Correction of non-random mutational biases along a linear bacterial chromosome by the mismatch repair endonuclease NucS.

The linear chromosome of Streptomyces exhibits a highly compartmentalized structure with a conserved central region flanked by variable arms. As double strand break (DSB) repair mechanisms play a crucial role in shaping the genome plasticity of Streptomyces, we investigated the role of EndoMS/NucS, a recently characterized endonuclease involved in a non-canonical mismatch repair (MMR) mechanism in archaea and actinobacteria, that singularly corrects mismatches by creating a DSB. We showed that Streptomyces mutants lacking NucS display a marked colonial phenotype and a drastic increase in spontaneous mutation rate. In vitro biochemical assays revealed that NucS cooperates with the replication clamp to efficiently cleave G/T, G/G and T/T mismatched DNA by producing DSBs. These findings are consistent with the transition-shifted mutational spectrum observed in the mutant strains and reveal that NucS-dependent MMR specific task is to eliminate G/T mismatches generated by the DNA polymerase during replication. Interestingly, our data unveil a crescent-shaped distribution of the transition frequency from the replication origin towards the chromosomal ends, shedding light on a possible link between NucS-mediated DSBs and Streptomyces genome evolution.

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.

The cytosolic Arabidopsis thaliana cysteine desulfurase ABA3 delivers sulfur to the sulfurtransferase STR18.

The biosynthesis of many sulfur-containing molecules depends on cysteine as a sulfur source. Both the cysteine desulfurase (CD) and rhodanese (Rhd) domain-containing protein families participate in the trafficking of sulfur for various metabolic pathways in bacteria and human, but their connection is not yet described in plants. The existence of natural chimeric proteins containing both CD and Rhd domains in specific bacterial genera, however, suggests a general interaction between these proteins. We report here the biochemical relationships between two cytosolic proteins from Arabidopsis thaliana, a Rhd domain-containing protein, the sulfurtransferase 18 (STR18), and a CD isoform referred to as ABA3, and compare these biochemical features to those of a natural CD-Rhd fusion protein from the bacterium Pseudorhodoferax sp. We observed that the bacterial enzyme is bifunctional exhibiting both CD and STR activities using l-cysteine and thiosulfate as sulfur donors but preferentially using l-cysteine to catalyze transpersulfidation reactions. In vitro activity assays and mass spectrometry analyses revealed that STR18 stimulates the CD activity of ABA3 by reducing the intermediate persulfide on its catalytic cysteine, thereby accelerating the overall transfer reaction. We also show that both proteins interact in planta and form an efficient sulfur relay system, whereby STR18 catalyzes transpersulfidation reactions from ABA3 to the model acceptor protein roGFP2. In conclusion, the ABA3-STR18 couple likely represents an uncharacterized pathway of sulfur trafficking in the cytosol of plant cells, independent of ABA3 function in molybdenum cofactor maturation.

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.

Diversity and roles of cysteine desulfurases in photosynthetic organisms.

As sulfur is part of many essential protein cofactors such as iron-sulfur clusters, molybdenum cofactors, or lipoic acid, its mobilization from cysteine represents a fundamental process. The abstraction of the sulfur atom from cysteine is catalysed by highly conserved pyridoxal 5'-phosphate-dependent enzymes called cysteine desulfurases. The desulfuration of cysteine leads to the formation of a persulfide group on a conserved catalytic cysteine and the concomitant release of alanine. Sulfur is then transferred from cysteine desulfurases to different targets. Numerous studies have focused on cysteine desulfurases as sulfur-extracting enzymes for iron-sulfur cluster synthesis in mitochondria and chloroplasts but also for molybdenum cofactor sulfuration in the cytosol. Despite this, knowledge about the involvement of cysteine desulfurases in other pathways is quite rudimentary, particularly in photosynthetic organisms. In this review, we summarize current understanding of the different groups of cysteine desulfurases and their characteristics in terms of primary sequence, protein domain architecture, and subcellular localization. In addition, we review the roles of cysteine desulfurases in different fundamental pathways and highlight the gaps in our knowledge to encourage future work on unresolved issues especially in photosynthetic organisms.

Arabidopsis thaliana 3-mercaptopyruvate sulfurtransferases interact with and are protected by reducing systems.

The formation of a persulfide group (-SSH) on cysteine residues has gained attention as a reversible posttranslational modification contributing to protein regulation or protection. The widely distributed 3-mercaptopyruvate sulfurtransferases (MSTs) are implicated in the generation of persulfidated molecules and H2S biogenesis through transfer of a sulfane sulfur atom from a suitable donor to an acceptor. Arabidopsis has two MSTs, named STR1 and STR2, but they are poorly characterized. To learn more about these enzymes, we conducted a series of biochemical experiments including a variety of possible reducing systems. Our kinetic studies, which used a combination of sulfur donors and acceptors revealed that both MSTs use 3-mercaptopyruvate efficiently as a sulfur donor while thioredoxins, glutathione, and glutaredoxins all served as high-affinity sulfane sulfur acceptors. Using the redox-sensitive GFP (roGFP2) as a model acceptor protein, we showed that the persulfide-forming MSTs catalyze roGFP2 oxidation and more generally trans-persulfidation reactions. However, a preferential interaction with the thioredoxin system and glutathione was observed in case of competition between these sulfur acceptors. Moreover, we observed that MSTs are sensitive to overoxidation but are protected from an irreversible inactivation by their persulfide intermediate and subsequent reactivation by thioredoxins or glutathione. This work provides significant insights into Arabidopsis STR1 and STR2 catalytic properties and more specifically emphasizes the interaction with cellular reducing systems for the generation of H2S and glutathione persulfide and reactivation of an oxidatively modified form.

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.

[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.

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.

Coumarin accumulation and trafficking in Arabidopsis thaliana: a complex and dynamic process.

Iron (Fe) is a major micronutrient and is required for plant growth and development. Nongrass species have evolved a reduction-based strategy to solubilize and take up Fe. The secretion of Fe-mobilizing coumarins (e.g. fraxetin, esculetin and sideretin) by plant roots plays an important role in this process. Although the biochemical mechanisms leading to their biosynthesis have been well described, very little is known about their cellular and subcellular localization or their mobility within plant tissues. Spectral imaging was used to monitor, in Arabidopsis thaliana, the in planta localization of Fe-mobilizing coumarins and scopolin. Molecular, genetic and biochemical approaches were also used to investigate the dynamics of coumarin accumulation in roots. These approaches showed that root hairs play a major role in scopoletin secretion, whereas fraxetin and esculetin secretion occurs through all epidermis cells. The findings of this study also showed that the transport of coumarins from the cortex to the rhizosphere relies on the PDR9 transporter under Fe-deficient conditions. Additional experiments support the idea that coumarins move throughout the plant body via the xylem sap and that several plant species can take up coumarins present in the surrounding media. Altogether, the data presented here demonstrate that coumarin storage and accumulation in roots is a highly complex and dynamic process.

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.

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.

Novel insights into the diversity of the sulfurtransferase family in photosynthetic organisms with emphasis on oak.

Sulfurtransferases (STRs) constitute a large and complex protein family characterized by the presence of a rhodanese domain and implicated in diverse molecular and signaling processes as sulfur carriers. Although sulfurtransferases are present in the three domains of life and share evolutionary relationships, a high variability exists at different levels including the protein length and active site sequence, the presence of an indispensable catalytic cysteine residue, the domain arrangement and the subcellular localization. Because only Arabidopsis thaliana sequences have been inventoried so far, this paper aims at providing a detailed classification and inventory of evolutionary features of this family in photosynthetic organisms using comparative genomics, focusing on the oak genome. Based on the expansion of STRs in higher photosynthetic organisms, we classified the STR family in nine clusters depending on their primary sequence and domain arrangement. We found that oak possesses at least one isoform in all defined clusters and that clusters IV, V and VI contain plant-specific isoforms that are located mostly in chloroplasts. The novel classification proposed here provides the basis for functional genomics approaches in order to dissect the biochemical characteristics and physiological functions of individual STR representatives.

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.

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.

A Global Proteomic Approach Sheds New Light on Potential Iron-Sulfur Client Proteins of the Chloroplastic Maturation Factor NFU3.

Iron-sulfur (Fe-S) proteins play critical functions in plants. Most Fe-S proteins are synthetized in the cytosol as apo-proteins and the subsequent Fe-S cluster incorporation relies on specific protein assembly machineries. They are notably formed by a scaffold complex, which serves for the de novo Fe-S cluster synthesis, and by transfer proteins that insure cluster delivery to apo-targets. However, scarce information is available about the maturation pathways of most plastidial Fe-S proteins and their specificities towards transfer proteins of the associated SUF machinery. To gain more insights into these steps, the expression and protein localization of the NFU1, NFU2, and NFU3 transfer proteins were analyzed in various Arabidopsis thaliana organs and tissues showing quite similar expression patterns. In addition, quantitative proteomic analysis of an nfu3 loss-of-function mutant allowed to propose novel potential client proteins for NFU3 and to show that the protein accumulation profiles and thus metabolic adjustments differ substantially from those established in the nfu2 mutant. By clarifying the respective roles of the three plastidial NFU paralogs, these data allow better delineating the maturation process of plastidial Fe-S proteins.

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.

Function and maturation of the Fe-S center in dihydroxyacid dehydratase from Arabidopsis.

Dihydroxyacid dehydratase (DHAD) is the third enzyme required for branched-chain amino acid biosynthesis in bacteria, fungi, and plants. DHAD enzymes contain two distinct types of active-site Fe-S clusters. The best characterized examples are Escherichia coli DHAD, which contains an oxygen-labile [Fe4S4] cluster, and spinach DHAD, which contains an oxygen-resistant [Fe2S2] cluster. Although the Fe-S cluster is crucial for DHAD function, little is known about the cluster-coordination environment or the mechanism of catalysis and cluster biogenesis. Here, using the combination of UV-visible absorption and circular dichroism and resonance Raman and electron paramagnetic resonance, we spectroscopically characterized the Fe-S center in DHAD from Arabidopsis thaliana (At). Our results indicated that AtDHAD can accommodate [Fe2S2] and [Fe4S4] clusters. However, only the [Fe2S2] cluster-bound form is catalytically active. We found that the [Fe2S2] cluster is coordinated by at least one non-cysteinyl ligand, which can be replaced by the thiol group(s) of dithiothreitol. In vitro cluster transfer and reconstitution reactions revealed that [Fe2S2] cluster-containing NFU2 protein is likely the physiological cluster donor for in vivo maturation of AtDHAD. In summary, AtDHAD binds either one [Fe4S4] or one [Fe2S2] cluster, with only the latter being catalytically competent and capable of substrate and product binding, and NFU2 appears to be the physiological [Fe2S2] cluster donor for DHAD maturation. This work represents the first in vitro characterization of recombinant AtDHAD, providing new insights into the properties, biogenesis, and catalytic role of the active-site Fe-S center in a plant DHAD.

Oxidative protein folding: state-of-the-art and current avenues of research in plants.

Contents Summary 1230 I. Introduction 1230 II. Formation and isomerization of disulfides in the ER and the Golgi apparatus 1231 III. The disulfide relay in the mitochondrial intermembrane space: why are plants different? 1236 IV. Disulfide bond formation on luminal proteins in thylakoids 1240 V. Conclusion 1242 Acknowledgements 1242 References 1242 SUMMARY: Disulfide bonds are post-translational modifications crucial for the structure and function of thousands of proteins. Their formation and isomerization, referred to as oxidative folding, require specific protein machineries found in oxidizing subcellular compartments, namely the endoplasmic reticulum and the associated endomembrane system, the intermembrane space of mitochondria and the thylakoid lumen of chloroplasts. At least one protein component is required for transferring electrons from substrate proteins to an acceptor that is usually molecular oxygen. For oxidation reactions, incoming reduced substrates are oxidized by thiol-oxidoreductase proteins (or domains in case of chimeric proteins), which are usually themselves oxidized by a single thiol oxidase, the enzyme generating disulfide bonds de novo. By contrast, the description of the molecular actors and pathways involved in proofreading and isomerization of misfolded proteins, which require a tightly controlled redox balance, lags behind. Herein we provide a general overview of the knowledge acquired on the systems responsible for oxidative protein folding in photosynthetic organisms, highlighting their particularities compared to other eukaryotes. Current research challenges are discussed including the importance and specificity of these oxidation systems in the context of the existence of reducing systems in the same compartments.