AtGRXcp, an Arabidopsis chloroplastic glutaredoxin, is critical for protection against protein oxidative damage

Glutaredoxins (Grxs) are ubiquitous small heat-stable disulfide oxidoreductases and members of the thioredoxin (Trx) fold protein family. In bacterial, yeast, and mammalian cells, Grxs appear to be involved in maintaining cellular redox homeostasis. However, in plants, the physiological roles of Grxs have not been fully characterized. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs) has been identified but not well characterized. Here we demonstrate that a plant protein, AtGRXcp, is a chloroplast-localized monothiol Grx with high similarity to yeast Grx5. In yeast expression assays, AtGRXcp localized to the mitochondria and suppressed the sensitivity of yeast grx5 cells to H2O2 and protein oxidation. AtGRXcp expression can also suppress iron accumulation and partially rescue the lysine auxotrophy of yeast grx5 cells. Analysis of the conserved monothiol motif suggests that the cysteine residue affects AtGRXcp expression and stability. In planta, AtGRXcp expression was elevated in young cotyledons, green tissues, and vascular bundles. Analysis of atgrxcp plants demonstrated defects in early seedling growth under oxidative stresses. In addition, atgrxcp lines displayed increased protein carbonylation within chloroplasts. Thus, this work describes the initial functional characterization of a plant monothiol Grx and suggests a conserved biological function in protecting cells against protein oxidative damage.     

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.     

Corynebacterium glutamicum Methionine Sulfoxide Reductase A Uses both Mycoredoxin and Thioredoxin for Regeneration and Oxidative Stress Resistance

Oxidation of methionine leads to the formation of the S and R diastereomers of methionine sulfoxide (MetO), which can be reversed by the actions of two structurally unrelated classes of methionine sulfoxide reductase (Msr), MsrA and MsrB, respectively. Although MsrAs have long been demonstrated in numerous bacteria, their physiological and biochemical functions remain largely unknown in Actinomycetes. Here, we report that a Corynebacterium glutamicum methionine sulfoxide reductase A (CgMsrA) that belongs to the 3-Cys family of MsrAs plays important roles in oxidative stress resistance. Deletion of the msrA gene in C. glutamicum resulted in decrease of cell viability, increase of ROS production, and increase of protein carbonylation levels under various stress conditions. The physiological roles of CgMsrA in resistance to oxidative stresses were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic-function (ECF) sigma factor SigH. Activity assays performed with various regeneration pathways showed that CgMsrA can reduce MetO via both the thioredoxin/thioredoxin reductase (Trx/TrxR) and mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/Mtr/MSH) pathways. Site-directed mutagenesis confirmed that Cys56 is the peroxidatic cysteine that is oxidized to sulfenic acid, while Cys204 and Cys213 are the resolving Cys residues that form an intramolecular disulfide bond. Mrx1 reduces the sulfenic acid intermediate via the formation of an S-mycothiolated MsrA intermediate (MsrA-SSM) which is then recycled by mycoredoxin and the second molecule of mycothiol, similarly to the glutathione/glutaredoxin/glutathione reductase (GSH/Grx/GR) system. However, Trx reduces the Cys204-Cys213 disulfide bond in CgMsrA produced during MetO reduction via the formation of a transient intermolecular disulfide bond between Trx and CgMsrA. While both the Trx/TrxR and Mrx1/Mtr/MSH pathways are operative in reducing CgMsrA under stress conditions in vivo, the Trx/TrxR pathway alone is sufficient to reduce CgMsrA under normal conditions. Based on these results, a catalytic model for the reduction of CgMsrA by Mrx1 and Trx is proposed.     

Structure-function analysis of yeast Grx5 monothiol glutaredoxin defines essential amino acids for the function of the protein

Grx5 defines a family of yeast monothiol glutaredoxins that also includes Grx3 and Grx4. All three proteins display significant sequence homology with proteins found from bacteria to humans. Grx5 is involved in iron/sulfur cluster assembly at the mitochondria, but the function of Grx3 and Grx4 is unknown. Three-dimensional modeling based on known dithiol glutaredoxin structures predicted a thioredoxin fold structure for Grx5. Positionally conserved amino acids in this glutaredoxin family were replaced in Grx5, and the effect on the biological function of the protein has been tested. For all changes studied, there was a correlation between the effects on several different phenotypes: sensitivity to oxidants, constitutive protein oxidation, ability for respiratory growth, auxotrophy for a number of amino acids, and iron accumulation. Cys(60) and Gly(61) are essential for Grx5 function, whereas other single or double substitutions in the same region had no phenotypic effects. Gly(115) and Gly(116) could be important for the formation of a glutathione cleft on the Grx5 surface, in contrast to adjacent Cys(117). Substitution of Phe(50) alters the beta-sheet in the thioredoxin fold structure and inhibits Grx5 function. None of the substitutions tested affect the structure at a significant enough level to reduce protein stability.     

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.     

Evolution and cellular function of monothiol glutaredoxins: involvement in iron-sulphur cluster assembly

A number of bacterial species, mostly proteobacteria, possess monothiol glutaredoxins homologous to the Saccharomyces cerevisiae mitochondrial protein Grx5, which is involved in iron-sulphur cluster synthesis. Phylogenetic profiling is used to predict that bacterial monothiol glutaredoxins also participate in the iron-sulphur cluster (ISC) assembly machinery, because their phylogenetic profiles are similar to the profiles of the bacterial homologues of yeast ISC proteins. High evolutionary co-occurrence is observed between the Grx5 homologues and the homologues of the Yah1 ferredoxin, the scaffold proteins Isa1 and Isa2, the frataxin protein Yfh1 and the Nfu1 protein. This suggests that a specific functional interaction exists between these ISC machinery proteins. Physical interaction analyses using low-definition protein docking predict the formation of strong and specific complexes between Grx5 and several components of the yeast ISC machinery. Two-hybrid analysis has confirmed the in vivo interaction between Grx5 and Isa1. Sequence comparison techniques and cladistics indicate that the other two monothiol glutaredoxins of S. cerevisiae, Grx3 and Grx4, have evolved from the fusion of a thioredoxin gene with a monothiol glutaredoxin gene early in the eukaryotic lineage, leading to differential functional specialization. While bacteria do not contain these chimaeric glutaredoxins, in many eukaryotic species Grx5 and Grx3/4-type monothiol glutaredoxins coexist in the cell.     

Cloning, functional analysis, and mitochondrial localization of Trypanosoma brucei monothiol glutaredoxin-1

African trypanosomes encode three monothiol glutaredoxins (1-C-Grx1 to 3). 1-C-Grx1 has a putative CAYS active site and Cys181 as single additional cysteine. The recombinant protein forms non-covalent homodimers. As observed for other monothiol glutaredoxins, Trypanosoma brucei 1-C-Grx1 was not active in the glutaredoxin assay with hydroxyethyl disulfide and glutathione nor catalyzed the reduction of insulin disulfide. In addition, it lacked peroxidase activity and did not catalyze protein (de)glutathionylation. Upon oxidation, 1-C-Grx1 forms an intramolecular disulfide bridge and, to a minor degree, covalent dimers. Both disulfide forms are reduced by the parasite trypanothione/tryparedoxin system. 1-C-Grx1 shows mitochondrial localization. The total cellular concentration is at least 5 microm. Thus, 1-C-Grx1 is an abundant protein especially in the rudimentary organelle of the mammalian form of the parasite. Expression of 1-C-Grx1 in Grx5-deficient yeast cells with its authentic presequence targeted the protein to the mitochondria and partially restored the growth phenotype and aconitase activity of the mutant, and conferred resistance against hydroperoxides and diamide. The parasite Grx2 and 3 failed to substitute for Grx5. This is surprising because even bacterial and plant 1-Cys-glutaredoxins efficiently revert the defects, and may be due to the lack of two basic residues conserved in all but the trypanosomatid proteins.     

Both thioredoxin 2 and glutaredoxin 2 contribute to the reduction of the mitochondrial 2-Cys peroxiredoxin Prx3

The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (K(m), 23.8 μmol·liter(-1); V(max), 1.2 μmol·(mg·min)(-1)) similar to Trx2 (K(m), 11.2 μmol·liter(-1); V(max), 1.1 μmol·(mg·min)(-1)). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms.     

A novel group of glutaredoxins in the cis-Golgi critical for oxidative stress resistance

Glutaredoxins represent a ubiquitous family of proteins that catalyze the reduction of disulfide bonds in their substrate proteins by use of reduced glutathione. In an attempt to identify the full complement of glutaredoxins in baker's yeast, we found three so-far uncharacterized glutaredoxin-like proteins that we named Grx6, Grx7, and Grx8. Grx6 and Grx7 represent closely related monothiol glutaredoxins that are synthesized with N-terminal signal sequences. Both proteins are located in the cis-Golgi, thereby representing the first glutaredoxins found in a compartment of the secretory pathway. In contrast to formerly described monothiol glutaredoxins, Grx6 and Grx7, showed a high glutaredoxin activity in vitro. Grx6 and Grx7 overlap in their activity and deletion mutants lacking both proteins show growth defects and a strongly increased sensitivity toward oxidizing agents such as hydrogen peroxide or diamide. Our observations suggest that Grx6 and Grx7 do not play a general role in the oxidative folding of proteins in the early secretory pathway but rather counteract the oxidation of specific thiol groups in substrate proteins.     

Binding specificity and mechanistic insight into glutaredoxin-catalyzed protein disulfide reduction.

The reduction equivalents necessary for the ribonucleotide reductase (RNR)-catalyzed production of deoxyribonucleotides are provided by glutaredoxin (Grx) or thioredoxin (Trx). The initial location for transfer of reducing equivalents to RNR is located at the C terminus of the B1 subunit and involves the reduction of a disulfide between Cys754 and Cys759. We have used a 25-mer peptide corresponding to residues 737-761 of RNR B1 (C754-->S) to synthesize a stable mixed disulfide with Escherichia coli Grx-1 (C14-->S) resembling the structure of an intermediate in the reaction. The high-resolution solution structure of the mixed disulfide has been obtained by NMR with an RMSD of 0.56 A for all the backbone atoms of the protein and the well-defined portion of the peptide. The binding interactions responsible for specificity have been identified demonstrating the importance of electrostatic interactions in this system and providing a rationale for the specificity of the Grx-RNR interaction. The disulfide is buried in this complex, implying a solely intra-molecular mechanism of reduction in contrast to the previously determined structure of the glutathione complex where the disulfide was exposed; mutagenesis studies have shown the relevance of intermolecular reduction processes. Substantial conformational changes in the helices of the protein are associated with peptide binding which have significant mechanistic implications for protein disulfide reduction by glutaredoxins.     

A genome-wide survey of human thioredoxin and glutaredoxin family pseudogenes

The thioredoxin/glutaredoxin family consists of small heat-stable proteins that have a highly conserved CXXC active site and that participate in the regulation of many redox reactions. We have searched the human genome sequence to find putative pseudogenes (non-functional copies of protein-coding genes) for all known members of this family. This survey has resulted in the identification of seven processed pseudogenes for human Trx1 and two more for human Grx1. No evidence for the presence of processed pseudogenes has been found for the remaining members of this family. In addition, we have been unable to detect any non-processed pseudogenes derived from any member of the family in the human genome. The seven thioredoxin pseudogenes can be divided into two groups: Trx1-psi2, -psi3, -psi4, -psi5 and -psi6 arose from the functional ancestor, whereas Trx1-psi1 and -psi7 originated from Trx1-psi2 and -psi6, respectively. In all cases, the pseudogenes originated after the human/rodent radiation as shown by phylogenetic analysis. This is also the case for Grx1-psi1 and Grx1-psi2, which are placed between rodent and human sequences in the phylogenetic tree. Our study provides a molecular record of the recent evolution of these two genes in the hominid lineage.     

Glutaredoxin-dependent peroxiredoxin from poplar: protein-protein interaction and catalytic mechanism

Recently, a poplar phloem peroxiredoxin (Prx) was found to accept both glutaredoxin (Grx) and thioredoxin (Trx) as proton donors. To investigate the catalytic mechanism of the Grx-dependent reduction of hydroperoxides catalyzed by Prx, a series of cysteinic mutants was constructed. Mutation of the most N-terminal conserved cysteine of Prx (Cys-51) demonstrates that it is the catalytic one. The second cysteine (Cys-76) is not essential for peroxiredoxin activity because the C76A mutant retained approximately 25% of the wild type Prx activity. Only one cysteine of the Grx active site (Cys-27) is essential for peroxiredoxin catalysis, indicating that Grx can act in this reaction either via a dithiol or a monothiol pathway. The creation of covalent heterodimers between Prx and Grx mutants confirms that Prx Cys-51 and Grx Cys-27 are the two residues involved in the catalytic mechanism. The integration of a third cysteine in position 152 of the Prx, making it similar in sequence to the Trx-dependent human Prx V, resulted in a protein that had no detectable activity with Grx but kept activity with Trx. Based on these experimental results, a catalytic mechanism is proposed to explain the Grx- and Trx-dependent activities of poplar Prx.     

Redox regulation of 3'-phosphoadenylylsulfate reductase from Escherichia coli by glutathione and glutaredoxins

Inorganic sulfate (SO42-, S+VI) is reduced in vivo to sulfite (SO32-, S+IV) via phosphoadenylylsulfate (PAPS) reductase. Escherichia coli lacking glutathione reductase and glutaredoxins (gor-grxA-grxB-grxC-) barely grows on sulfate. We found that incubation of PAPS reductase with oxidized glutathione leads to enzyme inactivation with simultaneous formation of a mixed disulfide between glutathione and the active site Cys-239. A newly developed method based on thiol-specific fluorescent alkylation and gel electrophoresis showed that glutathionylated PAPS reductase is reduced by glutaredoxins via a monothiol mechanism. This glutathionylated species was also observed in poorly growing gor-grxA-grxB-grxC- cells expressing inactive glutaredoxin 2 (Grx2) C9S/C12S. However, it was absent in better growing cells expressing monothiol Grx2 C12S or wild type Grx2. Reversible glutathionylation may thus regulate the activity of PAPS reductase in vivo.     

Structural and Biochemical Characterization of Yeast Monothiol Glutaredoxin Grx6

Glutaredoxins (Grxs) are a ubiquitous family of proteins that reduce disulfide bonds in substrate proteins using electrons from reduced glutathione (GSH). The yeast Saccharomyces cerevisiae Grx6 is a monothiol Grx that is localized in the endoplasmic reticulum and Golgi compartments. Grx6 consists of three segments, a putative signal peptide (M1-I36), an N-terminal domain (K37-T110), and a C-terminal Grx domain (K111-N231, designated Grx6C). Compared to the classic dithiol glutaredoxin Grx1, Grx6 has a lower glutathione disulfide reductase activity but a higher glutathione S-transferase activity. In addition, similar to human Grx2, Grx6 binds GSH via an iron-sulfur cluster in vitro. The N-terminal domain is essential for noncovalent dimerization, but not required for either of the above activities. The crystal structure of Grx6C at 1.5 Å resolution revealed a novel two-strand antiparallel β-sheet opposite the GSH binding groove. This extra β-sheet might also exist in yeast Grx7 and in a group of putative Grxs in lower organisms, suggesting that Grx6 might represent the first member of a novel Grx subfamily.     

Nuclear monothiol glutaredoxins of Saccharomyces cerevisiae can function as mitochondrial glutaredoxins

Glutaredoxins are thiol oxidoreductases that regulate protein redox state. In Saccharomyces cerevisiae, Grx1 and Grx2 are cytosolic dithiol glutaredoxins, whereas Grx3, Grx4, and Grx5 are monothiol glutaredoxins. Grx5 locates at the mitochondrial matrix and is needed for iron/sulfur cluster biogenesis. Its absence causes phenotypes such as inactivation of iron/sulfur enzymes and sensitivity to oxidative stress. Whereas Grx5 contains a single glutaredoxin domain, in Grx3 and Grx4 a thioredoxin-like domain is fused to the glutaredoxin domain. Here we have shown that Grx3 locates at the nucleus and that the thioredoxin-like domain is required for such location. We have addressed the functional divergence among glutaredoxins by targeting Grx2/3/4 molecules to the mitochondrial matrix using the Grx5 targeting sequence. The mitochondrial forms of Grx3 and Grx4 partially rescue the defects of a grx5 null mutant. On the contrary, mitochondrially targeted Grx2 does not suppress the mutant phenotype. Both the thioredoxin-like and glutaredoxin domains are needed for the mitochondrial activity of Grx3, although none of the cysteine residues at the thioredoxin-like domain is required for rescue of the grx5 phenotypes. We have concluded that dithiol glutaredoxins are functionally divergent from monothiol ones, but the latter can interchange their biological activities when compartment barriers are surpassed.