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Reversible redox post-translational modifications such as oxido-reduction of disulfide bonds, S-nitrosylation, and S-glutathionylation, play a prominent role in the regulation of cell metabolism and signaling in all organisms. These modifications are mainly controlled by members of the thioredoxin and glutaredoxin families. Early studies in photosynthetic organisms have identified the Calvin-Benson cycle, the photosynthetic pathway responsible for carbon assimilation, as a redox regulated process. Indeed, 4 out of 11 enzymes of the cycle were shown to have a low activity in the dark and to be activated in the light through thioredoxin-dependent reduction of regulatory disulfide bonds. The underlying molecular mechanisms were extensively studied at the biochemical and structural level. Unexpectedly, recent biochemical and proteomic studies have suggested that all enzymes of the cycle and several associated regulatory proteins may undergo redox regulation through multiple redox post-translational modifications including glutathionylation and nitrosylation. The aim of this review is to detail the well-established mechanisms of redox regulation of Calvin-Benson cycle enzymes as well as the most recent reports indicating that this pathway is tightly controlled by multiple interconnected redox post-translational modifications. This redox control is likely allowing fine tuning of the Calvin-Benson cycle required for adaptation to varying environmental conditions, especially during responses to biotic and abiotic stresses. [ABSTRACT FROM AUTHOR]

Plants contain both cytosolic and chloroplastic GAPDHs (glyceraldehyde-3-phosphate dehydrogenases). In Arabidopsis thaliana, cytosolic GAPDH is involved in the glycolytic pathway and is represented by two differentially expressed isoforms (GapC1 and GapC2) that are 98% identical in amino acid sequence. In the present study we show that GapC1 is a phosphorylating NAD-specific GAPDH with enzymatic activity strictly dependent on Cys149. Catalytic Cys149 is the only solventexposed cysteine of the protein and its thiol is relatively acidic (pKa =5.7). This property makes GapC1 sensitive to oxidation by H2O2, which appears to inhibit enzyme activity by converting the thiolate of Cys149 (-S- ) into irreversible oxidized forms (-SO2 - and -SO3 - ) via a labile sulfenate intermediate (-SO--SSG), as demonstrated by both MS and biotinylated GSH. Glutathionylated GapC1 can be fully reactivated either by cytosolic glutaredoxin, via a GSHdependent monothiol mechanism, or, less efficiently, by cytosolic thioredoxins physiologically reduced by NADPH:thioredoxin reductase. The potential relevance of these findings is discussed in the light of the multiple functions of GAPDH in eukaryotic cells (e.g. glycolysis, control of gene expression and apoptosis) that appear to be influenced by the redox state of the catalytic Cys149 sulfenates to give rise to a mixed disulfide (Cys149-SSG), as demonstrated by both MS and biotinylated GSH. Glutathionylated GapC1 can be fully reactivated either by cytosolic glutaredoxin, via a GSHdependent monothiol mechanism, or, less efficiently, by cytosolic thioredoxins physiologically reduced by NADPH:thioredoxin reductase. The potential relevance of these findings is discussed in the light of the multiple functions of GAPDH in eukaryotic cells (e.g. glycolysis, control of gene expression and apoptosis) that appear to be influenced by the redox state of the catalytic Cys149. [ABSTRACT FROM AUTHOR]

Abstract: Protein deglutathionylation is mainly catalyzed by glutaredoxins (GRXs). We have analyzed the biochemical properties of four of the six different GRXs of Chlamydomonas reinhardtii. Kinetic parameters were determined for disulfide and dehydroascorbate reduction but also for deglutathionylation of artificial and protein substrates. The results indicate that GRXs exhibit striking differences in their catalytic properties, mainly linked to the class of GRX considered but also to the pKa of the N-terminal catalytic cysteine. Furthermore, glutathionylated proteins were found to exhibit distinct reactivities with GRXs. These results suggest that glutathionylation may allow a fine tuning of cell metabolism under stress conditions. Structured summary: MINT-7761120: GRX6 (uniprotkb:A8HN52) and GRX6 (uniprotkb:A8HN52) bind (MI:0408) by comigration in non denaturing gel electrophoresis (MI:0404) MINT-7761098:GRX5 (uniprotkb:A8I7Q4) and GRX5 (uniprotkb:A8I7Q4) bind (MI:0408) by comigration in non denaturing gel electrophoresis (MI:0404) [Copyright &y& Elsevier]

Protein S-glutathionylation, the reversible formation of a mixed-disulfide between glutathione and protein thiols, is involved in protection of protein cysteines from irreversible oxidation, but also in protein redox regulation. Recent studies have implicated S-glutathionylation as a cellular response to oxidative/nitrosative stress, likely playing an important role in signaling. Considering the potential importance of glutathionylation, a number of methods have been developed for identifying proteins undergoing glutathionylation. These methods, ranging from analysis of purified proteins in vitro to large-scale proteomic analyses in vivo, allowed identification of nearly 200 targets in mammals. By contrast, the number of known glutathionylated proteins is more limited in photosynthetic organisms, although they are severely exposed to oxidative stress. The aim of this review is to detail the methods available for identification and analysis of glutathionylated proteins in vivo and in vitro. The advantages and drawbacks of each technique will be discussed as well as their application to photosynthetic organisms. Furthermore, an overview of known glutathionylated proteins in photosynthetic organisms is provided and the physiological importance of this post-translational modification is discussed. [ABSTRACT FROM PUBLISHER]

Glutathione, a tripeptide with the sequence γ-Glu-Cys-Gly, exists either in a reduced form with a free thiol group or in an oxidized form with a disulfide between two identical molecules. We describe here briefly the pathways involved in the synthesis, reduction, polymerization, and degradation of glutathione, as well as its distribution throughout the plant and its redox buffering capacities. The function of glutathione in xenobiotic and heavy metal detoxification, plant development, and plant-pathogen interactions is also briefly discussed. Several lines of evidence indicate that glutathione and glutaredoxins (GRXs) are implicated in the response to oxidative stress through the regeneration of enzymes involved in peroxide and methionine sulfoxide reduction. Finally, emerging functions for plant GRXs and glutathione concern the regulation of protein activity via glutathionylation and the capacity of some GRXs to bind iron sulfur centers and for some of them to transfer FeS clusters into apoproteins. [ABSTRACT FROM AUTHOR]

Glutaredoxins (GRXs) are thioredoxin superfamily members exhibiting thiol-disulfide oxidoreductase activity and/or iron-sulfur (Fe-S) cluster binding capacities. These properties are determined by specific structural factors. In this study, we examined the capacity of the class I Chlamydomonas reinhardtii GRX2 recombinant protein to catalyze both protein glutathionylation and deglutathionylation reactions using a redox sensitive fluorescent protein as a model protein substrate. We observed that the catalytic cysteine of the CPYC active site motif of GRX2 was sufficient for catalyzing both reactions in the presence of glutathione. Unexpectedly, spectroscopic characterization of the protein purified under anaerobiosis showed the presence of a [2Fe-2S] cluster despite having a presumably inadequate active site signature, based on past mutational analyses. The spectroscopic characterization of cysteine mutated variants together with modeling of the Fe–S cluster-bound GRX homodimer from the structure of an apo-GRX2 indicate the existence of an atypical Fe–S cluster environment and ligation mode. Overall, the results further delineate the biochemical and structural properties of conventional GRXs, pointing to the existence of multiple factors more complex than anticipated, sustaining the capacity of these proteins to bind Fe–S clusters. [ABSTRACT FROM AUTHOR]

Abstract: Reactive oxygen species play important roles in redox signaling mainly through a set of reversible post-translational modifications of cysteine thiol residues in proteins, including glutathionylation and dithiol/disulfide exchange. Protein glutathionylation has been extensively studied in mammals but emerging evidence suggests that it can play important roles in plants and in chloroplast in particular. This redox modification involves protein thiols and glutathione and is mainly controlled by glutaredoxins, oxidoreductases belonging to the thioredoxin superfamily. In this review, we first present the possible mechanisms of protein glutathionylation and then introduce the chloroplast systems of glutaredoxins and thioredoxins, in order to pinpoint the biochemical properties that make some glutaredoxin isoforms the master enzymes in deglutathionylation. Finally, we discuss the possible roles of glutathionylation in thiol protection, protein regulation, reactive oxygen species scavenging and redox signaling in chloroplasts, with emphasis on the crosstalk between thioredoxin- and glutaredoxin-mediated signaling pathways. [Copyright &y& Elsevier]

In a continuing effort to analyze the selectivity/redundancy of the three glutaredoxin (Grx) enzymes of the model cyanobacte- rium Synechocystis PCC6803, we have characterized an enzyme system that plays a crucial role in protection against two toxic metal pollutants, mercury and uranium. The present data show that Grxl (Sir1562 in CyanoBase) selectively interacts with the presumptive mercuric reductase protein (S1r1849). This MerA enzyme plays a crucial role in cell defense against both mercuric and uranyl ions, in catalyzing their NADPH-driven reduction. Like MerA, Grxl operates in cell protection against both mercury and uranium. The Grxl-MerA interaction requires cysteine 86 (C86) of Grxl and C78 of MerA, which is critical for its reductase activity. MerA can be inhibited by glutathionylation and subsequently reactivated by Grxl, likely through deglutathionylation. The two Grxl residues C31, which belongs to the redox active site (CX2C), and C86, which operates in MerA interactions, are both required for reactivation of MerA. These novel findings emphasize the role of glutaredoxins in tolerance to metal stress as well as the evolutionary conservation of the glutathionylation process, so far described mostly for eukaryotes [ABSTRACT FROM AUTHOR]

Methionine oxidation leads to the formation of S- and R-diastereomers of methionine sulfoxide (MetSO), which are reduced back to methionine by methionine sulfoxide reductases (MSRs) A and B, respectively. MSRBs are classified in two groups depending on the conservation of one or two redox-active Cys; 2-Cys MSRBs possess a catalytic Cys-reducing MetSO and a resolving Cys, allowing regeneration by thioredoxins. The second type, 1-Cys MSRBs, possess only the catalytic Cys. The biochemical mechanisms involved in activity regeneration of 1-Cys MSRBs remain largely elusive. In the present work we used recombinant plastidial Arabidopsis thaliana MSRB1 and MSRB2 as models for 1-Cys and 2-Cys MSRBs, respectively, to delineate the Trxand glutaredoxin-dependent reduction mechanisms. Activity assays carried out using a series of cysteine mutants and various reductants combined with measurements of free thiols under distinct oxidation conditions and mass spectrometry experiments show that the 2-Cys MSRB2 is reduced by Trx through a dithiol-disulfide exchange involving both redox-active Cys of the two partners. Regarding 1-Cys MSRB1, oxidation of the enzyme after substrate reduction leads to the formation of a stable sulfenic acid on the catalytic Cys, which is subsequently glutathionylated. The deglutathionylation of MSRB1 is achieved by both monoand dithiol glutaredoxins and involves only their N-terminal conserved catalytic Cys. This study proposes a detailed mechanism of the regeneration of 1-Cys MSRB activity by glutaredoxins, which likely constitute physiological reductants for this type of MSR. [ABSTRACT FROM AUTHOR]

Glutaredoxins (Grxs) are efficient catalysts for the reduction of mixed disulfides in glutathionylated proteins, using glutathione or thioredoxin reductases for their regeneration. Using GFP fusion, we have shown that poplar GrxSl2, which possesses a monothiol [sup28]WCSYS[sup32] active site, is localized in chloroplasts. In the presence of reduced glutathione, the recombinant protein is able to reduce in vitro substrates, such as hydroxyethyldisulfide and dehydroascorbate, and to regenerate the glutathionylated glyceraldehyde-3-phosphate dehydrogenase. Although the protein possesses two conserved cysteines, it is functioning through a monothiol mechanism, the conserved C terminus cysteine (Cys[sup87]) being dispensable, since the C87S variant is fully active in all activity assays. Biochemical and crystallographic studies revealed that Cys[sup87] exhibits a certain reactivity, since its PK[suba] is around 5.6. Coupled with thiol titration, fluorescence, and mass spectrometry analyses, the resolution of poplar GrxSl2 x-ray crystal structure shows that the only oxidation state is a glutathionylated derivative of the active site cysteine (Cyss[sup29]) and that the enzyme does not form interor intramolecular disulfides. Contrary to some plant Grxs, GrxSl2 does not incorporate an iron-sulfur cluster in its wild-type form, but when the active site is mutated into YCSYS, it binds a [2Fe-2S] cluster, indicating that the single Trp residue prevents this incorporation. [ABSTRACT FROM AUTHOR]

Glutaredoxins (GRXs) are small ubiquitous disulfide oxidoreductases known to use GSH as electron donor. In photosynthetic organisms, little is known about the biochemical properties of GRXs despite the existence of ~30 different isoforms in higher plants. We report here the biochemical characterization of Chlamydomonas GRX1 and GRX3, the major cytosolic and chloroplastic isoforms, respectively. Glutaredoxins are classified on the basis of the amino acid sequence of the active site. GRX1 is a typical CPYC-type GRX, which is reduced by GSH and exhibits disulfide reductase, dehydroascorbate reductase, and deglutathionylation activities. In contrast, GRX3 exhibits unique properties. This chloroplastic CGFS-type GRX is not reduced by GSH and has an atypically low redox potential (-323 ± 4 mV at pH 7.9). Remarkably, GRX3 can be reduced in the light by photoreduced ferredoxin and ferredoxin-thioredoxin reductase. Both GRXs proved to be very efficient catalysts of A4-glyceraldehyde-3-phosphate dehydrogenase deglutathionylation, whereas cytosolic and chloroplastic thioredoxins were inefficient. Glutathionylated A4-glyceraldehyde-3-phosphate dehydrogenase is the first physiological substrate identified for a CGFS-type GRX. [ABSTRACT FROM AUTHOR]