The reducing activity of glutaredoxin 3 toward cytoplasmic substrate proteins is restricted by methionine 43

The reducing proteins glutaredoxin 3 (Grx3) and glutaredoxin 1 (Grx1) are structurally similar but exhibit different specificities toward substrates. Grx1 efficiently reduces ribonucleotide reductase and PAPS reductase, while Grx3 reduces these enzymes inefficiently or not at all. We previously described a selection for Grx3 mutants with increased activity toward substrates of Grx1 in vivo. Remarkably, we repeatedly isolated mutants with changes in only one of the amino acids of Grx3, methionine 43, converting it to either valine, leucine, or isoleucine. In this paper we present additional genetic studies and a biochemical characterization of Grx3-Met43Val, the most efficient mutant. We show that Grx3-Met43Val is able to reduce ribonucleotide reductae and PAPS reductase much more efficiently than the wild-type protein in vitro. The altered protein has an increased Vmax over that of Grx3, nearly the same Vmax as Grx1 while the Km remains high. Molecular dynamics simulations suggest that the Met43Val substitution results in changes in properties of the N-terminal cysteine of the active site leading to a considerably lower pKa. Furthermore, Grx3-Met43Val shows an 11 mV lower redox potential than the wild-type Grx3. These findings provide biochemical and structural explanations for the increased reductive efficiency of the mutant Grx3.     

Structure, dynamics and electrostatics of the active site of glutaredoxin 3 from Escherichia coli: comparison with functionally related proteins.

The chemistry of active-site cysteine residues is central to the activity of thiol-disulfide oxidoreductases of the thioredoxin superfamily. In these reactions, a nucleophilic thiolate is required, but the associated pK(a) values differ vastly in the superfamily, from less than 4 in DsbA to greater than 7 in Trx. The factors that stabilize this thiolate are, however, not clearly established. The glutaredoxins (Grxs), which are members of this superfamily, contain a Cys-Pro-Tyr-Cys motif in their active site. In reduced Grxs, the pK(a) of the N-terminal active-site nucleophilic cysteine residue is lowered significantly, and the stabilization of the corresponding thiolate is expected to influence the redox potential of these enzymes. Here, we use a combination of long molecular dynamics (MD) simulations, pK(a) calculations, and experimental investigations to derive the structure and dynamics of the reduced active site from Escherichia coli Grx3, and investigate the factors that stabilize the thiolate. Several different MD simulations converged toward a consensus conformation for the active-site cysteine residues (Cys11 and Cys14), after a number of local conformational changes. Key features of the model were tested experimentally by measurement of NMR scalar coupling constants, and determination of pK(a) values of selected residues. The pK(a) values of the Grx3 active-site residues were calculated during the MD simulations, and support the underlying structural model. The structure of Grx3, in combination with the pK(a) calculations, indicate that the pK(a) of the N-terminal active-site cysteine residue in Grx3 is intermediate between that of its counterpart in DsbA and Trx. The pK(a) values in best agreement with experiment are obtained with a low (<4) protein dielectric constant. The calculated pK(a) values fluctuate significantly in response to protein dynamics, which underscores the importance of the details of the underlying structures when calculating pK(a) values. The thiolate of Cys11 is stabilized primarily by direct hydrogen bonding with the amide protons of Tyr13 and Cys14 and the thiol proton of Cys14, rather than by long-range interactions from charged groups or from a helix macrodipole. From the comparison of reduced Grx3 with other members of the thioredoxin superfamily, a unifying theme for the structural basis of thiol pK(a) differences in this superfamily begins to emerge.     

Redox properties and evolution of human glutaredoxins

Glutaredoxins (Grxs) are glutathione-dependent oxidoreductases that belong to the thioredoxin superfamily catalyzing thiol-disulfide exchange reactions via active site cysteine residues. Focusing on the human dithiol glutaredoxins having a C-X-Y-C active site sequence motif, the redox potentials of hGrx1 and hGrx2 were determined to be -232 and -221 mV, respectively, using a combination of redox buffers, protein-protein equilibrium and thermodynamic linkage. In addition, a nonactive site disulfide was identified between Cys28 and Cys113 in hGrx2 using redox buffers and chemical digestion. This disulfide confers nearly five kcal mol(-1) additional stability by linking the C-terminal helix to the bulk of the protein. The redox potential of this nonactive site disulfide was determined to be -317 mV and is thus expected to be present in all but the most reducing conditions in vivo. As all human glutaredoxins contain additional nonactive site cysteine residues, a full phylogenetic analysis was performed to help elucidate their structural and functional roles. Three distinct groups were found: Grx1, Grx2, and Grx5, the latter representing a highly conserved group of monothiol glutaredoxins having a C-G-F-S active site sequence, with clear homologs from bacteria to human. Grx1 and Grx2 diverged from a common ancestor before the origin of vertebrates, possibly even earlier in animal evolution. The highly stabilizing nonactive site disulfide observed in hGrx2 is found to be a conserved feature within the deuterostomes and appears to be the only additional conserved intramolecular disulfide within the glutaredoxins.     

Stabilization of the catalytic thiolate in a mammalian glutaredoxin: structure, dynamics and electrostatics of reduced pig glutaredoxin and its mutants

The variety of functions performed by proteins of the thioredoxin superfamily, including glutaredoxins, involves the wide range of redox potential associated with the -Cys-X-X-Cys- motif found in their active sites. The determinants of these differences in redox potential are still obscure. A better understanding requires a detailed characterization of the reduced state of these enzymes, especially because the lowered pK(a) of the reduced N-terminal active-site cysteine is a key feature of these enzymes' chemistry, including their redox potential. Analysis of the factors controlling this pK(a) is complicated by the apparent structural heterogeneity of the reduced active sites across glutaredoxins. In this family, pig glutaredoxin (pGrx) was one of the first to be functionally characterized, including some intriguing mutagenesis data, but a structure of its reduced state has been lacking. We used long molecular dynamics simulations and electrostatic calculations to analyze the structure, dynamics and electrostatics of reduced pGrx and some of its mutants. Comparison with experimental data is drawn whenever possible. It is shown that a dynamic model is essential to capture the structural properties of the cationic side-chains around the -Cys22-Pro23-Phe24-Cys25- sequence in the pGrx active site. Examples include Arg26, which can swing to stack on this sequence, and Lys19 which can contact the thiolate. However, contrary to a commonly held hypothesis, these cationic side-chains provide little stabilization for the thiolate, implying that they affect the enzymatic activity via other mechanisms. The pK(a) value of nucleophilic cysteine 22 (pK(a)(22)) is dominated by local hydrogen-bonds, formed only in a well-defined active-site conformation, supported by a comparison between the calculated and experimental values of pK(a)(22). The edge of the aromatic ring of Phe24 is polar enough to contribute to stabilize the thiolate, consistent with the conserved aromatic side-chain at this position in the glutaredoxin motif. The locality and directionality of the hydrogen bonds in the active site suffice to explain the vast difference between the pK(a) values of its two cysteine residues. A control of the cysteine pK(a) values by local hydrogen bonds implies that the peripheral ionized side-chains can evolve independently of the maintenance of these pK(a) values, maybe guided instead by substrate recognition. Comparison with other glutaredoxins indicates that the calculated pK(a) values of the N-terminal cysteine are better conserved than those of the C-terminal cysteine. Overall, a methodological strategy to systematically compare all reduced enzymes of this family emerges.     

Catalysis of thiol/disulfide exchange. Glutaredoxin 1 and protein-disulfide isomerase use different mechanisms to enhance oxidase and reductase activities

Glutaredoxin (Grx) and protein-disulfide isomerase (PDI) are members of the thioredoxin superfamily of thiol/disulfide exchange catalysts. Thermodynamically, rat PDI is a 600-fold better oxidizing agent than Grx1 from Escherichia coli. Despite that, Grx1 is a surprisingly good protein oxidase. It catalyzes protein disulfide formation in a redox buffer with an initial velocity that is 30-fold faster than PDI. Catalysis of protein and peptide oxidation by the individual catalytic domains of PDI and by a Grx1-PDI chimera show that differences in active site chemistry are fundamental to their oxidase activity. Mutations in the active site cysteines reveal that Grx1 needs only one cysteine to catalyze rapid substrate oxidation, whereas PDI requires both cysteines. Grx1 is a good oxidase because of the high reactivity of a Grx1-glutathione mixed disulfide, and PDI is a good oxidase because of the high reactivity of the disulfide between the two active site cysteines. As a protein disulfide reductase, Grx1 is also superior to PDI. It catalyzes the reduction of nonnative disulfides in scrambled ribonuclease and protein-glutathione mixed disulfides 30-180 times faster than PDI. A multidomain structure is necessary for PDI to catalyze effective protein reduction; however, placing Grx1 into the PDI multidomain structure does not enhance its already high reductase activity. Grx1 and PDI have both found mechanisms to enhance active site reactivity toward proteins, particularly in the kinetically difficult direction: Grx1 by providing a reactive glutathione mixed disulfide to supplement its oxidase activity and PDI by utilizing its multidomain structure to supplement its reductase activity.     

Direct NMR observation of the Cys-14 thiol proton of reduced Escherichia coli glutaredoxin-3 supports the presence of an active site thiol-thiolate hydrogen bond.

The active site of Escherichia coli glutaredoxin-3 (Grx3) consists of two redox active cysteine residues in the sequence -C11-P-Y-C14-H-. The 1H NMR resonance of the cysteine thiol proton of Cys-14 in reduced Grx3 is observed at 7.6 ppm. The large downfield shift and NOEs observed with this thiol proton resonance suggest the presence of a hydrogen bond with the Cys-11 thiolate, which is shown to have an abnormally low pKa value. A hydrogen bond would also agree with activity data of Grx3 active site mutants. Furthermore, the activity is reduced in a Grx3 H15V mutant, indicating electrostatic contributions to the stabilization of the Cys-11 thiolate.     

Human mitochondrial glutaredoxin reduces S-glutathionylated proteins with high affinity accepting electrons from either glutathione or thioredoxin reductase

Glutaredoxins catalyze glutathione-dependent thiol disulfide oxidoreductions via a GSH-binding site and active cysteines. Recently a second human glutaredoxin (Grx2), which is targeted to either mitochondria or the nucleus, was cloned. Grx2 contains the active site sequence CSYC, which is different from the conserved CPYC motif present in the cytosolic Grx1. Here we have compared the activity of Grx2 and Grx1 using glutathionylated substrates and active site mutants. The kinetic studies showed that Grx2 catalyzes the reduction of glutathionylated substrates with a lower rate but higher affinity compared with Grx1, resulting in almost identical catalytic efficiencies (k(cat)/K(m)). Permutation of the active site motifs of Grx1 and Grx2 revealed that the CSYC sequence of Grx2 is a prerequisite for its high affinity toward glutathionylated proteins, which comes at the price of lower k(cat). Furthermore Grx2 was a substrate for NADPH and thioredoxin reductase, which efficiently reduced both the active site disulfide and the GSH-glutaredoxin intermediate formed in the reduction of glutathionylated substrates. Using this novel electron donor pathway, Grx2 reduced low molecular weight disulfides such as CoA but with particular high efficiency glutathionylated substrates including GSSG. These results suggest an important role for Grx2 in protection and recovery from oxidative stress.     

Characterization of the human monothiol glutaredoxin 3 (PICOT) as iron-sulfur protein

Mammalian glutaredoxin 3 (Grx3/PICOT) is an essential protein involved in the regulation of signal transduction, for instance during immune cell activation and development of cardiac hypertrophy, presumably in response to redox signals. This function requires the sensing of such stresses by a hitherto unknown mechanism. Here, we characterized Grx3/PICOT as iron-sulfur protein. The protein binds two bridging [2Fe-2S] clusters in a homodimeric complex with the active site cysteinyl residues of its two monothiol glutaredoxin domains and glutathione bound non-covalently to the Grx domains. Co-immunoprecipitation of 55-iron with Grx3/PICOT from Jurkat cells suggested the presence of these cofactors under physiological conditions. The [2Fe-2S]²⁺ clusters were not redox active, instead they were lost upon treatment of the holo protein with ferricyanide or S-nitroso glutathione. This redox-induced dissociation of the Grx3/PICOT holo complex may be a mechanism of Grx3/PICOT activation in response to reactive oxygen and nitrogen species.     

A novel monothiol glutaredoxin (Grx4) from Escherichia coli can serve as a substrate for thioredoxin reductase

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750-2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3',5'-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.     

Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae

Glutaredoxins are members of a superfamily of thiol disulfide oxidoreductases involved in maintaining the redox state of target proteins. In Saccharomyces cerevisiae, two glutaredoxins (Grx1 and Grx2) containing a cysteine pair at the active site had been characterized as protecting yeast cells against oxidative damage. In this work, another subfamily of yeast glutaredoxins (Grx3, Grx4, and Grx5) that differs from the first in containing a single cysteine residue at the putative active site is described. This trait is also characteristic for a number of glutaredoxins from bacteria to humans, with which the Grx3/4/5 group has extensive homology over two regions. Mutants lacking Grx5 are partially deficient in growth in rich and minimal media and also highly sensitive to oxidative damage caused by menadione and hydrogen peroxide. A significant increase in total protein carbonyl content is constitutively observed in grx5 cells, and a number of specific proteins, including transketolase, appear to be highly oxidized in this mutant. The synthetic lethality of the grx5 and grx2 mutations on one hand and of grx5 with the grx3 grx4 combination on the other points to a complex functional relationship among yeast glutaredoxins, with Grx5 playing a specially important role in protection against oxidative stress both during ordinary growth conditions and after externally induced damage. Grx5-deficient mutants are also sensitive to osmotic stress, which indicates a relationship between the two types of stress in yeast cells.     

Oxidation and S-nitrosylation of cysteines in human cytosolic and mitochondrial glutaredoxins: effects on structure and activity

Glutathione (GSH) is the major intracellular thiol present in 1-10-mm concentrations in human cells. However, the redox potential of the 2GSH/GSSG (glutathione disulfide) couple in cells varies in association with proliferation, differentiation, or apoptosis from -260 mV to -200 or -170 mV. Hydrogen peroxide is transiently produced as second messenger in receptor-mediated growth factor signaling. To understand oxidation mechanisms by GSSG or nitric oxide-related nitrosylation we studied effects on glutaredoxins (Grx), which catalyze GSH-dependent thiol-disulfide redox reactions, particularly reversible glutathionylation of protein sulfhydryl groups. Human Grx1 and Grx2 contain Cys-Pro-Tyr-Cys and Cys-Ser-Tyr-Cys active sites and have three and two additional structural Cys residues, respectively. We analyzed the redox state and disulfide pairing of Cys residues upon GSSG oxidation and S-nitrosylation. Cytosolic/nuclear Grx1 was partly inactivated by both S-nitrosylation and oxidation. Inhibition by nitrosylation was reversible under anaerobic conditions; aerobically it was stronger and irreversible, indicating inactivation by nitration. Oxidation of Grx1 induced a complex pattern of disulfide-bonded dimers and oligomers formed between Cys-8 and either Cys-79 or Cys-83. In addition, an intramolecular disulfide between Cys-79 and Cys-83 was identified, predicted to have a profound effect on the three-dimensional structure. In contrast, mitochondrial Grx2 retains activity upon oxidation, did not form disulfide-bonded dimers or oligomers, and could not be S-nitrosylated. The dimeric iron sulfur cluster-coordinating inactive form of Grx2 dissociated upon nitrosylation, leading to activation of the protein. The striking differences between Grx1 and Grx2 reflect their diverse regulatory functions in vivo and also adaptation to different subcellular localization.     

Identification and characterization of a new mammalian glutaredoxin (thioltransferase), Grx2

A thiol/disulfide oxidoreductase component of the GSH system, glutaredoxin (Grx), is involved in the reduction of GSH-based mixed disulfides and participates in a variety of cellular redox pathways. A single cytosolic Grx (Grx1) was previously described in mammals. We now report identification and characterization of a second mammalian Grx, designated Grx2. Grx2 exhibited 36% identity with Grx1 and had a disulfide active center containing the Cys-Ser-Tyr-Cys motif. Grx2 was encoded in the genomes of mammals and birds and expressed in a variety of cell types. The gene for human Grx2 consisted of four exons and three introns, spanned 10 kilobase pairs, and localized to chromosome 1q31.2-31.3. The coding sequence was present in all exons, with the first exon encoding a mitochondrial signal peptide. The mitochondrial leader sequence was also present in mouse and rat Grx2 sequences and was shown to direct either Grx2 or green fluorescent protein to mitochondria. Alternative splicing forms of mammalian Grx2 mRNAs were identified that differed in sequences upstream of exon 2. To functionally characterize the new protein, human and mouse Grx2 proteins were expressed in Escherichia coli, and the purified proteins were shown to reduce mixed disulfides formed between GSH and S-sulfocysteine, hydroxyethyldisulfide, or cystine. Grx1 and Grx2 were sensitive to inactivation by iodoacetamide and H(2)O(2) and exhibited similar pH dependence of catalytic activity. However, H(2)O(2)-inactivated Grx2 could only be reactivated with 5 mm GSH, whereas Grx1 could also be reactivated with dithiothreitol or thioredoxin/thioredoxin reductase. The Grx2 structural model suggested a common reaction mechanism for this class of proteins. The data provide the first example of a mitochondrial Grx and also indicate the occurrence of a second functional Grx in mammals.     

The Synechocystis PCC6803 MerA-Like Enzyme Operates in the Reduction of Both Mercury and Uranium under the Control of the Glutaredoxin 1 Enzyme

In a continuing effort to analyze the selectivity/redundancy of the three glutaredoxin (Grx) enzymes of the model cyanobacterium 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 Grx1 (Slr1562 in CyanoBase) selectively interacts with the presumptive mercuric reductase protein (Slr1849). This MerA enzyme plays a crucial role in cell defense against both mercuric and uranyl ions, in catalyzing their NADPH-driven reduction. Like MerA, Grx1 operates in cell protection against both mercury and uranium. The Grx1-MerA interaction requires cysteine 86 (C86) of Grx1 and C78 of MerA, which is critical for its reductase activity. MerA can be inhibited by glutathionylation and subsequently reactivated by Grx1, likely through deglutathionylation. The two Grx1 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.     

Structural basis for the different activities of yeast Grx1 and Grx2

Yeast glutaredoxins Grx1 and Grx2 catalyze the reduction of both inter- and intra-molecular disulfide bonds using glutathione (GSH) as the electron donor. Although sharing the same dithiolic CPYC active site and a sequence identity of 64%, they have been proved to play different roles during oxidative stress and to possess different glutathione-disulfide reductase activities. To address the structural basis of these differences, we solved the crystal structures of Grx2 in oxidized and reduced forms, at 2.10 Å and 1.50 Å, respectively. With the Grx1 structures we previously reported, comparative structural analyses revealed that Grx1 and Grx2 share a similar GSH binding site, except for a single residue substitution from Asp89 in Grx1 to Ser123 in Grx2. Site-directed mutagenesis in combination with activity assays further proved this single residue variation is critical for the different activities of yeast Grx1 and Grx2.     

A mammalian monothiol glutaredoxin, Grx3, is critical for cell cycle progression during embryogenesis

Glutaredoxins (Grxs) have been shown to be critical in maintaining redox homeostasis in living cells. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs) has been identified. However, the biological and physiological functions of this group of proteins have not been well characterized. Here, we characterize a mammalian monothiol Grx (Grx3, also termed TXNL2/PICOT) with high similarity to yeast ScGrx3/ScGrx4. In yeast expression assays, mammalian Grx3s were localized to the nuclei and able to rescue growth defects of grx3grx4 cells. Furthermore, Grx3 inhibited iron accumulation in yeast grx3gxr4 cells and suppressed the sensitivity of mutant cells to exogenous oxidants. In mice, Grx3 mRNA was ubiquitously expressed in developing embryos, adult tissues and organs, and was induced during oxidative stress. Mouse embryos absent of Grx3 grew smaller with morphological defects and eventually died at 12.5 days of gestation. Analysis in mouse embryonic fibroblasts revealed that Grx3(-/-) cells had impaired growth and cell cycle progression at the G(2) /M phase, whereas the DNA replication during the S phase was not affected by Grx3 deletion. Furthermore, Grx3-knockdown HeLa cells displayed a significant delay in mitotic exit and had a higher percentage of binucleated cells. Therefore, our findings suggest that the mammalian Grx3 has conserved functions in protecting cells against oxidative stress and deletion of Grx3 in mice causes early embryonic lethality which could be due to defective cell cycle progression during late mitosis.     

A dithiol glutaredoxin cDNA from sweet potato (Ipomoea batatas [L.] Lam): enzyme properties and kinetic studies

Glutaredoxins (Grx) play an important role in reduction of protein glutathione mixed disulphides. An IbGrx cDNA (561 bp, EF362614 ) encoding a putative dithiol Grx was cloned from sweet potato (Ipomoea batatas [L.] Lam). The deduced amino acid sequence is conserved among the reported dithiol Grx, having a CGYC dithiol motif at the active site. A 3‐D structural model was created based on the known crystal structure of a poplar Grx (GrxC1). To characterise the IbGrx protein, the coding region was subcloned into an expression vector and transformed into Escherichia coli. The recombinant His₆‐tagged IbGrx was expressed and purified by metal affinity chromatography. The purified enzyme showed a monomeric band, as demonstrated with 15% SDS‐PAGE. The Michaelis constant (K_M) for ß‐hydroxyethyl disulphide (HED) was 0.50 ± 0.08 Mm . The enzyme retained 60% activity at 80 °C for 16 min. The enzyme was active over a broad pH range from 6.0 to 11.0, and in the presence of imidazole up to 0.4 M . The enzyme was susceptible to protease.     

Protective effects of the thioredoxin and glutaredoxin systems in dopamine-induced cell death

Although the etiology of sporadic Parkinson disease (PD) is unknown, it is well established that oxidative stress plays an important role in the pathogenic mechanism. The thioredoxin (Trx) and glutaredoxin (Grx) systems are two central systems upholding the sulfhydryl homeostasis by reducing disulfides and mixed disulfides within the cell and thereby protecting against oxidative stress. By examining the expression of redox proteins in human postmortem PD brains, we found the levels of Trx1 and thioredoxin reductase 1 (TrxR1) to be significantly decreased. The human neuroblastoma cell line SH-SY5Y and the nematode Caenorhabditis elegans were used as model systems to explore the potential protective effects of the redox proteins against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. 6-OHDA is highly prone to oxidation, resulting in the formation of the quinone of 6-OHDA, a highly reactive species and powerful neurotoxin. Treatment of human cells with 6-OHDA resulted in an increased expression of Trx1, TrxR1, Grx1, and Grx2, and small interfering RNA for these genes significantly increased the cytotoxic effects exerted by the 6-OHDA neurotoxin. Evaluation of the dopaminergic neurons in C. elegans revealed that nematodes lacking trxr-1 were significantly more sensitive to 6-OHDA, with significantly increased neuronal degradation. Importantly, both the Trx and the Grx systems were also found to directly mediate reduction of the 6-OHDA-quinone in vitro and thus render its cytotoxic effects. In conclusion, our results suggest that the two redox systems are important for neuronal survival in dopamine-induced cell death.     

Glutathionylation of the Active Site Cysteines of Peroxiredoxin 2 and Recycling by Glutaredoxin

Peroxiredoxin 2 (Prx2) is a thiol protein that functions as an antioxidant, regulator of cellular peroxide concentrations, and sensor of redox signals. Its redox cycle is widely accepted to involve oxidation by a peroxide and reduction by thioredoxin/thioredoxin reductase. Interactions of Prx2 with other thiols are not well characterized. Here we show that the active site Cys residues of Prx2 form stable mixed disulfides with glutathione (GSH). Glutathionylation was reversed by glutaredoxin 1 (Grx1), and GSH plus Grx1 was able to support the peroxidase activity of Prx2. Prx2 became glutathionylated when its disulfide was incubated with GSH and when the reduced protein was treated with H₂O₂ and GSH. The latter reaction occurred via the sulfenic acid, which reacted sufficiently rapidly (k = 500 m⁻¹ s⁻¹) for physiological concentrations of GSH to inhibit Prx disulfide formation and protect against hyperoxidation to the sulfinic acid. Glutathionylated Prx2 was detected in erythrocytes from Grx1 knock-out mice after peroxide challenge. We conclude that Prx2 glutathionylation is a favorable reaction that can occur in cells under oxidative stress and may have a role in redox signaling. GSH/Grx1 provide an alternative mechanism to thioredoxin and thioredoxin reductase for Prx2 recycling.