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 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.

Relationships between the Reversible Oxidation of the Single Cysteine Residue and the Physiological Function of the Mitochondrial Glutaredoxin S15 from Arabidopsis thaliana.

Glutaredoxins (GRXs) are widespread proteins catalyzing deglutathionylation or glutathionylation reactions or serving for iron-sulfur (Fe-S) protein maturation. Previous studies highlighted a role of the Arabidopsis thaliana mitochondrial class II GRXS15 in Fe-S cluster assembly, whereas only a weak glutathione-dependent oxidation activity was detected with the non-physiological roGFP2 substrate in vitro. Still, the protein must exist in a reduced form for both redox and Fe-S cluster binding functions. Therefore, this study aimed at examining the redox properties of AtGRXS15. The acidic pKa of the sole cysteine present in AtGRXS15 indicates that it should be almost totally under a thiolate form at mitochondrial pH and thus possibly subject to oxidation. Oxidizing treatments revealed that this cysteine reacts with H2O2 or with oxidized glutathione forms. This leads to the formation of disulfide-bridge dimers and glutathionylated monomers which have redox midpoint potentials of -304 mV and -280 mV, respectively. Both oxidized forms are reduced by glutathione and mitochondrial thioredoxins. In conclusion, it appears that AtGRXS15 is prone to oxidation, forming reversible oxidation forms that may be seen either as a catalytic intermediate of the oxidoreductase activity and/or as a protective mechanism preventing irreversible oxidation and allowing Fe-S cluster binding upon reduction.

Glutaredoxins with iron-sulphur clusters in eukaryotes - Structure, function and impact on disease.

Among the thioredoxin superfamily of proteins, the observation that numerous glutaredoxins bind iron-sulphur (Fe/S) clusters is one of the more recent and major developments concerning their functional properties. Glutaredoxins are present in most organisms. All members of the class II subfamily (including most monothiol glutaredoxins), but also some members of the class I (mostly dithiol glutaredoxins) and class III (land plant-specific monothiol or dithiol glutaredoxins) are Fe/S proteins. In glutaredoxins characterised so far, the [2Fe2S] cluster is coordinated by two active-site cysteine residues and two molecules of non-covalently bound glutathione in homo-dimeric complexes bridged by the cluster. In contrast to dithiol glutaredoxins, monothiol glutaredoxins possess no or very little oxidoreductase activity, but have emerged as important players in cellular iron metabolism. In this review we summarise the recent developments of the most prominent Fe/S glutaredoxins in eukaryotes, the mitochondrial single domain monothiol glutaredoxin 5, the chloroplastic single domain monothiol glutaredoxin S14 and S16, the nuclear/cytosolic multi-domain monothiol glutaredoxin 3, and the mitochondrial/cytosolic dithiol glutaredoxin 2.