Jérémy Couturier

Glutaredoxins: roles in iron homeostasis

Glutaredoxins, proteins traditionally involved in redox reactions, are also required for iron-sulfur cluster assembly and haem biosynthesis. These new roles are probably related to the ability of some glutaredoxins to bind labile [2Fe-2S] clusters and to transfer them rapidly and efficiently to acceptor proteins. Recent results point to putative roles for glutaredoxins in the sensing of cellular iron and in iron-sulfur cluster biogenesis, either as scaffold proteins for the de novo synthesis of iron-sulfur clusters or as carrier proteins for the transfer of preformed iron-sulfur clusters. Based on prokaryote genome analysis and in vivo studies of iron regulation in yeast, we propose putative new roles and binding partners for glutaredoxins in the assembly of metalloproteins.

Evolution and diversity of glutaredoxins in photosynthetic organisms

The genome sequencing of prokaryotic and eukaryotic photosynthetic organisms enables a comparative genomic study of the glutaredoxin (Grx) family. The analysis of 58 genomes, using a specific motif composed of the active site sequence and of amino acids involved in glutathione binding, led to an updated classification of Grxs into six classes. Only two classes (I and II) are common to all photosynthetic organisms. Eukaryotes and cyanobacteria have two specific Grx classes (classes III and IV and classes V and VI, respectively). The classes IV, V and VI have not yet been identified and contain multimodular Grx fusions. In addition, putative Grx partners were identified from the presence of fusion proteins, the conservation of gene order in bacterial operons, and the gene co-occurrence. The genes encoding class II Grxs and BolA/YrbA proteins are frequently adjacent, in the same transcriptional orientation in prokaryote genomes and present in the same organisms.

The iron-sulfur cluster assembly machineries in plants: current knowledge and open questions

Many metabolic pathways and cellular processes occurring in most sub-cellular compartments depend on the functioning of iron-sulfur (Fe-S) proteins, whose cofactors are assembled through dedicated protein machineries. Recent advances have been made in the knowledge of the functions of individual components through a combination of genetic, biochemical and structural approaches, primarily in prokaryotes and non-plant eukaryotes. Whereas most of the components of these machineries are conserved between kingdoms, their complexity is likely increased in plants owing to the presence of additional assembly proteins and to the existence of expanded families for several assembly proteins. This review focuses on the new actors discovered in the past few years, such as glutaredoxin, BOLA and NEET proteins as well as MIP18, MMS19, TAH18, DRE2 for the cytosolic machinery, which are integrated into a model for the plant Fe-S cluster biogenesis systems. It also discusses a few issues currently subjected to an intense debate such as the role of the mitochondrial frataxin and of glutaredoxins, the functional separation between scaffold, carrier and iron-delivery proteins and the crosstalk existing between different organelles.

Cysteine–based redox regulation and signaling in plants

Living organisms are subjected to oxidative stress conditions which are characterized by the production of reactive oxygen, nitrogen, and sulfur species. In plants as in other organisms, many of these compounds have a dual function as they damage different types of macromolecules but they also likely fulfil an important role as secondary messengers. Owing to the reactivity of their thiol groups, some protein cysteine residues are particularly prone to oxidation by these molecules. In the past years, besides their recognized catalytic and regulatory functions, the modification of cysteine thiol group was increasingly viewed as either protective or redox signaling mechanisms. The most physiologically relevant reversible redox post-translational modifications (PTMs) are disulfide bonds, sulfenic acids, S-glutathione adducts, S-nitrosothiols and to a lesser extent S-sulfenyl-amides, thiosulfinates and S-persulfides. These redox PTMs are mostly controlled by two oxidoreductase families, thioredoxins and glutaredoxins. This review focuses on recent advances highlighting the variety and physiological roles of these PTMs and the proteomic strategies used for their detection.

Structure-function relationship of the chloroplastic glutaredoxin S12 with an atypical WCSYS active site.

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 GrxS12, which possesses a monothiol 28WCSYS32 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 (Cys87) being dispensable, since the C87S variant is fully active in all activity assays. Biochemical and crystallographic studies revealed that Cys87 exhibits a certain reactivity, since its pKa is around 5.6. Coupled with thiol titration, fluorescence, and mass spectrometry analyses, the resolution of poplar GrxS12 x-ray crystal structure shows that the only oxidation state is a glutathionylated derivative of the active site cysteine (Cys29) and that the enzyme does not form inter- or intramolecular disulfides. Contrary to some plant Grxs, GrxS12 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.

Glutathionylation of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the model plant Arabidopsis thaliana is reversed by both glutaredoxins and thioredoxins in vitro.

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 Cys(149). Catalytic Cys(149) is the only solvent-exposed cysteine of the protein and its thiol is relatively acidic (pK(a)=5.7). This property makes GapC1 sensitive to oxidation by H(2)O(2), which appears to inhibit enzyme activity by converting the thiolate of Cys(149) (-S-) into irreversible oxidized forms (-SO(2)(-) and -SO(3)(-)) via a labile sulfenate intermediate (-SO(-)). GSH (reduced glutathione) prevents this irreversible process by reacting with Cys(149) sulfenates to give rise to a mixed disulfide (Cys(149)-SSG), as demonstrated by both MS and biotinylated GSH. Glutathionylated GapC1 can be fully reactivated either by cytosolic glutaredoxin, via a GSH-dependent 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 Cys(149).

Monothiol glutaredoxins and A-type proteins: Partners in Fe-S cluster trafficking

Monothiol glutaredoxins (Grxs) are proposed to function in Fe-S cluster storage and delivery, based on their ability to exist as apo monomeric forms and dimeric forms containing a subunit-bridging [Fe(2)S(2)](2+) cluster, and to accept [Fe(2)S(2)](2+) clusters from primary scaffold proteins. In addition yeast cytosolic monothiol Grxs interact with Fra2 (Fe repressor of activation-2), to form a heterodimeric complex with a bound [Fe(2)S(2)](2+) cluster that plays a key role in iron sensing and regulation of iron homeostasis. In this work, we report on in vitro UV-visible CD studies of cluster transfer between homodimeric monothiol Grxs and members of the ubiquitous A-type class of Fe-S cluster carrier proteins ((Nif)IscA and SufA). The results reveal rapid, unidirectional, intact and quantitative cluster transfer from the [Fe(2)S(2)](2+) cluster-bound forms of A. thaliana GrxS14, S. cerevisiae Grx3, and A. vinelandii Grx-nif homodimers to A. vinelandii(Nif)IscA and from A. thaliana GrxS14 to A. thaliana SufA1. Coupled with in vivo evidence for interaction between monothiol Grxs and A-type Fe-S cluster carrier proteins, the results indicate that these two classes of proteins work together in cellular Fe-S cluster trafficking. However, cluster transfer is reversed in the presence of Fra2, since the [Fe(2)S(2)](2+) cluster-bound heterodimeric Grx3-Fra2 complex can be formed by intact [Fe(2)S(2)](2+) cluster transfer from (Nif)IscA. The significance of these results for Fe-S cluster biogenesis or repair and the cellular regulation of the Fe-S cluster status are discussed.

Arabidopsis Chloroplastic Glutaredoxin C5 as a Model to Explore Molecular Determinants for Iron-Sulfur Cluster Binding into Glutaredoxins

Unlike thioredoxins, glutaredoxins are involved in iron-sulfur cluster assembly and in reduction of specific disulfides (i.e. protein-glutathione adducts), and thus they are also important redox regulators of chloroplast metabolism. Using GFP fusion, AtGrxC5 isoform, present exclusively in Brassicaceae, was shown to be localized in chloroplasts. A comparison of the biochemical, structural, and spectroscopic properties of Arabidopsis GrxC5 (WCSYC active site) with poplar GrxS12 (WCSYS active site), a chloroplastic paralog, indicated that, contrary to the solely apomonomeric GrxS12 isoform, AtGrxC5 exists as two forms when expressed in Escherichia coli. The monomeric apoprotein possesses deglutathionylation activity mediating the recycling of plastidial methionine sulfoxide reductase B1 and peroxiredoxin IIE, whereas the dimeric holoprotein incorporates a [2Fe-2S] cluster. Site-directed mutagenesis experiments and resolution of the x-ray crystal structure of AtGrxC5 in its holoform revealed that, although not involved in its ligation, the presence of the second active site cysteine (Cys32) is required for cluster formation. In addition, thiol titrations, fluorescence measurements, and mass spectrometry analyses showed that, despite the presence of a dithiol active site, AtGrxC5 does not form any inter- or intramolecular disulfide bond and that its activity exclusively relies on a monothiol mechanism.

The roles of glutaredoxins ligating Fe-S clusters: Sensing, transfer or repair functions?

Glutaredoxins (Grxs) are major oxidoreductases involved in the reduction of glutathionylated proteins. Owing to the capacity of several class I Grxs and likely all class II Grxs to incorporate iron-sulfur (Fe-S) clusters, they are also linked to iron metabolism. Most Grxs bind [2Fe-2S] clusters which are oxidatively- and reductively-labile and have identical ligation, involving notably external glutathione. However, subtle differences in the structural organization explain that class II Fe-S Grxs, having more labile and solvent-exposed clusters, can accept Fe-S clusters and transfer them to client proteins, whereas class I Fe-S Grxs usually do not. From the observed glutathione disulfide-mediated Fe-S cluster degradation, the current view is that the more stable Fe-S clusters found in class I Fe-S Grxs might constitute a sensor of oxidative stress conditions by modulating their activity. Indeed, in response to an oxidative signal, inactive holoforms i.e., without disulfide reductase activity, should be converted to active apoforms. Among class II Fe-S Grxs, monodomain Grxs likely serve as carrier proteins for the delivery of preassembled Fe-S clusters to acceptor proteins in organelles. Another proposed function is the repair of Fe-S clusters. From their cytoplasmic and/or nuclear localization, multidomain Grxs function in signalling pathways. In particular, they regulate iron homeostasis in yeast species by modulating the activity of transcription factors and eventually forming heterocomplexes with BolA-like proteins in response to the cellular iron status. We provide an overview of the biochemical and structural properties of Fe-S cluster-loaded Grxs in relation to their hypothetical or confirmed associated functions. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

Glutaredoxin S12: Unique Properties for Redox Signaling

AIMS Cysteines (Cys) made acidic by the protein environment are generally sensitive to pro-oxidant molecules. Glutathionylation is a post-translational modification that can occur by spontaneous reaction of reduced glutathione (GSH) with oxidized Cys as sulfenic acids (-SOH). The reverse reaction (deglutathionylation) is strongly stimulated by glutaredoxins (Grx) and requires a reductant, often GSH. RESULTS Here, we show that chloroplast GrxS12 from poplar efficiently reacts with glutathionylated substrates in a GSH-dependent ping pong mechanism. The pK(a) of GrxS12 catalytic Cys is very low (3.9) and makes GrxS12 itself sensitive to oxidation by H(2)O(2) and to direct glutathionylation by nitrosoglutathione. Glutathionylated-GrxS12 (GrxS12-SSG) is temporarily inactive until it is deglutathionylated by GSH. The equilibrium between GrxS12 and glutathione (E(m(GrxS12-SSG))= -315 mV, pH 7.0) is characterized by K(ox) values of 310 at pH 7.0, as in darkened chloroplasts, and 69 at pH 7.9, as in illuminated chloroplasts. INNOVATION Based on thermodynamic data, GrxS12-SSG is predicted to accumulate in vivo under conditions of mild oxidation of the GSH pool that may occur under stress. Moreover, GrxS12-SSG is predicted to be more stable in chloroplasts in the dark than in the light. CONCLUSION These peculiar catalytic and thermodynamic properties could allow GrxS12 to act as a stress-related redox sensor, thus allowing glutathione to play a signaling role through glutathionylation of GrxS12 target proteins.