Reduction Of Dehydroascorbate Research Articles

Evolutionary insights into strategy shifts for the safe and effective accumulation of ascorbate in plants.

Plants accumulate high concentrations of ascorbate, commonly in their leaves, as a redox buffer. While ascorbate levels have increased during plant evolution, the mechanisms behind this phenomenon are unclear. Moreover, has the increase in ascorbate concentration been achieved without imposing any detrimental effects on the plants? In this review, we focus on potential transitions in two regulatory mechanisms related to ascorbate biosynthesis and the availability of cellular dehydroascorbate (DHA) during plant evolution. The first transition might be that the trigger for the transcriptional induction of VTC2, which encodes the rate-limiting enzyme in ascorbate biosynthesis, has shifted from oxidative stress (in green algae) to light/photosynthesis (in land plants), probably enabling the continuous accumulation of ascorbate under illumination. This could serve as a preventive system against the unpredictable occurrence of oxidative stress. The second transition might be that DHA-degrading enzymes, which protect cells from the highly reactive DHA in green algae and mosses, have been lost in ferns or flowering plants. Instead, flowering plants may have increased glutathione concentrations to reinforce the DHA reduction capacity, possibly allowing ascorbate accumulation and avoiding the toxicity of DHA. These potential transitions may have contributed to strategies for plants' safe and effective accumulation of ascorbate.

Drought Stress and High Temperature Affect the Antioxidant Metabolism of Cotton (Gossypium hirsutum L.) Anthers and Reduce Pollen Fertility

Both drought and high temperature can influence the antioxidant metabolism of crop reproductive organs in different ways, affecting the fertility of reproductive organs and yield formation. However, the combined effects of drought stress and high temperature on the crop reproductive physiology have not yet been widely considered. In order to broaden our understanding of this mechanism of influence, a pond experiment was conducted using a cotton variety Yuzaomian 9110 divided into four treatment groups: control (CK), drought stress (DS), high temperature (HT), and drought stress coupled with high temperature (DS+HT). Results showed a significant negative correlation between pollen viability and superoxide anion (O2−) content, as well as hydrogen peroxide (H2O2). Compared with CK, DS did not alter O2− content in anthers, but HT treatment resulted in higher anther O2−. Compared with single-stress groups, DS+HT further promoted the formation of O2− in anthers, leading to more malondialdehyde in anthers. Moreover, a higher H2O2 content in anthers was found in DS and HT than in CK. DS+HT did not show altered H2O2 content relative to HT treatment, although its H2O2 was higher than in DS. Further analyses of the antioxidant enzyme system showed that DS had no significant effect on superoxide dismutase gene (GhCu/ZnSOD) expression, but HT and DS+HT significantly downregulated its expression. The expression of GhCu/ZnSOD was lower under DS+HT than HT, which might be why O2− content was not altered under DS treatment compared with CK and was higher in DS+HT than HT. DS and HT significantly downregulated the expression of the peroxidase gene (GhPOD) and catalase gene (GhCAT), which should be the main reason for the larger accumulation of H2O2 under drought stress and high-temperature conditions. Compared with single-stress groups, DS+HT had lower expression of GhCAT, resulting in a larger H2O2 content. Regarding the ascorbic acid–glutathione (AsA–GSH) cycle, DS and HT significantly downregulated the expression of monodehydroascorbate reductase gene (GhMDHAR) to hinder the production of AsA and upregulated the expression of ascorbate oxidase gene (GhAAO) to promote the oxidation of AsA, which was theoretically detrimental to AsA accumulation. However, HT downregulated the expression of the ascorbate peroxidase gene (GhAPX), hindering the reduction of H2O2 by AsA, which was the reason for AsA and H2O2 accumulation. Moreover, DS also significantly upregulated the expression of the dehydroascorbate reductase gene (GhDHAR2) to enhance the reduction of dehydroascorbate to form AsA, leading to a higher content of AsA under DS than HT. The combined stress significantly downregulated the expression of GhAAO to inhibit the oxidation of AsA but significantly upregulated the expression of GhMDHAR and GhDHAR2, promoting the AsA production, and downregulated the expression of GhAPX, hindering the reduction of H2O2 by AsA. All these resulted in increased AsA content under combined stresses. In addition, HT significantly downregulated the glutathione reductase gene (GhGR) expression, hindering the reduction of oxidized glutathione (GSSG), which led to the reduction of GSH. However, DS and DS+HT significantly downregulated the glutathione peroxidase gene (GhGPX) expression, resulting in the accumulation of GSH. Overall, compared with single-stress treatments, the effects of DS+HT on cotton pollen fertility and peroxide accumulation were more significant. The effects of DS+HT on the antioxidant enzyme system were mainly caused by high temperature, while the mechanism of abnormal accumulation of AsA and GSH caused by DS+HT was different from those of single-stress groups.

Redox Properties of Human Erythrocytes Are Adapted for Vitamin C Recycling.

Ascorbic acid (AA; or vitamin C) is an important physiological antioxidant and radical scavenger. Some mammalian species, including homo sapiens, have lost the ability to synthetize AA and depend on its nutritional uptake. Erythrocytes from AA-auxotroph mammals express high amounts of the glucose transporter GLUT1. This isoform enables rapid uptake of glucose as well as dehydroascorbate (DHA), the fully oxidized form of AA. Here, we explored the effects of DHA uptake on the redox metabolism of human erythrocytes. DHA uptake enhanced plasma membrane electron transport (PMET) activity. This process is mediated by DCytb, a membrane bound cytochrome catalyzing extracellular reduction of Fe3+ and ascorbate free radical (AFR), the first oxidized form of AA. DHA uptake also decreased cellular radical oxygen species (ROS) levels. Both effects were massively enhanced in the presence of physiological glucose concentrations. Reduction of DHA to AA largely depleted intracellular glutathione (GSH) and induced the efflux of its oxidized form, GSSG. GSSG efflux could be inhibited by MK-571 (IC50 = 5 μM), indicating involvement of multidrug resistance associated protein (MRP1/4). DHA-dependent GSH depletion and GSSG efflux were completely rescued in the presence of 5 mM glucose and, partially, by 2-deoxy-glucose (2-DG), respectively. These findings indicate that human erythrocytes are physiologically adapted to recycle AA both intracellularly via GLUT1-mediated DHA uptake and reduction and extracellularly via DCytb-mediated AFR reduction. We discuss the possibility that this improved erythrocyte-mediated AA recycling was a prerequisite for the emergence of AA auxotrophy which independently occurred at least twice during mammalian evolution.

Molecular characterization, redox regulation, and immune responses of monothiol and dithiol glutaredoxins from disk abalone (Haliotis discus discus)

Glutaredoxins (Grxs) are well-known oxidoreductases involved in a wide range of redox activities in organisms. In this study, two invertebrate Grxs (AbGrx1-like and AbGrx2) from disk abalone were identified and characterized in an effort to gain a deeper understanding into their immune and redox regulatory roles. Both AbGrxs share typical thioredoxin/Grx structures. AbGrx1-like and AbGrx2 were identified as monothiol and diothiol Grxs, respectively. AbGrxs were significantly expressed at the egg and 16-cell stage of early abalone development. Although the expression of both AbGrxs demonstrated similar patterns, the expression of AbGrx1-like was higher than AbGrx2 during development stages. In contrast, AbGrx2 expression was significantly higher than that of AbGrx1-like in adult tissues. Highest AbGrx1-like expression was observed in the hepatopancreas and digestive tract, while highest AbGrx2 expression was found in the gills, followed by the mantle, in healthy adult abalone tissues. The highest expression of AbGrx1-like was observed in the gills at 12 h and 6 h post injection (p.i) of Vibrio parahemolyticus and other stimulants, respectively. The highest expression of AbGrx2 in the gills were observed at 120 h, 6 h, 24 h, and 12 h post injection of V. parahaemolyticus, Listeria monocytogenes, Viral hemorrhagic septicemia virus, and Polyinosinic:polycytidylic acid, respectively. AbGrxs possessed significant 2-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) reduction activity, but AbGrx2 exhibited higher redox activity than AbGrx1-like. Altogether, our results suggest an important role of AbGrx1-like and AbGrx2 in redox homeostasis, as well as in the invertebrate immune defense system. Our findings will aid the development of new disease management strategies for this economically valuable species.

The pivotal function of dehydroascorbate reductase in glutathione homeostasis in plants.

Under natural conditions, plants are exposed to various abiotic and biotic stresses that trigger rapid changes in the production and removal of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). The ascorbate-glutathione pathway has been recognized to be a key player in H2O2 metabolism, in which reduced glutathione (GSH) regenerates ascorbate by reducing dehydroascorbate (DHA), either chemically or via DHA reductase (DHAR), an enzyme belonging to the glutathione S-transferase (GST) superfamily. Thus, DHAR has been considered to be important in maintaining the ascorbate pool and its redox state. Although some GSTs and peroxiredoxins may contribute to GSH oxidation, analysis of Arabidopsis dhar mutants has identified the key role of DHAR in coupling H2O2 to GSH oxidation. The reaction of DHAR has been proposed to proceed by a ping-pong mechanism, in which binding of DHA to the free reduced form of the enzyme is followed by binding of GSH. Information from crystal structures has shed light on the formation of sulfenic acid at the catalytic cysteine of DHAR that occurs with the reduction of DHA. In this review, we discuss the molecular properties of DHAR and its importance in coupling the ascorbate and glutathione pools with H2O2 metabolism, together with its functions in plant defense, growth, and development.

Glutaredoxin 2 from big belly seahorse (Hippocampus abdominalis) and its potential involvement in cellular redox homeostasis and host immune responses

Glutaredoxins are oxidoreductases present in almost all living organisms. They belong to the thioredoxin superfamily and share the thioredoxin structure and catalytic motif. Glutaredoxin 2 has been identified as a mitochondrial protein in vertebrates. In this study, the sequence of Glutaredoxin 2 from Hippocampus abdominalis (HaGrx2) was analyzed by molecular, transcriptional, and functional assays. In-silico analysis revealed that HaGrx2 shows the highest homology with Hippocampus comes, while distinctly cluster with fish Grx2 orthologs. Tissue distribution analysis showed that HaGrx2 is ubiquitously expressed in all tissues tested, and the highest expression was observed in the brain and skin. Significant HaGrx2 transcript modulation was identified in blood and liver upon injecting bacterial and Pathogen Associated Molecular Patterns. The redox activity of HaGrx2 was revealed by Dehydroascorbic reduction and insulin disulfide reduction activity assays. Further, the deglutathionylation activity of 1 nM HaGrx2 was found to be equivalent to that of 0.84 nM HaGrx1. HaGrx2 exhibited antiapoptotic activity against H2O2-induced oxidative stress in FHM cells. Altogether, the results of this study suggest that HaGrx2 plays a role in redox homeostasis and innate immune responses in fish.

Functional, Structural and Biochemical Features of Plant Serinyl-Glutathione Transferases.

Glutathione transferases (GSTs) belong to a ubiquitous multigenic family of enzymes involved in diverse biological processes including xenobiotic detoxification and secondary metabolism. A canonical GST is formed by two domains, the N-terminal one adopting a thioredoxin (TRX) fold and the C-terminal one an all-helical structure. The most recent genomic and phylogenetic analysis based on this domain organization allowed the classification of the GST family into 14 classes in terrestrial plants. These GSTs are further distinguished based on the presence of the ancestral cysteine (Cys-GSTs) present in TRX family proteins or on its substitution by a serine (Ser-GSTs). Cys-GSTs catalyze the reduction of dehydroascorbate and deglutathionylation reactions whereas Ser-GSTs catalyze glutathione conjugation reactions and eventually have peroxidase activity, both activities being important for stress tolerance or herbicide detoxification. Through non-catalytic, so-called ligandin properties, numerous plant GSTs also participate in the binding and transport of small heterocyclic ligands such as flavonoids including anthocyanins, and polyphenols. So far, this function has likely been underestimated compared to the other documented roles of GSTs. In this review, we compiled data concerning the known enzymatic and structural properties as well as the biochemical and physiological functions associated to plant GSTs having a conserved serine in their active site.

Glutaredoxin 1 from big-belly seahorse (Hippocampus abdominalis): Molecular, transcriptional, and functional evidence in teleost immune responses

Glutaredoxins (Grx) are redox enzymes conserved in viruses, eukaryotes, and prokaryotes. In this study, we characterized glutaredoxin 1 (HaGrx1) from big-belly seahorse, Hippocampus abdominalis. In-silico analysis showed that HaGrx1 contained the classical glutaredoxin 1 structure with a CSYC thioredoxin active site motif. According to multiple sequence alignment and phylogenetic reconstruction, HaGrx1 presented the highest homology to the Grx1 ortholog from Hippocampus comes. Transcriptional studies demonstrated the ubiquitous distribution of HaGrx1 transcripts in all the seahorse tissues tested. Significant modulation (p < 0.05) of HaGrx1 transcripts were observed in blood upon stimulation with pathogen-associated molecular patterns and live pathogens. The β-hydroxyethyl disulfide reduction assay confirmed the antioxidant activity of recombinant HaGrx1. Further, dehydroascorbate reduction and insulin disulfide reduction assays revealed the oxidoreductase activity of HaGrx1. HaGrx1 utilized 1,4-dithiothreitol, l-cysteine, 2-mercaptoethanol, and reduced l-glutathione as reducing agent with different dehydroascorbate reduction activity levels. Altogether, our results suggested a vital role of HaGrx1 in redox homeostasis as well as the host innate immune defense system.

Spectroscopic determination of intracellular quercetin uptake using erythrocyte model and its implications in human aging.

The present study was carried out to detect intracellular quercetin uptake by RBCs during human aging. The study was carried out on 95 normal healthy subjects of both the sexes. Intracellular quercetin uptake was estimated by performing ethyl acetate extraction. A significant (p < 0.001) decline in intracellular quercetin uptake by human RBCs was observed in elderly as compared to young population, while plasma membrane redox system (PMRS) activity was significantly decreasing as a function of human age. To the best of our knowledge, we are the first to present quercetin uptake by erythrocytes during aging in humans with this study. It is hypothesized that intracellular uptake of quercetin may serve as an intracellular electron donor for plasma membrane redox system in red blood cells during cellular aging which plays an important role in extracellular dehydroascorbate reduction and ascorbate recycling.

Enzymatic characterization and crystal structure analysis of Chlamydomonas reinhardtii dehydroascorbate reductase and their implications for oxidative stress

Dehydroascorbate reductase (DHAR) is a key enzyme for glutathione (GSH)-dependent reduction of dehydroascorbate (DHA) to recycled ascorbate (AsA) in plants, and plays a major role against the toxicity of reactive oxygen species (ROS). Previously, we proposed that the increase of AsA regeneration via enhanced DHAR activity modulates the ascorbate-glutathione cycle activity against photooxidative stress in Chlamydomonas reinhardtii. In the present work, we use site-directed mutagenesis and crystal structure analysis to elucidate the molecular basis of how C. reinhardtii DHAR (CrDHAR1) is involved in the detoxification mechanisms. Mutagenesis data show that the D21A, D21N and C22A mutations result in severe loss of the enzyme's function, suggesting crucial roles of Asp-21 and Cys-22 in substrate binding and catalysis. The mutant K11A also exhibits reduced redox activity (∼50%). The crystal structure of apo CrDHAR1 further provides insights into the proposed mechanism centering on the strictly conserved Cys-22, which is suggested to initiate the redox reactions of DHA and GSH. Furthermore, in vitro oxidation of the recombinant CrDHAR1 in the presence of 1 mM H2O2 has minor effects on the Km for the substrates but significantly reduces the kcat. The enzyme's activity and its mRNA abundance in the C. reinhardtii cells are increased by treatment with 0.2–1 mM H2O2 but decreased when H2O2 is ≥ 1.5 mM. The latter decrease is accompanied by oxidative damage and lower AsA concentrations. These biochemical and physiological data provide new insights into the catalytic mechanism of CrDHAR1, which protects the C. reinhardtii cells from oxidative stress-induced toxicity.

P 012 - The Enzymatic Nature of Ascorbate Recycling

Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.

Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis

Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.

Insights into ascorbate regeneration in plants: investigating the redox and structural properties of dehydroascorbate reductases from Populus trichocarpa.

Dehydroascorbate reductases (DHARs), enzymes belonging to the GST superfamily, catalyse the GSH-dependent reduction of dehydroascorbate into ascorbate in plants. By maintaining a reduced ascorbate pool, they notably participate to H2O2 detoxification catalysed by ascorbate peroxidases (APXs). Despite this central role, the catalytic mechanism used by DHARs is still not well understood and there is no supportive 3D structure. In this context, we have performed a thorough biochemical and structural analysis of the three poplar DHARs and coupled this to the analysis of their transcript expression patterns and subcellular localizations. The transcripts for these genes are mainly detected in reproductive and green organs and the corresponding proteins are expressed in plastids, in the cytosol and in the nucleus, but not in mitochondria and peroxisomes where ascorbate regeneration is obviously necessary. Comparing the kinetic properties and the sensitivity to GSSG-mediated oxidation of DHAR2 and DHAR3A, exhibiting 1 or 3 cysteinyl residues respectively, we observed that the presence of additional cysteines in DHAR3A modifies the regeneration mechanism of the catalytic cysteine by forming different redox states. Finally, from the 3D structure of DHAR3A solved by NMR, we were able to map the residues important for the binding of both substrates (GSH and DHA), showing that DHAR active site is very selective for DHA recognition and providing further insights into the catalytic mechanism and the roles of the additional cysteines found in some DHARs.

Noninvasive in vivo imaging of diabetes-induced renal oxidative stress and response to therapy using hyperpolarized 13C dehydroascorbate magnetic resonance.

Oxidative stress has been proposed to be a unifying cause for diabetic nephropathy and a target for novel therapies. Here we apply a new endogenous reduction-oxidation (redox) sensor, hyperpolarized (HP) 13C dehydroascorbate (DHA), in conjunction with MRI to noninvasively interrogate the renal redox capacity in a mouse diabetes model. The diabetic mice demonstrate an early decrease in renal redox capacity, as shown by the lower in vivo HP 13C DHA reduction to the antioxidant vitamin C (VitC), prior to histological evidence of nephropathy. This correlates with lower tissue reduced glutathione (GSH) concentration and higher NADPH oxidase 4 (Nox4) expression, consistent with increased superoxide generation and oxidative stress. ACE inhibition restores the HP 13C DHA reduction to VitC with concomitant normalization of GSH concentration and Nox4 expression in diabetic mice. HP 13C DHA enables rapid in vivo assessment of altered redox capacity in diabetic renal injury and after successful treatment.

The still mysterious roles of cysteine-containing glutathione transferases in plants.

Glutathione transferases (GSTs) represent a widespread multigenic enzyme family able to modify a broad range of molecules. These notably include secondary metabolites and exogenous substrates often referred to as xenobiotics, usually for their detoxification, subsequent transport or export. To achieve this, these enzymes can bind non-substrate ligands (ligandin function) and/or catalyze the conjugation of glutathione onto the targeted molecules, the latter activity being exhibited by GSTs having a serine or a tyrosine as catalytic residues. Besides, other GST members possess a catalytic cysteine residue, a substitution that radically changes enzyme properties. Instead of promoting GSH-conjugation reactions, cysteine-containing GSTs (Cys-GSTs) are able to perform deglutathionylation reactions similarly to glutaredoxins but the targets are usually different since glutaredoxin substrates are mostly oxidized proteins and Cys-GST substrates are metabolites. The Cys-GSTs are found in most organisms and form several classes. While Beta and Omega GSTs and chloride intracellular channel proteins (CLICs) are not found in plants, these organisms possess microsomal ProstaGlandin E-Synthase type 2, glutathionyl hydroquinone reductases, Lambda, Iota and Hemerythrin GSTs and dehydroascorbate reductases (DHARs); the four last classes being restricted to the green lineage. In plants, whereas the role of DHARs is clearly associated to the reduction of dehydroascorbate to ascorbate, the physiological roles of other Cys-GSTs remain largely unknown. In this context, a genomic and phylogenetic analysis of Cys-GSTs in photosynthetic organisms provides an updated classification that is discussed in the light of the recent literature about the functional and structural properties of Cys-GSTs. Considering the antioxidant potencies of phenolic compounds and more generally of secondary metabolites, the connection of GSTs with secondary metabolism may be interesting from a pharmacological perspective.

Structural modelling and molecular characterization of omega‐class glutathione S‐transferase 2 from Drosophila melanogaster

Glutathione S-transferase omega (GSTO) is a recently identified Glutathione S-transferase (GST), and it has several known functions and variable distribution patterns in many organisms. In Drosophila, GstO2 exists as two isoforms, GstO2A and GstO2B. Despite the high sequence homology between the two GstO2 isoforms, they have different physiological functions. In the present study, we characterized the structural and molecular properties of Drosophila melanogaster GstO2 isoforms. Homology modelling of GstO2s using I-TASSER servers for protein structure and function prediction revealed that the two GstO2s have different electropotential surface distributions and different shapes of the substrate-binding sites. The recombinant GstO2s have native molecular weights of ∼60 kDa. GstO2s have similar optimum conditions for enzymatic reactions at pH 8.0 and 40 °C. The kinetic parameters of the reduction of dehydroascorbate by these two GstO2s were determined. Collectively, our results provide structural insights into the different substrate profiles of the GstO2 isoforms.

The role of ascorbate in protein folding

Ascorbate was linked to protein folding a long time ago. At the first level of this connection, it had been shown that ascorbate functions as an essential cofactor in the hydroxylation enzymes involved in collagen synthesis. Although the hydroxylation reactions catalyzed by the members of the prolyl 4-hydroxylase family are considered to be ascorbate dependent, the hydroxylation of proline alone does not need ascorbate. Prolyl 4-hydroxylases participate in two catalytic reactions: one in which proline residues are hydroxylated, while 2-oxoglutarate is decarboxylated and molecular oxygen is consumed. This reaction is ascorbate independent. However, in another reaction, prolyl 4-hydroxylases catalyze the decarboxylation of 2-oxoglutarate uncoupled from proline hydroxylation but still needing molecular oxygen. At this time, ferrous iron is oxidized and the protein is rendered catalytically inactive until reduced by ascorbate. At the second level of the connection, the oxidation and the oxidized form of ascorbate, dehydroascorbate, is involved in the formation of disulfide bonds of secretory proteins. The significance of the dehydroascorbate reductase activity of protein disulfide isomerase was debated because protein disulfide isomerase as a dehydroascorbate reductase was found to be too slow to be the major route for the reduction of dehydroascorbate (and formation of disulfides) in the endoplasmic reticulum lumen. However, very recently, low tissue ascorbate levels and a noncanonical scurvy were observed in endoplasmic reticulum thiol oxidase- and peroxiredoxin 4-compromised mice. This novel observation implies that ascorbate may be involved in oxidative protein folding and creates a link between the disulfide bond formation (oxidative protein folding) and hydroxylation.

Quantitative data on the contribution of GSH and Complex II dependent ascorbate recycling in plant mitochondria

The reduction of dehydroascorbate, the oxidized form of ascorbate plays important role in the maintenance of sufficient level of ascorbate. In plant mitochondria two DHA reducing mechanisms, the GSH-dependent and the mitochondrial electron transfer chain dependent ascorbate recycling have been characterized. Although both pathways have been extensively studied quantitative information about the electron fluxes from one or another direction for the reduction of DHA is not known. The cellular, mitochondrial glutathione pools and mitochondrial DHA reducing capacity was measured in BSO treated and control tobacco cells. While BSO caused dramatic decrease of cellular GSH content the difference was much smoother at mitochondrial level. The difference in DHA reduction capacity was even smoother affirming the existence of alternative, non-GSH dependent DHA reducing mechanism(s) in plant mitochondria. On the base of the parallel determination of mitochondrial GSH content and ascorbate production upon DHA addition, GSH (consumption) is responsible for the ~ 20 % of ascorbate production. Almost 90 % enhancement of ascorbate production could be provoked by the addition of Complex II substrate succinate which could be almost totally prevented by the concomitant addition of malonate or TTFA. On the base of these results, the importance of mitochondrial Complex II compared to GSH-dependent mechanisms in mitochondrial ascorbate recycling has been underestimated so far.

Toward a refined classification of class I dithiol glutaredoxins from poplar: biochemical basis for the definition of two subclasses

Glutaredoxins (Grxs) are small oxidoreductases particularly specialized in the reduction of protein-glutathione adducts. Compared to other eukaryotic organisms, higher plants present an increased diversity of Grxs which are organized into four classes. This work presents a thorough comparative analysis of the biochemical and catalytic properties of dithiol class I Grxs from poplar, namely GrxC1, GrxC2, GrxC3, and GrxC4. By evaluating the in vitro oxidoreductase activity of wild type and cysteine mutated variants and by determining their dithiol-disulfide redox potentials, pKa values of the catalytic cysteine, redox state changes in response to oxidative treatments, two subgroups can be distinguished. In accordance with their probable quite recent duplication, GrxC1 and GrxC2 are less efficient catalysts for the reduction of dehydroascorbate and hydroxyethyldisulfide compared to GrxC3 and GrxC4, and they can form covalent dimers owing to the presence of an additional C-terminal cysteine (CysC). Interestingly, the second active site cysteine (CysB) influences the reactivity of the catalytic cysteine (CysA) in GrxC1 and GrxC2 as already observed with GrxC5 (restricted to A. thaliana), but not in GrxC3 and C4. However, all proteins can form an intramolecular disulfide between the two active site cysteines (CysA-CysB) which could represent either a protective mechanism considering that this second cysteine is dispensable for deglutathionylation reaction or a true catalytic intermediate occurring during the reduction of particular disulfide substrates or in specific conditions or compartments where glutathione levels are insufficient to support Grx regeneration. Overall, in addition to their different sub-cellular localization and expression pattern, the duplication and maintenance along evolution of several class I Grxs in higher plants can be explained by the existence of differential biochemical and catalytic properties.

Structural Insights into the Dehydroascorbate Reductase Activity of Human Omega-Class Glutathione Transferases

The reduction of dehydroascorbate (DHA) to ascorbic acid (AA) is a vital cellular function. The omega-class glutathione transferases (GSTs) catalyze several reductive reactions in cellular biochemistry, including DHA reduction. In humans, two isozymes (GSTO1-1 and GSTO2-2) with significant DHA reductase (DHAR) activity are found, sharing 64% sequence identity. While the activity of GSTO2-2 is higher, it is significantly more unstable in vitro. We report the first crystal structures of human GSTO2-2, stabilized through site-directed mutagenesis and determined at 1.9 Å resolution in the presence and absence of glutathione (GSH). The structure of a human GSTO1-1 has been determined at 1.7 Å resolution in complex with the reaction product AA, which unexpectedly binds in the G-site, where the glutamyl moiety of GSH binds. The structure suggests a similar mode of ascorbate binding in GSTO2-2. This is the first time that a non-GSH-based reaction product has been observed in the G-site of any GST. AA stacks against a conserved aromatic residue, F34 (equivalent to Y34 in GSTO2-2). Mutation of Y34 to alanine in GSTO2-2 eliminates DHAR activity. From these structures and other biochemical data, we propose a mechanism of substrate binding and catalysis of DHAR activity.