Ascorbate Peroxidase 2 (APX2) of Chlamydomonas Binds Copper and Modulates the Copper Insertion into Plastocyanin.

Recent phylogenetic studies have unveiled a novel class of ascorbate peroxidases called "ascorbate peroxidase-related" (APX-R). These enzymes, found in green photosynthetic eukaryotes, lack the amino acids necessary for ascorbate binding. This study focuses on the sole APX-R from Chlamydomonas reinhardtii referred to as ascorbate peroxidase 2 (APX2). We used immunoblotting to locate APX2 within the chloroplasts and in silico analysis to identify key structural motifs, such as the twin-arginine transport (TAT) motif for lumen translocation and the metal-binding MxxM motif. We also successfully expressed recombinant APX2 in Escherichia coli. Our in vitro results showed that the peroxidase activity of APX2 was detected with guaiacol but not with ascorbate as an electron donor. Furthermore, APX2 can bind both copper and heme, as evidenced by spectroscopic, and fluorescence experiments. These findings suggest a potential interaction between APX2 and plastocyanin, the primary copper-containing enzyme within the thylakoid lumen of the chloroplasts. Predictions from structural models and evidence from 1H-NMR experiments suggest a potential interaction between APX2 and plastocyanin, emphasizing the influence of APX2 on the copper-binding abilities of plastocyanin. In summary, our results propose a significant role for APX2 as a regulator in copper transfer to plastocyanin. This study sheds light on the unique properties of APX-R enzymes and their potential contributions to the complex processes of photosynthesis in green algae.

Mitochondrial ferredoxin-like is essential for forming complex I-containing supercomplexes in Arabidopsis.

In eukaryotes, mitochondrial ATP is mainly produced by the oxidative phosphorylation (OXPHOS) system, which is composed of 5 multiprotein complexes (complexes I-V). Analyses of the OXPHOS system by native gel electrophoresis have revealed an organization of OXPHOS complexes into supercomplexes, but their roles and assembly pathways remain unclear. In this study, we characterized an atypical mitochondrial ferredoxin (mitochondrial ferredoxin-like, mFDX-like). This protein was previously found to be part of the bridge domain linking the matrix and membrane arms of the complex I. Phylogenetic analysis suggested that the Arabidopsis (Arabidopsis thaliana) mFDX-like evolved from classical mitochondrial ferredoxins (mFDXs) but lost one of the cysteines required for the coordination of the iron-sulfur (Fe-S) cluster, supposedly essential for the electron transfer function of FDXs. Accordingly, our biochemical study showed that AtmFDX-like does not bind an Fe-S cluster and is therefore unlikely to be involved in electron transfer reactions. To study the function of mFDX-like, we created deletion lines in Arabidopsis using a CRISPR/Cas9-based strategy. These lines did not show any abnormal phenotype under standard growth conditions. However, the characterization of the OXPHOS system demonstrated that mFDX-like is important for the assembly of complex I and essential for the formation of complex I-containing supercomplexes. We propose that mFDX-like and the bridge domain are required for the correct conformation of the membrane arm of complex I that is essential for the association of complex I with complex III2 to form supercomplexes.

Protein lipoylation in mitochondria requires Fe-S cluster assembly factors NFU4 and NFU5.

Plants have evolutionarily conserved NifU (NFU)-domain proteins that are targeted to plastids or mitochondria. "Plastid-type" NFU1, NFU2, and NFU3 in Arabidopsis (Arabidopsis thaliana) play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here, we confirmed that NFU4 and NFU5 are targeted to the mitochondria. The proteins were constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of NFU4 and NFU5 proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes or aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth improved with elevated CO2 treatment. In addition, pyruvate, 2-oxoglutarate, and branched-chain amino acids accumulated in nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, most likely providing Fe-S clusters to lipoyl synthase.

Occurrence, Evolution and Specificities of Iron-Sulfur Proteins and Maturation Factors in Chloroplasts from Algae.

Iron-containing proteins, including iron-sulfur (Fe-S) proteins, are essential for numerous electron transfer and metabolic reactions. They are present in most subcellular compartments. In plastids, in addition to sustaining the linear and cyclic photosynthetic electron transfer chains, Fe-S proteins participate in carbon, nitrogen, and sulfur assimilation, tetrapyrrole and isoprenoid metabolism, and lipoic acid and thiamine synthesis. The synthesis of Fe-S clusters, their trafficking, and their insertion into chloroplastic proteins necessitate the so-called sulfur mobilization (SUF) protein machinery. In the first part, we describe the molecular mechanisms that allow Fe-S cluster synthesis and insertion into acceptor proteins by the SUF machinery and analyze the occurrence of the SUF components in microalgae, focusing in particular on the green alga Chlamydomonas reinhardtii. In the second part, we describe chloroplastic Fe-S protein-dependent pathways that are specific to Chlamydomonas or for which Chlamydomonas presents specificities compared to terrestrial plants, putting notable emphasis on the contribution of Fe-S proteins to chlorophyll synthesis in the dark and to the fermentative metabolism. The occurrence and evolutionary conservation of these enzymes and pathways have been analyzed in all supergroups of microalgae performing oxygenic photosynthesis.

Genome-wide analysis in response to nitrogen and carbon identifies regulators for root AtNRT2 transporters.

In Arabidopsis (Arabidopsis thaliana), the High-Affinity Transport System (HATS) for root nitrate (NO3-) uptake depends mainly on four NRT2 NO3- transporters, namely NRT2.1, NRT2.2, NRT2.4, and NRT2.5. The HATS is the target of many regulations to coordinate nitrogen (N) acquisition with the N status of the plant and with carbon (C) assimilation through photosynthesis. At the molecular level, C and N signaling pathways control gene expression of the NRT2 transporters. Although several regulators of these transporters have been identified in response to either N or C signals, the response of NRT2 gene expression to the interaction of these signals has never been specifically investigated, and the underlying molecular mechanisms remain largely unknown. To address this question we used an original systems biology approach to model a regulatory gene network targeting NRT2.1, NRT2.2, NRT2.4, and NRT2.5 in response to N/C signals. Our systems analysis of the data identified three transcription factors, TGA3, MYC1, and bHLH093. Functional analysis of mutants combined with yeast one-hybrid experiments confirmed that all three transcription factors are regulators of NRT2.4 or NRT2.5 in response to N or C signals. These results reveal a role for TGA3, MYC1, and bHLH093 in controlling the expression of root NRT2 transporter genes.

Gene atlas of iron-containing proteins in Arabidopsis thaliana.

Iron (Fe) is an essential element for the development and physiology of plants, owing to its presence in numerous proteins involved in central biological processes. Here, we established an exhaustive, manually curated inventory of genes encoding Fe-containing proteins in Arabidopsis thaliana, and summarized their subcellular localization, spatiotemporal expression and evolutionary age. We have currently identified 1068 genes encoding potential Fe-containing proteins, including 204 iron-sulfur (Fe-S) proteins, 446 haem proteins and 330 non-Fe-S/non-haem Fe proteins (updates of this atlas are available at https://conf.arabidopsis.org/display/COM/Atlas+of+Fe+containing+proteins). A fourth class, containing 88 genes for which iron binding is uncertain, is indexed as 'unclear'. The proteins are distributed in diverse subcellular compartments with strong differences per category. Interestingly, analysis of the gene age index showed that most genes were acquired early in plant evolutionary history and have progressively gained regulatory elements, to support the complex organ-specific and development-specific functions necessitated by the emergence of terrestrial plants. With this gene atlas, we provide a valuable and updateable tool for the research community that supports the characterization of the molecular actors and mechanisms important for Fe metabolism in plants. This will also help in selecting relevant targets for breeding or biotechnological approaches aiming at Fe biofortification in crops.

Iron-sulfur proteins in plant mitochondria: roles and maturation.

Iron-sulfur (Fe-S) clusters are prosthetic groups ensuring electron transfer reactions, activating substrates for catalytic reactions, providing sulfur atoms for the biosynthesis of vitamins or other cofactors, or having protein-stabilizing effects. Hence, metalloproteins containing these cofactors are essential for numerous and diverse metabolic pathways and cellular processes occurring in the cytoplasm. Mitochondria are organelles where the Fe-S cluster demand is high, notably because the activity of the respiratory chain complexes I, II, and III relies on the correct assembly and functioning of Fe-S proteins. Several other proteins or complexes present in the matrix require Fe-S clusters as well, or depend either on Fe-S proteins such as ferredoxins or on cofactors such as lipoic acid or biotin whose synthesis relies on Fe-S proteins. In this review, we have listed and discussed the Fe-S-dependent enzymes or pathways in plant mitochondria including some potentially novel Fe-S proteins identified based on in silico analysis or on recent evidence obtained in non-plant organisms. We also provide information about recent developments concerning the molecular mechanisms involved in Fe-S cluster synthesis and trafficking steps of these cofactors from maturation factors to client apoproteins.

The Arabidopsis Mitochondrial Glutaredoxin GRXS15 Provides [2Fe-2S] Clusters for ISCA-Mediated [4Fe-4S] Cluster Maturation.

Iron-sulfur (Fe-S) proteins are crucial for many cellular functions, particularly those involving electron transfer and metabolic reactions. An essential monothiol glutaredoxin GRXS15 plays a key role in the maturation of plant mitochondrial Fe-S proteins. However, its specific molecular function is not clear, and may be different from that of the better characterized yeast and human orthologs, based on known properties. Hence, we report here a detailed characterization of the interactions between Arabidopsis thaliana GRXS15 and ISCA proteins using both in vivo and in vitro approaches. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that GRXS15 interacts with each of the three plant mitochondrial ISCA1a/1b/2 proteins. UV-visible absorption/CD and resonance Raman spectroscopy demonstrated that coexpression of ISCA1a and ISCA2 resulted in samples with one [2Fe-2S]2+ cluster per ISCA1a/2 heterodimer, but cluster reconstitution using as-purified [2Fe-2S]-ISCA1a/2 resulted in a [4Fe-4S]2+ cluster-bound ISCA1a/2 heterodimer. Cluster transfer reactions monitored by UV-visible absorption and CD spectroscopy demonstrated that [2Fe-2S]-GRXS15 mediates [2Fe-2S]2+ cluster assembly on mitochondrial ferredoxin and [4Fe-4S]2+ cluster assembly on the ISCA1a/2 heterodimer in the presence of excess glutathione. This suggests that ISCA1a/2 is an assembler of [4Fe-4S]2+ clusters, via two-electron reductive coupling of two [2Fe-2S]2+ clusters. Overall, the results provide new insights into the roles of GRXS15 and ISCA1a/2 in effecting [2Fe-2S]2+ to [4Fe-4S]2+ cluster conversions for the maturation of client [4Fe-4S] cluster-containing proteins in plants.

[4Fe-4S] cluster trafficking mediated by Arabidopsis mitochondrial ISCA and NFU proteins.

Numerous iron-sulfur (Fe-S) proteins with diverse functions are present in the matrix and respiratory chain complexes of mitochondria. Although [4Fe-4S] clusters are the most common type of Fe-S cluster in mitochondria, the molecular mechanism of [4Fe-4S] cluster assembly and insertion into target proteins by the mitochondrial iron-sulfur cluster (ISC) maturation system is not well-understood. Here we report a detailed characterization of two late-acting Fe-S cluster-carrier proteins from Arabidopsis thaliana, NFU4 and NFU5. Yeast two-hybrid and bimolecular fluorescence complementation studies demonstrated interaction of both the NFU4 and NFU5 proteins with the ISCA class of Fe-S carrier proteins. Recombinant NFU4 and NFU5 were purified as apo-proteins after expression in Escherichia coliIn vitro Fe-S cluster reconstitution led to the insertion of one [4Fe-4S]2+ cluster per homodimer as determined by UV-visible absorption/CD, resonance Raman and EPR spectroscopy, and analytical studies. Cluster transfer reactions, monitored by UV-visible absorption and CD spectroscopy, showed that a [4Fe-4S]2+ cluster-bound ISCA1a/2 heterodimer is effective in transferring [4Fe-4S]2+ clusters to both NFU4 and NFU5 with negligible back reaction. In addition, [4Fe-4S]2+ cluster-bound ISCA1a/2, NFU4, and NFU5 were all found to be effective [4Fe-4S]2+ cluster donors for maturation of the mitochondrial apo-aconitase 2 as assessed by enzyme activity measurements. The results demonstrate rapid, unidirectional, and quantitative [4Fe-4S]2+ cluster transfer from ISCA1a/2 to NFU4 or NFU5 that further delineates their respective positions in the plant ISC machinery and their contributions to the maturation of client [4Fe-4S] cluster-containing proteins.

Identification of client iron-sulfur proteins of the chloroplastic NFU2 transfer protein in Arabidopsis thaliana.

Iron-sulfur (Fe-S) proteins have critical functions in plastids, notably participating in photosynthetic electron transfer, sulfur and nitrogen assimilation, chlorophyll metabolism, and vitamin or amino acid biosynthesis. Their maturation relies on the so-called SUF (sulfur mobilization) assembly machinery. Fe-S clusters are synthesized de novo on a scaffold protein complex and then delivered to client proteins via several transfer proteins. However, the maturation pathways of most client proteins and their specificities for transfer proteins are mostly unknown. In order to decipher the proteins interacting with the Fe-S cluster transfer protein NFU2, one of the three plastidial representatives found in Arabidopsis thaliana, we performed a quantitative proteomic analysis of shoots, roots, and seedlings of nfu2 plants, combined with NFU2 co-immunoprecipitation and binary yeast two-hybrid experiments. We identified 14 new targets, among which nine were validated in planta using a binary bimolecular fluorescence complementation assay. These analyses also revealed a possible role for NFU2 in the plant response to desiccation. Altogether, this study better delineates the maturation pathways of many chloroplast Fe-S proteins, considerably extending the number of NFU2 clients. It also helps to clarify the respective roles of the three NFU paralogs NFU1, NFU2, and NFU3.

Tissue-specific isolation of Arabidopsis/plant mitochondria - IMTACT (isolation of mitochondria tagged in specific cell types).

Plant cells contain numerous subcompartments with clearly delineated metabolic functions. Mitochondria represent a very small fraction of the total cell volume and yet are the site of respiration and thus crucial for cells throughout all developmental stages of a plant's life. As such, their isolation from the rest of the cellular components is a basic requirement for numerous biochemical and physiological experiments. Although procedures exist to isolate plant mitochondria from different organs (i.e. leaves, roots, tubers, etc.), they are often tedious and do not provide resolution at the tissue level (i.e. phloem, mesophyll or pollen). Here, we present a novel method called IMTACT (isolation of mitochondria tagged in specific cell types), developed in Arabidopsis thaliana (Arabidopsis) that involves biotinylation of mitochondria in a tissue-specific manner using transgenic lines expressing a synthetic version of the OM64 (Outer Membrane 64) gene combined with BLRP and the BirA biotin ligase gene. Tissue specificity is achieved with cell-specific promoters (e.g. CAB3 and SUC2). Labeled mitochondria from crude extracts are retained by magnetic beads, allowing the simple and rapid isolation of highly pure and intact organelles from organs or specific tissues. For example, we could show that the mitochondrial population from mesophyll cells was significantly larger in size than the mitochondrial population isolated from leaf companion cells. To facilitate the applicability of this method in both wild-type and mutant Arabidopsis plants we generated a set of OM64-BLRP one-shot constructs with different selection markers and tissue-specific promoters.

The plastidial Arabidopsis thaliana NFU1 protein binds and delivers [4Fe-4S] clusters to specific client proteins.

Proteins incorporating iron-sulfur (Fe-S) co-factors are required for a plethora of metabolic processes. Their maturation depends on three Fe-S cluster assembly machineries in plants, located in the cytosol, mitochondria, and chloroplasts. After de novo formation on scaffold proteins, transfer proteins load Fe-S clusters onto client proteins. Among the plastidial representatives of these transfer proteins, NFU2 and NFU3 are required for the maturation of the [4Fe-4S] clusters present in photosystem I subunits, acting upstream of the high-chlorophyll fluorescence 101 (HCF101) protein. NFU2 is also required for the maturation of the [2Fe-2S]-containing dihydroxyacid dehydratase, important for branched-chain amino acid synthesis. Here, we report that recombinant Arabidopsis thaliana NFU1 assembles one [4Fe-4S] cluster per homodimer. Performing co-immunoprecipitation experiments and assessing physical interactions of NFU1 with many [4Fe-4S]-containing plastidial proteins in binary yeast two-hybrid assays, we also gained insights into the specificity of NFU1 for the maturation of chloroplastic Fe-S proteins. Using bimolecular fluorescence complementation and in vitro Fe-S cluster transfer experiments, we confirmed interactions with two proteins involved in isoprenoid and thiamine biosynthesis, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase, respectively. An additional interaction detected with the scaffold protein SUFD enabled us to build a model in which NFU1 receives its Fe-S cluster from the SUFBC2D scaffold complex and serves in the maturation of specific [4Fe-4S] client proteins. The identification of the NFU1 partner proteins reported here more clearly defines the role of NFU1 in Fe-S client protein maturation in Arabidopsis chloroplasts among other SUF components.

Iron-sulfur protein NFU2 is required for branched-chain amino acid synthesis in Arabidopsis roots.

Numerous proteins require a metallic co-factor for their function. In plastids, the maturation of iron-sulfur (Fe-S) proteins necessitates a complex assembly machinery. In this study, we focused on Arabidopsis thaliana NFU1, NFU2, and NFU3, which participate in the final steps of the maturation process. According to the strong photosynthetic defects observed in high chlorophyll fluorescence 101 (hcf101), nfu2, and nfu3 plants, we determined that NFU2 and NFU3, but not NFU1, act immediately upstream of HCF101 for the maturation of [Fe4S4]-containing photosystem I subunits. An additional function of NFU2 in the maturation of the [Fe2S2] cluster of a dihydroxyacid dehydratase was obvious from the accumulation of precursors of the branched-chain amino acid synthesis pathway in roots of nfu2 plants and from the rescue of the primary root growth defect by supplying branched-chain amino acids. The absence of NFU3 in roots precluded any compensation. Overall, unlike their eukaryotic and prokaryotic counterparts, which are specific to [Fe4S4] proteins, NFU2 and NFU3 contribute to the maturation of both [Fe2S2] and [Fe4S4] proteins, either as a relay in conjunction with other proteins such as HCF101 or by directly delivering Fe-S clusters to client proteins. Considering the low number of Fe-S cluster transfer proteins relative to final acceptors, additional targets probably await identification.

Mitochondrial Arabidopsis thaliana TRXo Isoforms Bind an Iron⁻Sulfur Cluster and Reduce NFU Proteins In Vitro.

In plants, the mitochondrial thioredoxin (TRX) system generally comprises only one or two isoforms belonging to the TRX h or o classes, being less well developed compared to the numerous isoforms found in chloroplasts. Unlike most other plant species, Arabidopsis thaliana possesses two TRXo isoforms whose physiological functions remain unclear. Here, we performed a structure⁻function analysis to unravel the respective properties of the duplicated TRXo1 and TRXo2 isoforms. Surprisingly, when expressed in Escherichia coli, both recombinant proteins existed in an apo-monomeric form and in a homodimeric iron⁻sulfur (Fe-S) cluster-bridged form. In TRXo2, the [4Fe-4S] cluster is likely ligated in by the usual catalytic cysteines present in the conserved Trp-Cys-Gly-Pro-Cys signature. Solving the three-dimensional structure of both TRXo apo-forms pointed to marked differences in the surface charge distribution, notably in some area usually participating to protein⁻protein interactions with partners. However, we could not detect a difference in their capacity to reduce nitrogen-fixation-subunit-U (NFU)-like proteins, NFU4 or NFU5, two proteins participating in the maturation of certain mitochondrial Fe-S proteins and previously isolated as putative TRXo1 partners. Altogether, these results suggest that a novel regulation mechanism may prevail for mitochondrial TRXs o, possibly existing as a redox-inactive Fe-S cluster-bound form that could be rapidly converted in a redox-active form upon cluster degradation in specific physiological conditions.

Conserved functions of Arabidopsis mitochondrial late-acting maturation factors in the trafficking of iron­sulfur clusters.

Numerous proteins require iron­sulfur (Fe-S) clusters as cofactors for their function. Their biogenesis is a multi-step process occurring in the cytosol and mitochondria of all eukaryotes and additionally in plastids of photosynthetic eukaryotes. A basic model of Fe-S protein maturation in mitochondria has been obtained based on studies achieved in mammals and yeast, yet some molecular details, especially of the late steps, still require investigation. In particular, the late-acting biogenesis factors in plant mitochondria are poorly understood. In this study, we expressed the factors belonging to NFU, BOLA, SUFA/ISCA and IBA57 families in the respective yeast mutant strains. Expression of the Arabidopsis mitochondrial orthologs was usually sufficient to rescue the growth defects observed on specific media and/or to restore the abundance or activity of the defective Fe-S or lipoic acid-dependent enzymes. These data demonstrate that the plant mitochondrial counterparts, including duplicated isoforms, likely retained their ancestral functions. In contrast, the SUFA1 and IBA57.2 plastidial isoforms cannot rescue the lysine and glutamate auxotrophies of the respective isa1-isa2Δ and iba57Δ strains or of the isa1-isa2-iba57Δ triple mutant when expressed in combination. This suggests a specialization of the yeast mitochondrial and plant plastidial factors in these late steps of Fe-S protein biogenesis, possibly reflecting substrate-specific interactions in these different compartments.

Correction to: Roles and maturation of iron-sulfur proteins in plastids.

With the author(s)' decision to opt for Open Choice the copyright of the article changed.

Roles and maturation of iron-sulfur proteins in plastids.

One reason why iron is an essential element for most organisms is its presence in prosthetic groups such as hemes or iron-sulfur (Fe-S) clusters, which are notably required for electron transfer reactions. As an organelle with an intense metabolism in plants, chloroplast relies on many Fe-S proteins. This includes those present in the electron transfer chain which will be, in fact, essential for most other metabolic processes occurring in chloroplasts, e.g., carbon fixation, nitrogen and sulfur assimilation, pigment, amino acid, and vitamin biosynthetic pathways to cite only a few examples. The maturation of these Fe-S proteins requires a complex and specific machinery named SUF (sulfur mobilisation). The assembly process can be split in two major steps, (1) the de novo assembly on scaffold proteins which requires ATP, iron and sulfur atoms, electrons, and thus the concerted action of several proteins forming early acting assembly complexes, and (2) the transfer of the preformed Fe-S cluster to client proteins using a set of late-acting maturation factors. Similar machineries, having in common these basic principles, are present in the cytosol and in mitochondria. This review focuses on the currently known molecular details concerning the assembly and roles of Fe-S proteins in plastids.

Fast and Efficient Cloning of Cis-Regulatory Sequences for High-Throughput Yeast One-Hybrid Analyses of Transcription Factors.

Yeast one-hybrid (Y1H) assay has been proven to be a powerful technique to characterize in vivo the interaction between a given transcription factor (TF), or its DNA-binding domain (DBD), and target DNA sequences. Comprehensive characterization of TF/DBD and DNA interactions should allow designing synthetic promoters that would undoubtedly be valuable for biotechnological approaches. Here, we use the ligation-independent cloning system (LIC) in order to enhance the cloning efficiency of DNA motifs into the pHISi Y1H vector. LIC overcomes important limitations of traditional cloning technologies, since any DNA fragment can be cloned into LIC compatible vectors without using restriction endonucleases, ligation, or in vitro recombination.

The mitochondrial monothiol glutaredoxin S15 is essential for iron-sulfur protein maturation in Arabidopsis thaliana.

The iron-sulfur cluster (ISC) is an ancient and essential cofactor of many proteins involved in electron transfer and metabolic reactions. In Arabidopsis, three pathways exist for the maturation of iron-sulfur proteins in the cytosol, plastids, and mitochondria. We functionally characterized the role of mitochondrial glutaredoxin S15 (GRXS15) in biogenesis of ISC containing aconitase through a combination of genetic, physiological, and biochemical approaches. Two Arabidopsis T-DNA insertion mutants were identified as null mutants with early embryonic lethal phenotypes that could be rescued by GRXS15. Furthermore, we showed that recombinant GRXS15 is able to coordinate and transfer an ISC and that this coordination depends on reduced glutathione (GSH). We found the Arabidopsis GRXS15 able to complement growth defects based on disturbed ISC protein assembly of a yeast Δgrx5 mutant. Modeling of GRXS15 onto the crystal structures of related nonplant proteins highlighted amino acid residues that after mutation diminished GSH and subsequently ISC coordination, as well as the ability to rescue the yeast mutant. When used for plant complementation, one of these mutant variants, GRXS15K83/A, led to severe developmental delay and a pronounced decrease in aconitase activity by approximately 65%. These results indicate that mitochondrial GRXS15 is an essential protein in Arabidopsis, required for full activity of iron-sulfur proteins.

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.