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.

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.

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.