2-Cys Prxs Research Articles

Unraveling the Peroxidase Activity in Peroxiredoxins: A Comprehensive Review of Mechanisms, Functions, and Biological Significance.

Peroxiredoxins (Prxs) are members of the antioxidant enzymes necessary for every living object in the three domains of life and play critical roles in controlling peroxide levels in cells. This comprehensive literature review aims to elucidate the peroxidase activity of Prxs, examining their roles and significance for organisms across various taxa. Ironically, the primary role of the Prxs is the peroxidase activity, which comprises the reduction of hydrogen peroxide and other organic hydroperoxides and decreases the risk of oxidative damage in the cells. The above enzymatic activity occurs through the reversible oxidation-reduction catalyzed by cysteine residues in the active site by forming sulfenic acid and reduction by intracellular reductants. Structurally and functionally, Prxs function as dimers or decamers and show different catalytic patterns according to their subfamilies or cellular compartments. Compared to the mechanisms of the other two subgroups of Prxs, including 2-Cys Prxs and atypical Prxs, the 1-Cys Prxs have monomer-dimer switch folding coupled with catalytic activity. In addition to their peroxidase activity, which is widely known, Prxs are becoming acknowledged to be involved in other signaling processes, including redox signaling and apoptosis. This aversion to oxidative stress and regulation by the cellular redox state places them at the heart of adaptive cellular responses to changes in the environment or manifestations of diseases. In conclusion, based on the data obtained and on furthering the knowledge of Prxs' structure and function, these enzymes may be classified as a diverse yet essential family of proteins that can effectively protect cells from the adverse effects of oxidative stress due to peroxidase activity. This indicates secondary interactions, summarized as peroxide detoxification or regulatory signaling, and identifies their applicability in multiple biological pathways. Such knowledge is valuable for enhancing the general comprehension of essential cellular functions and disclosing further therapeutic approaches to the diseases caused by the increased production of reactive oxygen species.

2-Cys peroxiredoxins contribute to thylakoid lipid unsaturation by affecting ω-3 fatty acid desaturase 8.

Fatty acid unsaturation levels affect chloroplast function and plant acclimation to environmental cues. However, the regulatory mechanism(s) controlling fatty acid unsaturation in thylakoid lipids is poorly understood. Here, we have investigated the connection between chloroplast redox homeostasis and lipid metabolism by focusing on 2-Cys peroxiredoxins (Prxs), which play a central role in balancing the redox state within the organelle. The chloroplast redox network relies on NADPH-dependent thioredoxin reductase C (NTRC), which controls the redox balance of 2-Cys Prxs to maintain the reductive activity of redox-regulated enzymes. Our results show that Arabidopsis (Arabidopsis thaliana) mutants deficient in 2-Cys Prxs contain decreased levels of trienoic fatty acids, mainly in chloroplast lipids, indicating that these enzymes contribute to thylakoid membrane lipids unsaturation. This function of 2-Cys Prxs is independent of NTRC, the main reductant of these enzymes, hence 2-Cys Prxs operates beyond the classic chloroplast regulatory redox system. Moreover, the effect of 2-Cys Prxs on lipid metabolism is primarily exerted through the prokaryotic pathway of glycerolipid biosynthesis and fatty acid desaturase 8 (FAD8). While 2-Cys Prxs and FAD8 interact in leaf membranes as components of a large protein complex, the levels of FAD8 were markedly decreased when FAD8 is overexpressed in 2-Cys Prxs-deficient mutant backgrounds. These findings reveal a function for 2-Cys Prxs, possibly acting as a scaffold protein, affecting the unsaturation degree of chloroplast membranes.

The Functional Relationship between NADPH Thioredoxin Reductase C, 2-Cys Peroxiredoxins, and m-Type Thioredoxins in the Regulation of Calvin-Benson Cycle and Malate-Valve Enzymes in Arabidopsis.

The concerted regulation of chloroplast biosynthetic pathways and NADPH extrusion via malate valve depends on f and m thioredoxins (Trxs). The finding that decreased levels of the thiol-peroxidase 2-Cys peroxiredoxin (Prx) suppress the severe phenotype of Arabidopsis mutants lacking NADPH-dependent Trx reductase C (NTRC) and Trxs f uncovered the central function of the NTRC-2-Cys-Prx redox system in chloroplast performance. These results suggest that Trxs m are also regulated by this system; however, the functional relationship between NTRC, 2-Cys Prxs, and m-type Trxs is unknown. To address this issue, we generated Arabidopsis thaliana mutants combining deficiencies in NTRC, 2-Cys Prx B, Trxs m1, and m4. The single trxm1 and trxm4 mutants showed a wild-type phenotype, growth retardation being noticed only in the trxm1m4 double mutant. Moreover, the ntrc-trxm1m4 mutant displayed a more severe phenotype than the ntrc mutant, as shown by the impaired photosynthetic performance, altered chloroplast structure, and defective light-dependent reduction in the Calvin-Benson cycle and malate-valve enzymes. These effects were suppressed by the decreased contents of 2-Cys Prx, since the quadruple ntrc-trxm1m4-2cpb mutant displayed a wild-type-like phenotype. These results show that the activity of m-type Trxs in the light-dependent regulation of biosynthetic enzymes and malate valve is controlled by the NTRC-2-Cys-Prx system.

Plastid 2-Cys peroxiredoxins are essential for embryogenesis in Arabidopsis

The redox couple formed by NADPH-dependent thioredoxin reductase C (NTRC) and 2-Cys peroxiredoxins (Prxs) allows fine-tuning chloroplast performance in response to light intensity changes. Accordingly, the Arabidopsis 2cpab mutant lacking 2-Cys Prxs shows growth inhibition and sensitivity to light stress. However, this mutant also shows defective post-germinative growth, suggesting a relevant role of plastid redox systems in seed development, which is so far unknown. To address this issue, we first analyzed the pattern of expression of NTRC and 2-Cys Prxs in developing seeds. Transgenic lines expressing GFP fusions of these proteins showed their expression in developing embryos, which was low at the globular stage and increased at heart and torpedo stages, coincident with embryo chloroplast differentiation, and confirmed the plastid localization of these enzymes. The 2cpab mutant produced white and abortive seeds, which contained lower and altered composition of fatty acids, thus showing the relevance of 2-Cys Prxs in embryogenesis. Most embryos of white and abortive seeds of the 2cpab mutant were arrested at heart and torpedo stages of embryogenesis suggesting an essential function of 2-Cys Prxs in embryo chloroplast differentiation. This phenotype was not recovered by a mutant version of 2-Cys Prx A replacing the peroxidatic Cys by Ser. Neither the lack nor the overexpression of NTRC had any effect on seed development indicating that the function of 2-Cys Prxs at these early stages of development is independent of NTRC, in clear contrast with the operation of these regulatory redox systems in leaves chloroplasts.

A chloroplast redox relay adapts plastid metabolism to light and affects cytosolic protein quality control.

In chloroplasts, thiol-dependent redox regulation is linked to light since the disulfide reductase activity of thioredoxins (Trxs) relies on photo-reduced ferredoxin (Fdx). Furthermore, chloroplasts harbor an NADPH-dependent Trx reductase (NTR) with a joint Trx domain, termed NTRC. The activity of these two redox systems is integrated by the redox balance of 2-Cys peroxiredoxin (Prx), which is controlled by NTRC. However, NTRC was proposed to participate in redox regulation of additional targets, prompting inquiry into whether the function of NTRC depends on its capacity to maintain the redox balance of 2-Cys Prxs or by direct redox interaction with chloroplast enzymes. To answer this, we studied the functional relationship of NTRC and 2-Cys Prxs by a comparative analysis of the triple Arabidopsis (Arabidopsis thaliana) mutant, ntrc-2cpab, which lacks NTRC and 2-Cys Prxs, and the double mutant 2cpab, which lacks 2-Cys Prxs. These mutants exhibit almost indistinguishable phenotypes: in growth rate, photosynthesis performance, and redox regulation of chloroplast enzymes in response to light and darkness. These results suggest that the most relevant function of NTRC is in controlling the redox balance of 2-Cys Prxs. A comparative transcriptomics analysis confirmed the phenotypic similarity of the two mutants and suggested that the NTRC-2-Cys Prxs system participates in cytosolic protein quality control. We propose that NTRC and 2-Cys Prxs constitute a redox relay, exclusive to photosynthetic organisms that fine-tunes the redox state of chloroplast enzymes in response to light and affects transduction pathways towards the cytosol.

Hyperoxidation of Peroxiredoxins and Effects on Physiology of Drosophila.

The catalytic activity of peroxiredoxins (Prx) is determined by the conserved peroxidatic cysteine (CysP), which reacts with peroxides to form sulfenic acid (Cys-SOH). Under conditions of oxidative stress, CysP is oxidized to catalytically inactive sulfinic (Cys-SO2) and sulfonic (Cys-SO3) forms. The Cys-SO2 form can be reduced in a reaction catalyzed by sulfiredoxin (Srx). To explore the physiological significance of peroxiredoxin overoxidation, we investigated daily variations in the oxidation state of 2-Cys peroxiredoxins in flies of different ages, or under conditions when the pro-oxidative load is high. We found no statistically significant changes in the 2-Cys Prxs monomer:dimer ratio, which indirectly reflects changes in the Prx catalytic activity. However, we found daily variations in Prx-SO2/3 that were more pronounced in older flies as well as in flies lacking Srx. Unexpectedly, the srx mutant flies did not exhibit a diminished survivorship under normal or oxidative stress conditions. Moreover, the srx mutant was characterized by a higher physiological activity. In conclusion, catalytically inactive forms of Prx-SO2/3 serve not only as a marker of cellular oxidative burden, but may also play a role in an adaptive response, leading to a positive effect on the physiology of Drosophila melanogaster.

Direct Observation of the Catalytically Relevant Fully Folded-Locally Unfolded Equilibrium in Peroxiredoxin Q of Xanthamonas Campestris

Peroxiredoxins (Prxs) are ubiquitious peroxidases found in all living organisms. All peroxiredoxins share a conserved catalytic site containing a ‘peroxidatic’ cysteine which reduces peroxide to water while being converted to a Cys-sulfenate. A subset of Prxs called 2-Cys Prxs then either locally unfold and form an internal disulfide between the peroxidatic and a second ‘resolving’ cysteine, or become hyperoxidized, forming a Cys-sulfinate which inactivates the protein. The balance between resolution and hyperoxidation is thought to be key to the hypothesized role of some 2-Cys Prxs as a ‘floodgate’ regulating peroxide concentration.

Redox regulation of chloroplast metabolism.

Regulation of enzyme activity based on thiol-disulfide exchange is a regulatory mechanism in which the protein disulfide reductase activity of thioredoxins (TRXs) plays a central role. Plant chloroplasts are equipped with a complex set of up to 20 TRXs and TRX-like proteins, the activity of which is supported by reducing power provided by photosynthetically reduced ferredoxin (FDX) with the participation of a FDX-dependent TRX reductase (FTR). Therefore, the FDX–FTR–TRXs pathway allows the regulation of redox-sensitive chloroplast enzymes in response to light. In addition, chloroplasts contain an NADPH-dependent redox system, termed NTRC, which allows the use of NADPH in the redox network of these organelles. Genetic approaches using mutants of Arabidopsis (Arabidopsis thaliana) in combination with biochemical and physiological studies have shown that both redox systems, NTRC and FDX-FTR-TRXs, participate in fine-tuning chloroplast performance in response to changes in light intensity. Moreover, these studies revealed the participation of 2-Cys peroxiredoxin (2-Cys PRX), a thiol-dependent peroxidase, in the control of the reducing activity of chloroplast TRXs as well as in the rapid oxidation of stromal enzymes upon darkness. In this review, we provide an update on recent findings regarding the redox regulatory network of plant chloroplasts, focusing on the functional relationship of 2-Cys PRXs with NTRC and the FDX–FTR–TRXs redox systems for fine-tuning chloroplast performance in response to changes in light intensity and darkness. Finally, we consider redox regulation as an additional layer of control of the signaling function of the chloroplast.

Exploring the Functional Relationship between y-Type Thioredoxins and 2-Cys Peroxiredoxins in Arabidopsis Chloroplasts.

Thioredoxins (Trxs) are small, ubiquitous enzymes that catalyze disulphide–dithiol interchange in target enzymes. The large set of chloroplast Trxs, including f, m, x and y subtypes, use reducing equivalents fueled by photoreduced ferredoxin (Fdx) for fine-tuning photosynthetic performance and metabolism through the control of the activity of redox-sensitive proteins. Although biochemical analyses suggested functional diversity of chloroplast Trxs, genetic studies have established that deficiency in a particular Trx subtype has subtle phenotypic effects, leading to the proposal that the Trx isoforms are functionally redundant. In addition, chloroplasts contain an NADPH-dependent Trx reductase with a joint Trx domain, termed NTRC. Interestingly, Arabidopsis mutants combining the deficiencies of x- or f-type Trxs and NTRC display very severe growth inhibition phenotypes, which are partially rescued by decreased levels of 2-Cys peroxiredoxins (Prxs). These findings indicate that the reducing capacity of Trxs f and x is modulated by the redox balance of 2-Cys Prxs, which is controlled by NTRC. In this study, we explored whether NTRC acts as a master regulator of the pool of chloroplast Trxs by analyzing its functional relationship with Trxs y. While Trx y interacts with 2-Cys Prxs in vitro and in planta, the analysis of Arabidopsis mutants devoid of NTRC and Trxs y suggests that Trxs y have only a minor effect, if any, on the redox state of 2-Cys Prxs.

Analyses of the expression, immunohistochemical properties and serodiagnostic potential of Schistosoma japonicum peroxiredoxin-4

BackgroundSchistosoma japonicum, which inhabits the mesenteric vein of the mammalian hosts for about 20 to 30 years, is subjected to the oxidative stresses from the host defense mechanism during their intra-mammalian stages. To counteract this host immune attack, the parasite utilizes their antioxidant system for survival inside the host. Peroxiredoxins (Prxs), thiol-specific antioxidant proteins, play an essential role for protecting the parasite against oxidative stress by reducing hydrogen peroxide to water. Only three types of 2-Cys Prxs have been previously characterized in S. japonicum whereas a fourth Prx has been identified for Schistosoma mansoni as Prx-4. A sequence coding homologous to this gene in the S. japonicum database was identified, characterized and expressed as recombinant SjPrx-4 protein (rSjPrx-4). Furthermore, rSjPrx-4 was evaluated in this study for its diagnostic potentials in detecting S. japonicum infection in humans.ResultsThe gene found in the parasite genome contained 2 active-site cysteines with conserved sequences in the predicted amino acid (AA) sequence and showed 75% identity with that of the previously characterized Prx (TPx-1) of S. japonicum. The gene was expressed in different stages of schistosome life-cycle with highest transcription level in the adult male. The gene was cloned into a plasmid vector and then transfected into Escherichia coli for expression of rSjPrx-4. Anti-rSjPrx-4 mouse sera recognized native SjPrx-4 in egg and adult worm lysate by western blotting. The result of a mixed function oxidation assay in which rSjPrx-4 prevented the nicking of DNA from hydroxyl radicals confirmed its antioxidant activity. Subsequently, immunolocalization analysis showed the localization of SjPrx-4 inside the egg, on the tegument and in the parenchyma of the adult worm. Enzyme-linked immunosorbent assay results showed that rSjPrx-4 has 83.3% sensitivity and 87.8% specificity. Its diagnostic potential was further evaluated in combination with recombinant SjTPx-1 protein, yielding an improved sensitivity and specificity of 90% and 92.7%, respectively.ConclusionsThese results suggest that SjPrx-4 plays a role as an antioxidant dealing with oxidative stresses of S. japonicum, and its diagnostic potential improved by coupling it with SjTPx-1 is a proof for developing a serological test with better diagnostic performance for human schistosomiasis.

Structural and biochemical analysis of 1-Cys peroxiredoxin ScPrx1 from Saccharomyces cerevisiae mitochondria

BackgroundScPrx1 is a yeast mitochondrial 1-Cys peroxiredoxins (Prx), a type of Prx enzyme which require thiol-containing reducing agents to resolve its peroxidatic cysteine. ScPrx1 plays important role in protection against oxidative stress. Mitochondrial thioredoxin ScTrx3 and glutathione have been reported to be the physiological electron donor for ScPrx1. However, the mechanism underlying their actions, especially the substrate recognition of ScPrx1 requires additional elucidation. METHODS: The structure of ScPrx1 was obtained through crystallization experiments. The oligomeric state of ScPrx1 was monitored by Blue-Native PAGE. Mutations were generated by the QuikChange PCR-based method. The ScPrx1 activity assay was carried out by measuring the change of 340 nm absorption of the NADPH oxidation. ResultsScPrx1 exist as a homodimer in solution. The structure adopts a typical Prx-fold core which is preceded by an N-terminal β-hairpin and has a C-terminal extension. Mutations (Glu94Ala, Arg198Ala and Trp126) close to the active site could enhance the catalytic efficiency of ScPrx1 while His83Ala and mutations on α4-β6 region exhibited reduced activity. The biochemical data also show that the deletion or mutations on ScPrx1 C-terminal have 2–4.56 fold increased activity. ConclusionWe inferred that conformational changes of ScPrx1 C-terminal segment were important for its reaction, and the α4-β6 loop regions around the ScPrx1 active sites were important for the catalytic function of ScPrx1. Collectively, these structural features provides a basis for understanding the diverse reductant species usage in different 1-Cys Prxs.

Role of Peroxiredoxins in Protecting Against Cardiovascular and Related Disorders.

Peroxiredoxin (Prx) refers to a family of thiol-dependent peroxidases that decompose hydrogen peroxide, lipid hydroperoxides, as well as peroxynitrite, and protect against oxidative and inflammatory stress. There are six mammalian Prx isozymes (Prx1-6), classified as typical 2-Cys, atypical 2-Cys, or 1-Cys Prxs based on the mechanism and the number of cysteine residues involved during catalysis. In addition to their well-established peroxide-scavenging activity, some Prxs also participate in the regulation of various cell signaling pathways. Extensive animal studies employing primarily gene knockout models provide substantial evidence supporting a critical protective role of Prxs in various disease processes involving oxidative and inflammatory stress. This review surveys recent research findings, published primarily in influential journals, on the involvement of various Prx isozymes in protecting against cardiovascular injury and related disorders, including diabetes, metabolic syndromes, and sepsis, whose pathophysiology all intimately involves oxidative stress and inflammation.

Structural insight into the biological functions of Arabidopsis thaliana ACHT1

The Arabidopsis thaliana atypical Cys His-rich thioredoxins (ACHTs) are a small class of atypical thioredoxins (TRXs) located in chloroplasts thylakoids and are characterized by a noncanonical motif at their redox active site, C (G/S)(S/G)C. Previous studies have reported that ACHT1 can interact with A. thaliana 2-Cys peroxiredoxins (2-Cys Prxs, including PrxA and PrxB) to transmit oxidation signals in response to illumination with normal light intensity. In this study, we reported the crystal structure of ACHT1 and show that ACHT1 adopts a canonical TRX fold. Comparison of the structures of ACHT1 in both reducing and oxidizing environments revealed that while the redox environment did not influence the overall structure of ACHT1, it did change the conformation of its catalytic residues. We found that the catalytic C125 of ACHT1 is the target residue for PrxA in vitro. In addition, we found that ACHT1 can reduce the peroxidase activity of PrxA, and further confirmed that the ability of ACHT1 to restore the peroxidase function of PrxA was due to the interaction between the two. Our results provide a structural basis for studying the function of atypical TRXs and the oxidative regulation mechanism of ACHT1 and 2-Cys Prxs in chloroplasts.

A peroxiredoxin of Thermus thermophilus HB27: Biochemical characterization of a new player in the antioxidant defence

To fight oxidative damage due to reactive oxygen species (ROS), cells are equipped of different enzymes, among which Peroxiredoxins (Prxs) (EC 1.11.1.15) play a key role. Prxs are thiol-based enzymes containing one (1-Cys Prx) or two (2-Cys Prx) catalytic cysteine residues. In 2-Cys Prxs the cysteine residues form a disulfide bridge following reduction of peroxide which is in turn reduced by Thioredoxin reductase (Tr) /Thioredoxin (Trx) disulfide reducing system to regenerate the enzyme. In this paper we investigated on Prxs of Thermus thermophilus whose genome contains an ORF TT_C0933 encoding a putative Prx, belonging to the subfamily of Bacterioferritin comigratory protein (Bcp): the synthetic gene was produced and expressed in E. coli and the recombinant protein, TtBcp, was biochemically characterized. TtBcp was active on both organic and inorganic peroxides and showed stability at high temperatures. To get insight into disulfide reducing system involved in the recycling of the enzyme we showed that TtBcp catalically eliminates hydrogen peroxide using an unusual partner, the Protein Disulfide Oxidoreductase (TtPDO) that could replace regeneration of the enzyme. Altogether these results highlight not only a new anti-oxidative pathway but also a promising molecule for possible future biotechnological applications.

The crystal structure of sulfiredoxin from Arabidopsis thaliana revealed a more robust antioxidant mechanism in plants

Typical 2-cysteine peroxiredoxins (2-Cys Prxs) are critical peroxidase sensors and could be deactivated by the hyperoxidation under oxidative stress. In plants, 2-Cys Prxs present at a high level in chloroplasts and are repaired by Sulfiredoxin. Whereas many studies have explored the mechanism of Sulfiredoxin from Homo sapiens (HsSrx), the molecular mechanism of Sulfiredoxin in plants with unique photosynthesis remains unclear. Here we report the crystal structure of Sulfiredoxin from Arabidopsis thaliana (AtSrx), which displayed a typical ParB/Srx fold with an ATP bound at a conservative nucleotide binding motif GCHR. Both the ADP binding pocket and the putative AtSrx-AtPrxA interaction surface of AtSrx are more positively charged comparing to HsSrx, suggesting a robust mechanism of AtSrx. These features illustrate the unique mechanisms of AtSrx, which are vital for figure out the strategies of plants to cope with oxidation stress.

The bicarbonate/carbon dioxide pair increases hydrogen peroxide-mediated hyperoxidation of human peroxiredoxin 1

2-Cys peroxiredoxins (Prxs) rapidly reduce H2O2, thereby acting as antioxidants and also as sensors and transmitters of H2O2 signals in cells. Interestingly, eukaryotic 2-Cys Prxs lose their peroxidase activity at high H2O2 levels. Under these conditions, H2O2 oxidizes the sulfenic acid derivative of the Prx peroxidatic Cys (CPSOH) to the sulfinate (CPSO2 -) and sulfonated (CPSO3 -) forms, redirecting the CPSOH intermediate from the catalytic cycle to the hyperoxidation/inactivation pathway. The susceptibility of 2-Cys Prxs to hyperoxidation varies greatly and depends on structural features that affect the lifetime of the CPSOH intermediate. Among the human Prxs, Prx1 has an intermediate susceptibility to H2O2 and was selected here to investigate the effect of a physiological concentration of HCO3 -/CO2 (25 mm) on its hyperoxidation. Immunoblotting and kinetic and MS/MS experiments revealed that HCO3 -/CO2 increases Prx1 hyperoxidation and inactivation both in the presence of excess H2O2 and during enzymatic (NADPH/thioredoxin reductase/thioredoxin) and chemical (DTT) turnover. We hypothesized that the stimulating effect of HCO3 -/CO2 was due to HCO4 -, a peroxide present in equilibrated solutions of H2O2 and HCO3 -/CO2 Indeed, additional experiments and calculations uncovered that HCO4 - oxidizes CPSOH to CPSO2 - with a second-order rate constant 2 orders of magnitude higher than that of H2O2 ((1.5 ± 0.1) × 105 and (2.9 ± 0.2) × 103 m-1·s-1, respectively) and that HCO4 - is 250 times more efficient than H2O2 at inactivating 1% Prx1 per turnover. The fact that the biologically ubiquitous HCO3 -/CO2 pair stimulates Prx1 hyperoxidation and inactivation bears relevance to Prx1 functions beyond its antioxidant activity.

Chloroplast Redox Regulatory Mechanisms in Plant Adaptation to Light and Darkness.

Light is probably the most important environmental stimulus for plant development. As sessile organisms, plants have developed regulatory mechanisms that allow the rapid adaptation of their metabolism to changes in light availability. Redox regulation based on disulfide-dithiol exchange constitutes a rapid and reversible post-translational modification, which affects protein conformation and activity. This regulatory mechanism was initially discovered in chloroplasts when it was identified that enzymes of the Calvin-Benson cycle (CBC) are reduced and active during the day and become rapidly inactivated by oxidation in the dark. At present, the large number of redox-sensitive proteins identified in chloroplasts extend redox regulation far beyond the CBC. The classic pathway of redox regulation in chloroplasts establishes that ferredoxin (Fdx) reduced by the photosynthetic electron transport chain fuels reducing equivalents to the large set of thioredoxins (Trxs) of this organelle via the activity of a Fdx-dependent Trx reductase (FTR), hence linking redox regulation to light. In addition, chloroplasts harbor an NADPH-dependent Trx reductase with a joint Trx domain, termed NTRC. The presence in chloroplasts of this NADPH-dependent redox system raises the question of the functional relationship between NTRC and the Fdx-FTR-Trx pathways. Here, we update the current knowledge of these two redox systems focusing on recent evidence showing their functional interrelationship through the action of the thiol-dependent peroxidase, 2-Cys peroxiredoxin (2-Cys Prx). The relevant role of 2-Cys Prxs in chloroplast redox homeostasis suggests that hydrogen peroxide may exert a key function to control the redox state of stromal enzymes. Indeed, recent reports have shown the participation of 2-Cys Prxs in enzyme oxidation in the dark, thus providing an explanation for the long-lasting question of photosynthesis deactivation during the light-dark transition.

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 CP49 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.

The NADPH-Dependent Thioredoxin Reductase C-2-Cys Peroxiredoxin Redox System Modulates the Activity of Thioredoxin x in Arabidopsis Chloroplasts.

The chloroplast redox network is composed of a complex set of thioredoxins (Trxs), reduced by ferredoxin (Fdx) via a Fdx-dependent Trx reductase (FTR), and an NADPH-dependent Trx reductase with a joint Trx domain, NTRC, which efficiently reduces 2-Cys peroxiredoxins (2-Cys Prxs). Recently, it was proposed that the redox balance of 2-Cys Prxs maintains the redox state of f-type Trxs, thus allowing the proper redox regulation of Calvin-Benson cycle enzymes such as fructose 1,6-bisphosphatase (FBPase). Here, we have addressed whether the action of 2-Cys Prxs is also exerted on Trx x. To that end, an Arabidopsis thaliana quadruple mutant, ntrc-trxx-Δ2cp, which is knocked out for NTRC and Trx x, and contains severely decreased levels of 2-Cys Prxs, was generated. In contrast to ntrc-trxx, which showed a severe growth inhibition phenotype and poor photosynthetic performance, the ntrc-trxx-Δ2cp mutant showed a significant recovery of growth rate and photosynthetic efficiency, indicating that the content of 2-Cys Prxs is critical for the performance of plants lacking both NTRC and Trx x. Light-dependent reduction of FBPase was severely impaired in mutant plants lacking NTRC or NTRC plus Trx x, despite the fact that neither NTRC nor Trx x is an effective reductant of this enzyme. However, FBPase reduction was recovered in the ntrc-trxx-Δ2cp mutant. Our results show that the redox balance of 2-Cys Prxs, which is mostly dependent on NTRC, modulates the activity of Trx x in a similar way as f-type Trxs, thus suggesting that the activity of these Trxs is highly interconnected.

P-366 - Identification and characterization of reduction agents of 1-Cys peroxiredoxins from Aspergillus fumigatus, a human opportunistic pathogen

Peroxiredoxins (Prxs) are Cys-based highly efficient peroxidases. Prxs can be classified as 2-Cys and 1-Cys, according to the number of cysteines residues involved in the catalysis. 1-Cys Prx enzymes are still poorly characterized and the identities of their biological reductants are still controversial. Some of the reductant candidates are thioredoxin (Trx) and ascorbate. A. fumigatus is a human pathogen fungus that possesses two 1-Cys Prxs (Prx1 and PrxC). We had shown before that these Prxs display high reactivity towards H2O2 (~107 M-1s-1). Here, we described our studies on the reduction step. Thus, the genes encoding for Trx system from this fungus were cloned, expressed and purified. The 4 Trxs were efficiently reduced by TrxR (kcat/Km ~106 M-1s-1). However, our preliminary data indicated that Prx1 and PrxC were poorly reduced by these oxidoreductases. We are now investigating the erythroascorbate (a 5-carbon ascorbate analogue) role in Prx reduction. Since erythroascorbate can be found glycosylated in several fungi species, we are trying to identify and quantitate this reducing agent by mass spectrometry. The biochemical characterization of these Prxs catalytic cycle can open new therapeutical strategies, since at least Prx1 is involved in fungus virulence.