AtSufE is an essential activator of plastidic and mitochondrial desulfurases in Arabidopsis

Iron-sulfur (Fe-S) clusters are vital prosthetic groups for Fe-S proteins involved in fundamental processes such as electron transfer, metabolism, sensing and signaling. In plants, sulfur (SUF) protein-mediated Fe-S cluster biogenesis involves iron acquisition and sulfur mobilization, processes suggested to be plastidic. Here we have shown that AtSufE in Arabidopsis rescues growth defects in SufE-deficient Escherichia coli. In contrast to other SUF proteins, AtSufE localizes to plastids and mitochondria interacting with the plastidic AtSufS and mitochondrial AtNifS1 cysteine desulfurases. AtSufE activates AtSufS and AtNifS1 cysteine desulfurization, and AtSufE activity restoration in either plastids or mitochondria is not sufficient to rescue embryo lethality in AtSufE loss-of-function mutants. AtSufE overexpression induces AtSufS and AtNifS1 expression, which in turn leads to elevated cysteine desulfurization activity, chlorosis and retarded development. Our data demonstrate that plastidic and mitochondrial Fe-S cluster biogenesis shares a common, essential component, and that AtSufE acts as an activator of plastidic and mitochondrial desulfurases in Arabidopsis.     

Biogenesis of Fe-S cluster by the bacterial Suf system: SufS and SufE form a new type of cysteine desulfurase

Biosynthesis of iron-sulfur clusters (Fe-S) depends on multiprotein systems. Recently, we described the SUF system of Escherichia coli and Erwinia chrysanthemi as being important for Fe-S biogenesis under stressful conditions. The SUF system is made of six proteins: SufC is an atypical cytoplasmic ABC-ATPase, which forms a complex with SufB and SufD; SufA plays the role of a scaffold protein for assembly of iron-sulfur clusters and delivery to target proteins; SufS is a cysteine desulfurase which mobilizes the sulfur atom from cysteine and provides it to the cluster; SufE has no associated function yet. Here we demonstrate that: (i) SufE and SufS are both cystosolic as all members of the SUF system; (ii) SufE is a homodimeric protein; (iii) SufE forms a complex with SufS as shown by the yeast two-hybrid system and by affinity chromatography; (iv) binding of SufE to SufS is responsible for a 50-fold stimulation of the cysteine desulfurase activity of SufS. This is the first example of a two-component cysteine desulfurase enzyme.     

Silencing of NbNAP1 encoding a plastidic SufB-like protein affects chloroplast development in Nicotiana benthamiana

It was previously shown that AtNAP1 is a plastidic SufB protein involved in Fe-S cluster assembly in Arabidopsis. In this study, we investigated the effects of depleting SufB protein from plant cells using virus-induced gene silencing (VIGS). VIGS of NbNAP1 encoding a Nicotiana benthamiana homolog of AtNAP1 resulted in a leaf yellowing phenotype. NbNAP1 was expressed ubiquitously in plant tissues with the highest level in roots. A GFP fusion protein of the N-terminal region (M1-V103) of NbNAP1 was targeted to chloroplasts. Depletion of NbNAP1 resulted in reduced numbers of chloroplasts of reduced size. Mitochondria also seemed to be affected. Despite the reduced number and size of the chloroplasts in the NbNAP1 VIGS lines, the expression of many nuclear genes encoding chloroplast-targeted proteins and chlorophyll biosynthesis genes remained unchanged.     

Hydrodynamic characterization of the SufBC and SufCD complexes and their interaction with fluorescent adenosine nucleotides

Bacteria, as well as the plastid organelles of algae and higher plants, utilize proteins of the suf operon. These are involved in Fe-S cluster assembly, particularly under conditions of iron limitation or oxidative stress. Genetic experiments in some organisms found that the ATPase SufC is essential, though its role in Fe-S biogenesis remains unclear. To ascertain how interactions with other individual Suf proteins affect the activity of SufC we coexpressed it with either SufB or SufD from Thermotoga maritima and purified the resulting SufBC and SufCD complexes. Analytical ultracentrifuge and multiangle light-scattering measurements showed that the SufBC complex exists in solution as the tetrameric SufB(2)C(2) species, whereas SufCD exists as an equilibrium mixture of SufCD and SufC(2)D(2). Transient kinetic studies of the complexes were made using fluorescent 2'(3')-O-(N-methylanthraniloyl-(mant) analogues of ATP and ADP. Both SufBC and SufCD bound mantATP and mantADP much more tightly than does SufC alone. Compared to the cleavage step of the mantATPase of SufC alone, that of SufBC was accelerated 180-fold and that of SufCD only fivefold. Given that SufB and SufD have 20% sequence identity and similar predicted secondary structures, the different hydrodynamic properties and kinetic mechanisms of the two complexes are discussed.     

The SufBCD protein complex is the scaffold for iron–sulfur cluster assembly in Thermus thermophiles HB8

Iron–sulfur (Fe–S) clusters are the oldest and most versatile inorganic cofactors that are required to sustain fundamental life processes. Bacteria have three systems of [Fe–S] cluster biogenesis, designated ISC, NIF, and SUF. In contrast, the Thermus thermophiles HB8 has only one system, formed mostly by SUF homologs that contain six proteins: SufA, SufB, SufC, SufD, SufS and SufE. The kinetics of SufC ATPase was studied using a linked enzyme assay method. In the presence of SufB, SufD or SufBD complexes, the activity of SufC was enhanced. The cysteine desulfurase activity of SufS was also stimulated by the presence of the SufBCD complex. The results obtained through enzymology revealed that aconitase activity was activated by [Fe–S] clusters reconstituted on the SufBCD complex. Consolidated results from spectral and enzymatic analysis suggest that the SufBCD complex is a novel type of Fe–S scaffold system that can assemble Fe/S clusters de novo.     

Crystal structure of Escherichia coli SufC, an ABC-type ATPase component of the SUF iron-sulfur cluster assembly machinery

SufC is an ATPase component of the SUF machinery, which is involved in the biosynthesis of Fe-S clusters. To gain insight into the function of this protein, we have determined the crystal structure of Escherichia coli SufC at 2.5A resolution. Despite the similarity of the overall structure with ABC-ATPases (nucleotide-binding domains of ABC transporters), some key differences were observed. Glu171, an invariant residue involved in ATP hydrolysis, is rotated away from the nucleotide-binding pocket to form a SufC-specific salt bridge with Lys152. Due to this salt bridge, D-loop that follows Glu171 is flipped out to the molecular surface, which may sterically inhibit the formation of an active dimer. Thus, the salt bridge may play a critical role in regulating ATPase activity and preventing wasteful ATP hydrolysis. Furthermore, SufC has a unique Q-loop structure on its surface, which may form a binding site for its partner proteins, SufB and/or SufD.     

Iron-Sulfur (Fe-S) Cluster Assembly: THE SufBCD COMPLEX IS A NEW TYPE OF Fe-S SCAFFOLD WITH A FLAVIN REDOX COFACTOR*

Assembly of iron-sulfur (Fe-S) clusters and maturation of Fe-S proteins in vivo require complex machineries. In Escherichia coli, under adverse stress conditions, this process is achieved by the SUF system that contains six proteins as follows: SufA, SufB, SufC, SufD, SufS, and SufE. Here, we provide a detailed characterization of the SufBCD complex whose function was so far unknown. Using biochemical and spectroscopic analyses, we demonstrate the following: (i) the complex as isolated exists mainly in a 1:2:1 (B:C:D) stoichiometry; (ii) the complex can assemble a [4Fe-4S] cluster in vitro and transfer it to target proteins; and (iii) the complex binds one molecule of flavin adenine nucleotide per SufBC₂D complex, only in its reduced form (FADH₂), which has the ability to reduce ferric iron. These results suggest that the SufBC₂D complex functions as a novel type of scaffold protein that assembles an Fe-S cluster through the mobilization of sulfur from the SufSE cysteine desulfurase and the FADH₂-dependent reductive mobilization of iron.     

CpNifS-dependent iron-sulfur cluster biogenesis in chloroplasts

Iron-sulfur (Fe-S) clusters are important prosthetic groups in all organisms. The biosynthesis of Fe-S clusters has been studied extensively in bacteria and yeast. By contrast, much remains to be discovered about Fe-S cluster biogenesis in higher plants. Plant plastids are known to make their own Fe-S clusters. Plastid Fe-S proteins are involved in essential metabolic pathways, such as photosynthesis, nitrogen and sulfur assimilation, protein import, and chlorophyll transformation. This review aims to summarize the roles of Fe-S proteins in essential metabolic pathways and to give an overview of the latest findings on plastidic Fe-S assembly. The plastidic Fe-S biosynthetic machinery contains many homologues of bacterial mobilization of sulfur (SUF) proteins, but there are additional components and properties that may be plant-specific. These additional features could make the plastidic machinery more suitable for assembling Fe-S clusters in the presence of oxygen, and may enable it to be regulated in response to oxidative stress, iron status and light.     

SufC: an unorthodox cytoplasmic ABC/ATPase required for [Fe-S] biogenesis under oxidative stress

Proteins containing [Fe-S] clusters perform essential functions in all domains of life. Previously, we identified the sufABCDSE operon as being necessary for virulence of the plant pathogen Erwinia chrysanthemi. In addition, we collected preliminary evidence that the sufABCDSE operon might be involved in the assembly of [Fe-S] clusters. Of particular interest are the sufB, sufC and sufD genes, which are conserved among Eubacteria, Archaea, plants and parasites. The present study establishes SufC as an unorthodox ATPase of the ABC superfamily that is located in the cytosol, wherein it interacts with both SufB and SufD. Moreover, under oxidative stress conditions, SufC was found to be necessary for the activity of enzymes containing oxygen-labile [Fe-S] clusters, but dispensable for glutamate synthase, which contains an oxidatively stable [Fe-S] cluster. Lastly, we have shown SufBCD to be essential for iron acquisition via chrysobactin, a siderophore of major importance in virulence. We discuss a model wherein the SufBCD proteins contribute to bacterial pathogenicity via their role in the assembly of [Fe-S] clusters under oxidative stress and iron limitation.     

Distinct roles for U‐type proteins in iron–sulfur cluster biosynthesis revealed by genetic analysis of the Bacillus subtilis sufCDSUB operon

The biosynthesis of iron–sulfur (Fe–S) clusters in Bacillus subtilis is mediated by the SUF‐like system composed of the sufCDSUB gene products. This system is unique in that it is a chimeric machinery comprising homologues of E. coli SUF components (SufS, SufB, SufC and SufD) and an ISC component (IscU). B. subtilis SufS cysteine desulfurase transfers persulfide sulfur to SufU (the IscU homologue); however, it has remained controversial whether SufU serves as a scaffold for Fe–S cluster assembly, like IscU, or acts as a sulfur shuttle protein, like E. coli SufE. Here we report that reengineering of the isoprenoid biosynthetic pathway in B. subtilis can offset the indispensability of the sufCDSUB operon, allowing the resultant Δsuf mutants to grow without detectable Fe–S proteins. Heterologous bidirectional complementation studies using B. subtilis and E. coli mutants showed that B. subtilis SufSU is interchangeable with E. coli SufSE but not with IscSU. In addition, functional similarity in SufB, SufC and SufD was observed between B. subtilis and E. coli. Our findings thus indicate that B. subtilis SufU is the protein that transfers sulfur from SufS to SufB, and that the SufBCD complex is the site of Fe–S cluster assembly.     

SufE transfers sulfur from SufS to SufB for iron-sulfur cluster assembly

Iron-sulfur (Fe-S) clusters are key metal cofactors of metabolic, regulatory, and stress response proteins in most organisms. The unique properties of these clusters make them susceptible to disruption by iron starvation or oxidative stress. Both iron and sulfur can be perturbed under stress conditions, leading to Fe-S cluster defects. Bacteria and higher plants contain a specialized system for Fe-S cluster biosynthesis under stress, namely the Suf pathway. In Escherichia coli the Suf pathway consists of six proteins with functions that are only partially characterized. Here we describe how the SufS and SufE proteins interact with the SufBCD protein complex to facilitate sulfur liberation from cysteine and donation for Fe-S cluster assembly. It was previously shown that the cysteine desulfurase SufS donates sulfur to the sulfur transfer protein SufE. We have found here that SufE in turn interacts with the SufB protein for sulfur transfer to that protein. The interaction occurs only if SufC is present. Furthermore, SufB can act as a site for Fe-S cluster assembly in the Suf system. This provides the first evidence of a novel site for Fe-S cluster assembly in the SufBCD complex.     

Ancient and essential: the assembly of iron-sulfur clusters in plants

In plants iron-sulfur (Fe-S) proteins are found in the plastids, mitochondria, cytosol and nucleus, where they are essential for numerous physiological and developmental processes. Recent mutant studies, mostly in Arabidopsis thaliana, have identified three pathways for the assembly of Fe-S clusters. The plastids harbor the SUF (sulfur mobilization) pathway and operate independently, whereas cluster assembly in the cytosol depends on the emerging CIA (cytosolic iron-sulfur cluster assembly) pathway and mitochondria. The latter organelles use the ISC (iron-sulfur cluster) assembly pathway. In all three pathways the assembly process can be divided into a first stage where S and Fe are combined on a scaffold protein, and a second stage in which the Fe-S cluster is transferred to a target protein. The second stage might involve different carrier proteins with specialized functions.     

Crystal structure of atypical cytoplasmic ABC-ATPase SufC from Thermus thermophilus HB8

SufC, a cytoplasmic ABC-ATPase, is one of the most conserved Suf proteins. SufC forms a stable complex with SufB and SufD, and the SufBCD complex interacts with other Suf proteins in the Fe-S cluster assembly. We have determined the crystal structure of SufC from Thermus thermophilus HB8 in nucleotide-free and ADP-Mg-bound states at 1.7A and 1.9A resolution, respectively. The overall architecture of the SufC structure is similar to other ABC ATPases structures, but there are several specific motifs in SufC. Three residues following the end of the Walker B motif form a novel 3(10) helix which is not observed in other ABC ATPases. Due to the novel 3(10) helix, a conserved glutamate residue involved in ATP hydrolysis is flipped out. Although this unusual conformation is unfavorable for ATP hydrolysis, salt-bridges formed by conserved residues and a strong hydrogen-bonding network around the novel 3(10) helix suggest that the novel 3(10) helix of SufC is a rigid conserved motif. Compared to other ABC-ATPase structures, a significant displacement occurs at a linker region between the ABC alpha/beta domain and the alpha-helical domain. The linker conformation is stabilized by a hydrophobic interaction between conserved residues around the Q loop. The molecular surfaces of SufC and the C-terminal helices of SufD (PDB code: 1VH4) suggest that the unusual linker conformation conserved among SufC proteins is probably suitable for interacting with SufB and SufD.     

Interaction between sulphur mobilisation proteins SufB and SufC: evidence for an iron-sulphur cluster biogenesis pathway in the apicoplast of Plasmodium falciparum

The plastid of Plasmodium falciparum, the apicoplast, performs metabolic functions essential to the parasite. Various reactions in the plastid require the assembly of [Fe-S] prosthetic groups on participating proteins as well as the reductant activity of ferredoxin that is converted from its apo-form by the assembly of [Fe-S] clusters inside the apicoplast. The [Fe-S] assembly pathway involving sulphur mobilising Suf proteins has been predicted to function in the apicoplast with one component (PfSufB) encoded by the plastid genome itself. We demonstrate the ATPase activity of recombinant P. falciparum nuclear-encoded SufC and its localisation in the apicoplast. Further, an internal region of apicoplast SufB was used to detect PfSufB-PfSufC interaction in vitro; co-elution of SufB from parasite lysate with recombinant PfSufC on an affinity column also indicated an interaction of the two proteins. As a departure from bacterial SufB and similar to reported plant plastid SufB, apicoplast SufB exhibited ATPase activity, suggesting the evolution of specialised functions in the plastid counterparts. Our results provide experimental evidence for an active Suf pathway in the Plasmodium apicoplast.     

Identification of the Mycobacterium tuberculosis SUF machinery as the exclusive mycobacterial system of [Fe-S] cluster assembly: evidence for its implication in the pathogen's survival

The worldwide recrudescence of tuberculosis and widespread antibiotic resistance have strengthened the need for the rapid development of new antituberculous drugs targeting essential functions of its etiologic agent, Mycobacterium tuberculosis. In our search for new targets, we found that the M. tuberculosis pps1 gene, which contains an intein coding sequence, belongs to a conserved locus of seven open reading frames. In silico analyses indicated that the mature Pps1 protein is orthologous to the SufB protein of many organisms, a highly conserved component of the [Fe-S] cluster assembly and repair SUF (mobilization of sulfur) machinery. We showed that the mycobacterial pps1 locus constitutes an operon which encodes Suf-like proteins. Interactions between these proteins were demonstrated, supporting the functionality of the M. tuberculosis SUF system. The noticeable absence of any alternative [Fe-S] cluster assembly systems in mycobacteria is in agreement with the apparent essentiality of the suf operon in Mycobacterium smegmatis. Altogether, these results establish that Pps1, as a central element of the SUF system, could play an essential function for M. tuberculosis survival virtually through its implication in the bacterial resistance to iron limitation and oxidative stress. As such, Pps1 may represent an interesting molecular target for new antituberculous drugs.     

Unique genetic compartmentalization of the SUF system in cryptophytes and characterization of a SufD mutant in Arabidopsis thaliana

The mobilization of sulfur (SUF) system is one of three systems involved in iron-sulfur cluster biosynthesis and maintenance. In eukaryotes the SUF system is specific for the plastid and therefore of symbiotic origin. Analyses in cryptophytes showed a unique genetic compartmentalization of the SUF system, which evolved by at least two different gene transfer events. We analyzed one of the components, SufD, in the cryptophyte Guillardia theta and in Arabidopsis thaliana. We demonstrated that SufD fulfils house keeping functions during embryogenesis and in adult plants in A. thaliana.     

The SufE protein and the SufBCD complex enhance SufS cysteine desulfurase activity as part of a sulfur transfer pathway for Fe-S cluster assembly in Escherichia coli

The sufABCDSE operon of the Gram-negative bacterium Escherichia coli is induced by oxidative stress and iron deprivation. To examine the biochemical roles of the Suf proteins, we purified all of the proteins and assayed their effect on SufS cysteine desulfurase activity. Here we report that the SufE protein can stimulate the cysteine desulfurase activity of the SufS enzyme up to 8-fold and accepts sulfane sulfur from SufS. This sulfur transfer process from SufS to SufE is sheltered from the environment based on its resistance to added reductants and on the analysis of available crystal structures of the proteins. We also found that the SufB, SufC, and SufD proteins associate in a stable complex and that, in the presence of SufE, the SufBCD complex further stimulates SufS activity up to 32-fold. Thus, the SufE protein and the SufBCD complex act synergistically to modulate the cysteine desulfurase activity of SufS. We propose that this sulfur transfer mechanism may be important for limiting sulfide release during oxidative stress conditions in vivo.     

Enterococcus faecalis SufU scaffold protein enhances SufS desulfurase activity by acquiring sulfur from its cysteine-153

Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups that play essential roles in all living organisms. In vivo [Fe-S] cluster biogenesis requires enzymes involved in iron and sulfur mobilization, assembly of clusters, and delivery to their final acceptor. In these systems, a cysteine desulfurase is responsible for the release of sulfide ions, which are incorporated into a scaffold protein for subsequent [Fe-S] cluster assembly. Although three machineries have been shown to be present in Proteobacteria for [Fe-S] cluster biogenesis (NIF, ISC, and SUF), only the SUF machinery has been found in Firmicutes. We have recently described the structural similarities and differences between Enterococcus faecalis and Escherichia coli SufU proteins, which prompted the proposal that SufU is the scaffold protein of the E. faecalis sufCDSUB system. The present work aims at elucidating the biological roles of E. faecalis SufS and SufU proteins in [Fe-S] cluster assembly. We show that SufS has cysteine desulfurase activity and cysteine-365 plays an essential role in catalysis. SufS requires SufU as activator to [4Fe-4S] cluster assembly, as its ortholog, IscU, in which the conserved cysteine-153 acts as a proximal sulfur acceptor for transpersulfurization reaction.     

The kinetic mechanism of the SufC ATPase: the cleavage step is accelerated by SufB

Protein products of the suf operon are involved in iron-sulfur metabolism. SufC is an ATPase that can interact with SufB in the absence of nucleotide. We have studied the transient kinetics of the SufC ATPase mechanism using the fluorescent ATP analogue, 2'(3')-O-N-methylanthraniloyl-ATP (mantATP). mantATP initially binds to SufC weakly. A conformational change of the SufC.mantATP complex then occurs followed by the very slow cleavage of mantATP to mantADP and the rapid release of Pi. In the presence of SufB, the cleavage step is accelerated and the release of mantADP is inhibited. Both of these effects promote the formation of a SufC.mantADP complex. In the absence and presence of SufB, mantADP remains more tightly bound to SufC than mantATP. These studies provide a basis for how the SufB and -C proteins interact in the processes involved in regulating iron-sulfur transfer.