C-terminal domain, which contains a "YF motif" that is exclusively found in eukaryotic 2-Cys Prxs. A C-terminally truncated hPrxII exists as low and oligomeric protein species and does not respond to oxidative stress. Moreover, this C-terminal deletion of hPrxII converted it from an oxidation-sensitive to a hyperoxidation-resistant form of peroxidase. When functioning as a chaperone, hPrxII protects HeLa cells from H(2)O(2)-induced cell death, as measured by a terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay and fluorescence-activated cell sorting analysis.     

Crystal structure of a novel Plasmodium falciparum 1-Cys peroxiredoxin

Plasmodium falciparum, the causative agent of malaria, is sensitive to oxidative stress and therefore the family of antioxidant enzymes, peroxiredoxins (Prxs) represent a target for antimalarial drug design. We present here the 1.8 A resolution crystal structure of P.falciparum antioxidant protein, PfAOP, a Prx that in terms of sequence groups with mammalian PrxV. The structure is compared to all 11 known Prx structures to gain maximal insight into its properties. We describe the common Prx fold and show that the dimeric PfAOP can be mechanistically categorized as a 1-Cys Prx. In the active site the peroxidatic Cys is over-oxidized to cysteine sulfonic acid, making this the first Prx structure seen in that state. Now with structures of Prxs in Cys-sulfenic, -sulfinic and -sulfonic acid oxidation states known, the structural steps involved in peroxide binding and over-oxidation are suggested. We also describe that PfAOP has an alpha-aneurism (a one residue insertion), a feature that appears characteristic of the PrxV-like group. In terms of crystallographic methodology, we enhance the information content of the model by identifying bound water sites based on peak electron densities, and we use that information to infer that the oxidized active site has suboptimal interactions that may influence catalysis. The dimerization interface of PfAOP is representative of an interface that is widespread among Prxs, and has sequence-dependent variation in geometry. The interface differences and the structural features (like the alpha-aneurism) may be used as markers to better classify Prxs and study their evolution.     

Crystal structure of alkyl hydroperoxide-reductase (AhpC) from Helicobacter pylori

The AhpC protein from H. pylori, a thioredoxin (Trx)-dependent alkyl hydroperoxide-reductase, is a member of the ubiquitous 2-Cys peroxiredoxins family (2-Cys Prxs), a group of thiol-specific antioxidant enzymes. Prxs exert the protective antioxidant role in cells through their peroxidase activity, whereby hydrogen peroxide, peroxynitrite and a wide range of organic hydroperoxides (ROOH) are reduced and detoxified (ROOH + 2e(-)-->ROH + H2O). In this study AhpC has been cloned and overexpressed in E. coli. After purification to homogeneity, crystals of the recombinant protein were grown. They diffract to 2.95 A resolution using synchrotron radiation. The crystal structure of AhpC has been determined using the molecular replacement method (R = 23.6%, R(free) = 25.9%). The model, similar in the overall to other members of the 2-Cys Prx family crystallized as toroide-shaped complexes, consists of a pentameric arrangement of homodimers [(alpha2)5 decamer]. The model of AhpC from H. pylori presents significant differences with respect to other members of the family: apart from some loop regions, alpha5-helix and the C-terminus is shifted, preventing the C-terminal tail of the second subunit from extending toward this region of the molecule. Oligomerization properties of AhpC have been also characterized by gel filtration chromatography.