SufA, IscA, and Nfu have been proposed to function as scaffolds in the assembly of Fe/S clusters in bacteria. To investigate the roles of these proteins further, single and double null-mutant strains of Synechococcus sp. strain PCC 7002 were constructed by insertional inactivation of genes homologous to sufA, iscA, and nfu. Demonstrating the nonessential nature of their products, the sufA, iscA, and sufA iscA mutants grew photoautotrophically with doubling times that were similar to the wild type under standard growth conditions. In contrast, attempts to inactivate the nfu gene only resulted in stable merodiploids. These results imply that Nfu, but not SufA or IscA, is the essential Fe/S scaffold protein in cyanobacteria. When cells were grown under iron-limiting conditions, the iscA and sufA mutant strains exhibited less chlorosis than the wild type. Under iron-sufficient growth conditions, isiA transcript levels, a marker for iron limitation in cyanobacteria, as well as transcript levels of genes in both the suf and isc regulons were significantly higher in the iscA mutant than in the wild type. Under photosynthesis-induced redox stress conditions, the transcript levels of the suf genes are notably higher in the sufA and the sufA iscA mutants than in the wild type. The growth phenotypes and mRNA abundance patterns of the mutant strains contradict the proposed scaffold function for the SufA and IscA proteins in generalized Fe/S cluster assembly and instead suggest that they play regulatory roles in iron homeostasis and the sensing of redox stress in cyanobacteria.
IscA is a key member of the iron-sulfur cluster assembly machinery in prokaryotic and eukaryotic organisms; however, the physiological function of IscA still remains elusive. Here we report the in vivo evidence demonstrating the iron binding activity of IscA in Escherichia coli cells. Supplement of exogenous iron (1μM) in the M9 minimal medium is sufficient to maximize the iron binding in IscA expressed in E. coli cells under aerobic growth conditions. In contrast, IscU, an iron-sulfur cluster assembly scaffold protein, or CyaY, a bacterial frataxin homologue, fails to bind any iron in E. coli cells under the same experimental conditions. Interestingly, the strong iron binding activity of IscA is greatly diminished in E. coli cells under anaerobic growth conditions. Additional studies reveal that oxygen in medium promotes the iron binding in IscA and that the iron binding in IscA in turn prevents formation of biologically inaccessible ferric hydroxide under aerobic conditions. Consistent with the differential iron binding activity of IscA under aerobic and anaerobic conditions, we find that IscA and its paralog SufA are essential for the iron-sulfur cluster assembly in E. coli cells under aerobic growth conditions but not under anaerobic growth conditions. The results provide the in vivo evidence that IscA may act as an iron chaperone for the biogenesis of iron-sulfur clusters in E. coli cells under aerobic conditions.
Iron-sulfur cluster biogenesis; human IscA homologue; intracellular iron content
Bacteria use three distinct systems for iron-sulfur (Fe/S) cluster biogenesis: the ISC, SUF, and NIF machineries. The ISC and SUF systems are widely distributed, and many bacteria possess both of them. In Escherichia coli, ISC is the major and constitutive system, whereas SUF is induced under iron starvation and/or oxidative stress. Genomic analysis of the Fe/S cluster biosynthesis genes in Bacillus subtilis suggests that this bacterium's genome encodes only a SUF system consisting of a sufCDSUB gene cluster and a distant sufA gene. Mutant analysis of the putative Fe/S scaffold genes sufU and sufA revealed that sufU is essential for growth under minimal standard conditions, but not sufA. The drastic growth retardation of a conditional mutant depleted of SufU was coupled with a severe reduction of aconitase and succinate dehydrogenase activities in total-cell lysates, suggesting a crucial function of SufU in Fe/S protein biogenesis. Recombinant SufU was devoid of Fe/S clusters after aerobic purification. Upon in vitro reconstitution, SufU bound an Fe/S cluster with up to ∼1.5 Fe and S per monomer. The assembled Fe/S cluster could be transferred from SufU to the apo form of isopropylmalate isomerase Leu1, rapidly forming catalytically active [4Fe-4S]-containing holo-enzyme. In contrast to native SufU, its D43A variant carried a Fe/S cluster after aerobic purification, indicating that the cluster is stabilized by this mutation. Further, we show that apo-SufU is an activator of the cysteine desulfurase SufS by enhancing its activity about 40-fold in vitro. SufS-dependent formation of holo-SufU suggests that SufU functions as an Fe/S cluster scaffold protein tightly cooperating with the SufS cysteine desulfurase.
Iron sulfur (Fe/S) proteins are ubiquitous and participate in multiple biological processes, from photosynthesis to DNA repair. Iron and sulfur are highly reactive chemical species, and the mechanisms allowing the multiprotein systems ISC and SUF to assist Fe/S cluster formation in vivo have attracted considerable attention. Here, A-Type components of these systems (ATCs for A-Type Carriers) are studied by phylogenomic and genetic analyses. ATCs that have emerged in the last common ancestor of bacteria were conserved in most bacteria and were acquired by eukaryotes and few archaea via horizontal gene transfers. Many bacteria contain multiple ATCs, as a result of gene duplication and/or horizontal gene transfer events. Based on evolutionary considerations, we could define three subfamilies: ATC-I, -II and -III. Escherichia coli, which has one ATC-I (ErpA) and two ATC-IIs (IscA and SufA), was used as a model to investigate functional redundancy between ATCs in vivo. Genetic analyses revealed that, under aerobiosis, E. coli IscA and SufA are functionally redundant carriers, as both are potentially able to receive an Fe/S cluster from IscU or the SufBCD complex and transfer it to ErpA. In contrast, under anaerobiosis, redundancy occurs between ErpA and IscA, which are both potentially able to receive Fe/S clusters from IscU and transfer them to an apotarget. Our combined phylogenomic and genetic study indicates that ATCs play a crucial role in conveying ready-made Fe/S clusters from components of the biogenesis systems to apotargets. We propose a model wherein the conserved biochemical function of ATCs provides multiple paths for supplying Fe/S clusters to apotargets. This model predicts the occurrence of a dynamic network, the structure and composition of which vary with the growth conditions. As an illustration, we depict three ways for a given protein to be matured, which appears to be dependent on the demand for Fe/S biogenesis.
Iron sulfur (Fe/S) proteins are found in all living organisms where they participate in a wide array of biological processes. Accordingly, genetic defects in Fe/S biogenesis yield pleiotropic phenotypes in bacteria and several syndromes in humans. Multiprotein systems that assist Fe/S cluster formation and insertion into apoproteins have been identified. Most systems include so-called A-type proteins (which we refer to as ATC proteins hereafter), which have an undefined role in Fe/S biogenesis. Phylogenomic analyses presented, here, reveal that the ATC gene is ancient, that it was already present in the last common ancestor of bacteria, and that it subsequently spread to eukaryotes via mitochondria or chloroplastic endosymbioses and to a few archaea via horizontal gene transfers. Proteobacteria are unusual in having multiple ATCs. We show by a genetic approach that the three ATC proteins of E. coli are potentially interchangeable, but that redundancy is limited in vivo, either because of gene expression control or because of inefficient Fe/S transfers between ATCs and other components within the Fe/S biogenesis pathway. The combined phylogenomic and genetic approaches allow us to propose that multiple ATCs enable E. coli to diversify the ways for conveying ready-made Fe/S clusters from components of the biogenesis systems to apotargets, and that environmental conditions influence which pathway is used.
In this study, the function of two established Fe-S cluster biogenesis pathways, Isc (Iron sulfur cluster) and Suf (Sulfur mobilization), was compared under aerobic and anaerobic growth conditions by measuring the activity of the Escherichia coli global anaerobic regulator FNR. A [4Fe-4S] cluster is required for activity of FNR under anaerobic conditions. Assaying expression of FNR-dependent promoters in strains containing various deletions of the iscSUAhscBAfdx operon, revealed that under anaerobic conditions FNR activity was reduced by 60% in the absence of the Isc pathway. In contrast, a mutant lacking the entire Suf pathway had normal FNR activity, although overexpression of the suf operon fully rescued the anaerobic defect in FNR activity in strains lacking the Isc pathway. Expression of the sufA promoter and levels of SufD protein were upregulated 2–3 fold in Isc− strains under anaerobic conditions, suggesting that increased expression of the Suf pathway may be partially responsible for the FNR activity remaining in strains lacking the Isc pathway. In contrast, use of the O2-stable [4Fe-4S] cluster FNR variant, FNR-L28H, showed that overexpression of the suf operon did not restore FNR activity to strains lacking the Isc pathway under aerobic conditions. In addition, activity of FNR-L28H was more impaired under aerobic conditions compared to anaerobic conditions. The greater requirement for the Isc pathway under aerobic conditions was not due to a change in the rate of Fe-S cluster acquisition by FNR-L28H between aerobic and anaerobic conditions as shown by 55Fe labelling experiments. Using 35S-methionine pulse-chase assays, we observed that the Isc pathway, but not the Suf pathway, is the major pathway required for conversion of O2-inactivated apo-FNR to [4Fe-4S]-FNR upon the onset of anaerobic growth conditions. Taken together, these findings indicate a major role for the Isc pathway in FNR Fe-S cluster biogenesis under both aerobic and anaerobic conditions.
FNR; Fe-S cluster biogenesis; Isc; Suf; oxygen
A-type carrier (ATC) proteins of the Isc (iron-sulfur cluster) and Suf (sulfur mobilization) iron-sulfur ([Fe-S]) cluster biogenesis pathways are proposed to traffic preformed [Fe-S] clusters to apoprotein targets. In this study, we analyzed the roles of the ATC proteins ErpA, IscA, and SufA in the maturation of the nitrate-inducible, multisubunit anaerobic respiratory enzymes formate dehydrogenase N (Fdh-N) and nitrate reductase (Nar). Mutants lacking SufA had enhanced activities of both enzymes. While both Fdh-N and Nar activities were strongly reduced in an iscA mutant, both enzymes were inactive in an erpA mutant and in a mutant unable to synthesize the [Fe-S] cluster scaffold protein IscU. It could be shown for both Fdh-N and Nar that loss of enzyme activity correlated with absence of the [Fe-S] cluster-containing small subunit. Moreover, a slowly migrating form of the catalytic subunit FdnG of Fdh-N was observed, consistent with impeded twin arginine translocation (TAT)-dependent transport. The highly related Fdh-O enzyme was also inactive in the erpA mutant. Although the Nar enzyme has its catalytic subunit NarG localized in the cytoplasm, it also exhibited aberrant migration in an erpA iscA mutant, suggesting that these modular enzymes lack catalytic integrity due to impaired cofactor biosynthesis. Cross-complementation experiments demonstrated that multicopy IscA could partially compensate for lack of ErpA with respect to Fdh-N activity but not Nar activity. These findings suggest that ErpA and IscA have overlapping roles in assembly of these anaerobic respiratory enzymes but demonstrate that ErpA is essential for the production of active enzymes.
Iron sulfur (Fe-S) clusters are versatile biological cofactors that require biosynthetic systems in vivo to be assembled. In Escherichia coli the Isc (iscRSUA-hscBA-fdx-iscX) and the Suf (sufABCDSE) pathways fulfill this function. Despite extensive biochemical and genetic analysis of both pathways, the physiological function of the A-type proteins of each pathway (IscA and SufA) is still unclear. Studies conducted in vitro suggest two possible functions for A-type proteins, as Fe-S scaffold/transfer proteins or as iron donors during cluster assembly. To resolve this issue, SufA was co-expressed in vivo with its cognate partner proteins from the suf operon, SufBCDSE. Native SufA purified anaerobically using this approach was unambiguously demonstrated to be a [2Fe-2S] protein by biochemical analysis and UV-Visible, Mössbauer, resonance Raman, and EPR spectroscopy. Furthermore, native [2Fe-2S] SufA can transfer its Fe-S cluster to both [2Fe-2S] and [4Fe-4S] apoproteins. These results clearly show that A-type proteins form Fe-S clusters in vivo and are competent to function as Fe-S transfer proteins as purified. This study resolves the contradictory results from previous in vitro studies and demonstrates the critical importance of providing in vivo partner proteins during protein over-expression to allow correct biochemical maturation of metalloproteins.
Iron-sulfur; Suf; Biosynthesis; Mösbbauer; A-type protein; Scaffold; Transfer; Ferredoxin; Aconitase
During anaerobic growth Escherichia coli synthesizes two membrane-associated hydrogen-oxidizing [NiFe]-hydrogenases, termed hydrogenase 1 and hydrogenase 2. Each enzyme comprises a catalytic subunit containing the [NiFe] cofactor, an electron-transferring small subunit with a particular complement of [Fe-S] (iron-sulfur) clusters and a membrane-anchor subunit. How the [Fe-S] clusters are delivered to the small subunit of these enzymes is unclear. A-type carrier (ATC) proteins of the Isc (iron-sulfur-cluster) and Suf (sulfur mobilization) [Fe-S] cluster biogenesis pathways are proposed to traffic pre-formed [Fe-S] clusters to apoprotein targets. Mutants that could not synthesize SufA had active hydrogenase 1 and hydrogenase 2 enzymes, thus demonstrating that the Suf machinery is not required for hydrogenase maturation. In contrast, mutants devoid of the IscA, ErpA or IscU proteins of the Isc machinery had no detectable hydrogenase 1 or 2 activities. Lack of activity of both enzymes correlated with the absence of the respective [Fe-S]-cluster-containing small subunit, which was apparently rapidly degraded. During biosynthesis the hydrogenase large subunits receive their [NiFe] cofactor from the Hyp maturation machinery. Subsequent to cofactor insertion a specific C-terminal processing step occurs before association of the large subunit with the small subunit. This processing step is independent of small subunit maturation. Using western blotting experiments it could be shown that although the amount of each hydrogenase large subunit was strongly reduced in the iscA and erpA mutants, some maturation of the large subunit still occurred. Moreover, in contrast to the situation in Isc-proficient strains, these processed large subunits were not membrane-associated. Taken together, our findings demonstrate that both IscA and ErpA are required for [Fe-S] cluster delivery to the small subunits of the hydrogen-oxidizing hydrogenases; however, delivery of the Fe atom to the active site might have different requirements.
Iron-sulfur (Fe-S) clusters are inorganic cofactors required for a variety of biological processes. In vivo biogenesis of Fe-S clusters proceeds via complex pathways involving multiple protein complexes. In the Suf Fe-S cluster biogenesis system, SufB may be a scaffold for nascent Fe-S cluster assembly whereas SufA is proposed to act as either a scaffold or an Fe-S cluster carrier from the scaffold to target apo-proteins. However, SufB can form multiple stable complexes with other Suf proteins, such as SufB2C2 and SufBC2D and the specific functions of these complexes in Fe-S cluster assembly are not clear. Here we compare the ability of the SufB2C2 and SufBC2D complexes as well as SufA to promote in vitro maturation of the [2Fe-2S] ferredoxin (Fdx). We found that SufB2C2 was most proficient as a scaffold for de novo assembly of holo-Fdx using sulfide and iron as freely available building blocks while SufA was best at direct transfer of a pre-formed Fe-S cluster to Fdx. Furthermore, cluster transfer from [4Fe-4S] SufB2C2 or SufBC2D to Fdx will proceed through a SufA intermediate to Fdx is SufA is present. Finally, addition of ATP repressed cluster transfer from [4Fe-4S] SufB2C2 to Fdx and from SufBC2D to [2Fe-2S] SufA or Fdx. These studies indicate that SufB2C2 can serve as a terminal scaffold to load the SufA Fe-S cluster carrier for in vitro maturation of [2Fe-2S] enzymes like Fdx. This work is the first to systematically compare the cluster transfer rates of a scaffold (SufB) to the transfer rates of a carrier (SufA) under the same conditions to the same target enzyme and is also the first to reconstitute the full transfer pathway (from scaffold to carrier to target enzyme) in a single reaction.
Iron-sulfur clusters are key iron cofactors in biological pathways ranging from nitrogen fixation to respiration. Due to the toxicity of ferrous iron and sulfide to the cell, in vivo Fe-S cluster assembly is carried out by multi-protein biosynthetic pathways. Fe-S cluster assembly proteins traffic iron and sulfide, assemble nascent Fe-S clusters, and correctly transfer Fe-S clusters to the appropriate target metalloproteins in vivo. The gram-negative bacterium E. coli contains a stress-responsive Fe-S cluster assembly system, the SufABCDSE pathway, that functions under iron starvation and oxidative stress conditions that compromise Fe-S homeostasis. Using a combination of protein-protein interaction and in vitro Fe-S cluster assembly assays, we have characterized the relative roles of the SufBCD complex and the SufA protein during Suf Fe-S cluster biosynthesis. These studies reveal that SufA interacts with SufBCD in order to accept Fe-S clusters formed de novo on the SufBCD complex. Our results represent the first biochemical evidence that the SufBCD complex within the Suf pathway functions as a novel Fe-S scaffold system to assemble nascent clusters and transfer them to the SufA Fe-S shuttle.
Iron-sulfur clusters are ubiquitous and evolutionarily ancient inorganic prosthetic groups, the biosynthesis of which depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in Gram-positive bacteria. Within the Firmicutes phylum, the Enterococcus spp. genus have recently assumed importance in clinical microbiology being considered as emerging pathogens for humans, wherein Enterococcus faecalis represents the major species associated with nosocomial infections. The aim of this study was to carry out a phylogenetic analysis in Enterococcus faecalis V583 and a structural and conformational characterisation of it SufU protein.
BLAST searches of the Enterococcus genome revealed a series of genes with sequence similarity to the Escherichia coli SUF machinery of [Fe-S] cluster biosynthesis, namely sufB, sufC, sufD and SufS. In addition, the E. coli IscU ortholog SufU was found to be the scaffold protein of Enterococcus spp., containing all features considered essential for its biological activity, including conserved amino acid residues involved in substrate and/or co-factor binding (Cys50,76,138 and Asp52) and, phylogenetic analyses showed a close relationship with orthologues from other Gram-positive bacteria. Molecular dynamics for structural determinations and molecular modeling using E. faecalis SufU primary sequence protein over the PDB:1su0 crystallographic model from Streptococcus pyogenes were carried out with a subsequent 50 ns molecular dynamic trajectory. This presented a stable model, showing secondary structure modifications near the active site and conserved cysteine residues. Molecular modeling using Haemophilus influenzae IscU primary sequence over the PDB:1su0 crystal followed by a MD trajectory was performed to analyse differences in the C-terminus region of Gram-positive SufU and Gram-negative orthologous proteins, in which several modifications in secondary structure were observed.
The data describe the identification of the SUF machinery for [Fe-S] cluster biosynthesis present in the Firmicutes genome, showing conserved sufB, sufC, sufD and sufS genes and the presence of the sufU gene coding for scaffold protein, instead of sufA; neither sufE nor sufR are present. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU nearby on the ISC machinery. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes denoting the Gram positive region (GPR), suggested as a possible candidate for interaction with other factors and/or regulators.
A human homologue of the iron-sulfur cluster assembly protein IscA (hIscA1) has been cloned and expressed in Escherichia coli cells. The UV-visible absorption and EPR (electron paramagnetic resonance) measurements reveal that hIscA1 purified from E. coli cells contains a mononuclear iron center and that the iron binding in hIscA1 expressed in E. coli cells can be further modulated by the iron content in the cell growth medium. Additional studies show that purified hIscA1 binds iron with an iron association constant of approx. 2.0 × 1019 M−1, and that the iron-bound hIscA1 is able to provide the iron for the iron-sulfur cluster assembly in a proposed scaffold protein IscU of E. coli in vitro. The complementation experiments indicate that hIscA1 can partially substitute for IscA in restoring the cell growth of E. coli in the M9 minimal medium under aerobic conditions. The results suggest that human IscA1, like E. coli IscA, is an iron binding protein that may act as an iron chaperone for biogenesis of iron-sulfur clusters.
Iron-sulfur cluster biogenesis; human IscA homologue; intracellular iron content
Protein-bound dinitrosyl iron complexes (DNICs) have been observed in prokaryotic and eukaryotic cells under nitric oxide (NO) stress. The identity of proteins that bind DNICs, however, still remains elusive. Here we demonstrate that iron-sulfur proteins are the major source of protein-bound DNICs formed in Escherichia coli cells under NO stress. Expression of recombinant iron-sulfur proteins, but not the proteins without iron-sulfur clusters, almost doubles the amount of protein-bound DNICs formed in E. coli cells after NO exposure. Purification of recombinant proteins from the NO-exposed E. coli cells further confirms that iron-sulfur proteins, but not the proteins without iron-sulfur clusters, are modified forming protein-bound DINCs. Deletion of the iron-sulfur cluster assembly proteins IscA and SufA to block the [4Fe-4S] cluster biogenesis in E. coli cells largely eliminates the NO-mediated formation of protein-bound DNICs, suggesting that iron-sulfur clusters are mainly responsible for the NO-mediated formation of protein-bound DNICs in cells. Furthermore, depletion of “chelatable iron pool” in the wild-type E. coli cells effectively removes iron-sulfur clusters from proteins and concomitantly diminishes the NO-mediated formation of protein-bound DNICs, indicating that iron-sulfur clusters in proteins constitute at least part of “chelatable iron pool” in cells.
nitric oxide; iron-sulfur clusters; chelatable iron pool; dinitrosyl iron complex
Environmental H2O2 creates several injuries in Escherichia coli, including the oxidative conversion of dehydratase [4Fe-4S] clusters to an inactive [3Fe-4S] form. To protect itself, H2O2-stressed E. coli activates the OxyR regulon. This regulon includes the suf operon, which encodes an alternative to the housekeeping Isc iron-sulfur-cluster assembly system. Previously studied [3Fe-4S] clusters are repaired by an Isc/Suf-independent pathway, so the rationale for Suf induction was not obvious. Using strains that cannot scavenge H2O2, we imposed chronic low-grade stress and found that suf mutants could not maintain the activity of isopropylmalate isomerase, a key iron-sulfur dehydratase. Experiments showed that its damaged cluster was degraded in vivo beyond the [3Fe-4S] state, presumably to an apoprotein form, and thus required a de novo assembly system for reactivation. Surprisingly, sub-micromolar H2O2 poisoned the Isc machinery, thereby creating a requirement for Suf both to repair the isomerase and to activate nascent Fe-S enzymes in general. The IscS and IscA components of the Isc system are H2O2-resistant, suggesting that oxidants disrupt Isc by oxidizing clusters as they are assembled on or transferred from the IscU scaffold. Consistent with these results, organisms that are routinely exposed to oxidants rely upon Suf rather than Isc for cluster assembly.
Iron-sulfur clusters; the Suf system; the Isc system; and oxidative stress
During oxidative stress in E. coli, the SufABCDSE stress response pathway mediates iron-sulfur (Fe-S) cluster biogenesis rather than the Isc pathway. To determine why the Suf pathway is favored under stress conditions, the stress response SufS-SufE sulfur transfer pathway and the basal housekeeping IscS-IscU pathway were directly compared. We found that SufS-SufE cysteine desulfurase activity is significantly higher than IscS-IscU at physiological cysteine concentrations and after exposure to H2O2. Mass spectrometry analysis demonstrated that IscS-IscU is more susceptible than SufS-SufE to oxidative modification by H2O2. These important results provide biochemical insight into the stress resistance of the Suf pathway.
The assembly of iron-sulfur (Fe-S) clusters involves several pathways and in prokaryotes the mobilization of the sulfur (SUF) system is paramount for Fe-S biogenesis and repair during oxidative stress. The prokaryotic SUF system consists of six proteins: SufC is an ABC/ATPase that forms a complex with SufB and SufD, SufA acts as a scaffold protein, and SufE and SufS are involved in sulfur mobilization from cysteine. Despite the importance of Fe-S proteins in higher plant plastids, little is known regarding plastidic Fe-S cluster assembly. We have recently shown that Arabidopsis harbors an evolutionary conserved plastidic SufC protein (AtNAP7) capable of hydrolyzing ATP and interacting with the SufD homolog AtNAP6. Based on this and the prokaryotic SUF system we speculated that a SufB-like protein may exist in plastids. Here we demonstrate that the Arabidopsis plastid-localized SufB homolog AtNAP1 can complement SufB deficiency in Escherichia coli during oxidative stress. Furthermore, we demonstrate that AtNAP1 can interact with AtNAP7 inside living chloroplasts suggesting the presence of a plastidic AtNAP1·AtNAP6·AtNAP7 complex and remarkable evolutionary conservation of the SUF system. However, in contrast to prokaryotic SufB proteins with no associated ATPase activity we show that AtNAP1 is an iron-stimulated ATPase and that AtNAP1 is capable of forming homodimers. Our results suggest that AtNAP1 represents an atypical plastidic SufB-like protein important for Fe-S cluster assembly and for regulating iron homeostasis in Arabidopsis.
Shigella flexneri, a causative agent of bacterial dysentery, possesses two predicted iron-sulfur cluster biosynthesis systems called Suf and Isc. S. flexneri strains containing deletion mutations in the entire suf operon (UR011) or the iscSUA genes (UR022) were constructed. Both mutants were defective in surviving exposure to oxidative stress. The suf mutant showed growth that was comparable to that of the parental strain in both iron-replete and iron-limiting media; however, the isc mutant showed reduced growth, relative to the parental strain, in both media. Although the suf mutant formed wild-type plaques on Henle cell monolayers, the isc mutant was unable to form plaques on Henle cell monolayers because the strain was noninvasive. Expression from both the suf and isc promoters increased in iron-limiting media and in the presence of hydrogen peroxide. Iron repression of the suf promoter was mediated by Fur, and increased suf expression in iron-limiting media was enhanced by the presence of IscR. Iron repression of the isc promoter was mediated by IscR. Hydrogen peroxide-dependent induction of suf expression, but not isc expression, was mediated by OxyR. Furthermore, IscR was a positive regulator of suf expression in the presence of hydrogen peroxide and a negative regulator of isc expression in the absence of hydrogen peroxide. Expression from the S. flexneri suf and isc promoters increased when Shigella was within Henle cells, and our data suggest that the intracellular signal mediating this increased expression is reduced iron levels.
The apicoplast organelle of the malaria parasite Plasmodium falciparum contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors, however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology. We investigated two putative FeS cluster synthesis pathways (Isc and Suf) focusing on the initial step of sulfur acquisition. In other eukaryotes, these proteins can be located in multiple subcellular compartments, raising the possibility of cross-talk between the pathways or redundant functions. In P. falciparum, SufS and its partner SufE were found exclusively the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome – a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. Taken together, these results demonstrate that the Suf pathway is essential for parasite survival and has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis.
Iron is essential for the survival of blood stage P. falciparum and is used primarily in the synthesis of iron-sulfur (FeS) cluster cofactors. We investigated the role that (FeS) clusters play in malaria parasites. We demonstrated that the synthesis of FeS clusters is partitioned between two organelles: the Isc pathway is mitochondrial while the Suf pathway is found exclusively in the apicoplast organelle. Attempts to interfere with the Suf pathway through a dominant negative approach were only successful when parasite cultures were supplemented with an isoprenoid product. This result demonstrates that isoprenoid biosynthesis depends on a functional Suf pathway. Unexpectedly, we also observed the complete loss of the apicoplast organelle when we disrupted the Suf pathway. This phenotype does not result from inhibition of isoprenoid biosynthesis; we treated parasites with high levels of the isoprenoid inhibitor fosmidomycin without any loss of the apicoplast organelle. These results demonstrate that the Suf pathway has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis. Inhibition of the Suf pathway, which is not found in humans, will block the growth of malaria parasites.
In vivo biogenesis of Fe-S cluster cofactors requires complex biosynthetic machinery to limit release of iron and sulfide, to protect the Fe-S cluster from oxidation, and to target the Fe-S cluster to the correct apo-enzyme. The SufABCDSE pathway for Fe-S cluster assembly in E. coli accomplishes these tasks under iron starvation and oxidative stress conditions that disrupt Fe-S cluster metabolism. Although SufB, SufC, and SufD are all required for in vivo Suf function, their exact roles are unclear. Here we show that SufB, SufC, and SufD, co-expressed with the SufS-SufE sulfur transfer pair, purify as two distinct complexes (SufBC2D and SufB2C2) that contain Fe-S clusters and FADH2. These studies also show that SufC and SufD are required for in vivo Fe-S cluster formation on SufB. Furthermore, while SufD is dispensable for in vivo sulfur transfer, it is absolutely required for in vivo iron acquisition. Finally, we demonstrate for the first time that the ATPase activity of SufC is necessary for in vivo iron acquisition during Fe-S cluster assembly.
Iron-sulfur [Fe-S] clusters are ubiquitous and critical cofactors in diverse biochemical processes. They are assembled by distinct [Fe-S] cluster biosynthesis pathways, typically in organelles of endosymbiotic origin. Apicomplexan parasites, including Plasmodium, the causative agent of malaria, harbor two separate [Fe-S] cluster biosynthesis pathways in the their mitochondrion and apicoplast. In this study, we systematically targeted the five nuclear-encoded sulfur utilization factors (SUF) of the apicoplast [Fe-S] cluster biosynthesis pathway by experimental genetics in the murine malaria model parasite Plasmodium berghei. We show that four SUFs, namely SUFC, D, E, and S are refractory to targeted gene deletion, validating them as potential targets for antimalarial drug development. We achieved targeted deletion of SUFA, which encodes a potential [Fe-S] transfer protein, indicative of a dispensable role during asexual blood stage growth in vivo. Furthermore, no abnormalities were observed during Plasmodium life cycle progression in the insect and mammalian hosts. Fusion of a fluorescent tag to the endogenous P. berghei SUFs demonstrated that all loci were accessible to genetic modification and that all five tagged SUFs localize to the apicoplast. Together, our experimental genetics analysis identifies the key components of the SUF [Fe-S] cluster biosynthesis pathway in the apicoplast of a malarial parasite and shows that absence of SUFC, D, E, or S is incompatible with Plasmodium blood infection in vivo.
The human mitochondrial proteins ISCA1, ISCA2, and IBA57 are essential for the generation of mitochondrial [4Fe-4S] proteins in a late step of Fe/S protein biogenesis. This process is important for mitochondrial physiology, as documented by drastic enlargement of the organelles and the loss of cristae membranes in the absence of these proteins.
Members of the bacterial and mitochondrial iron–sulfur cluster (ISC) assembly machinery include the so-called A-type ISC proteins, which support the assembly of a subset of Fe/S apoproteins. The human genome encodes two A-type proteins, termed ISCA1 and ISCA2, which are related to Saccharomyces cerevisiae Isa1 and Isa2, respectively. An additional protein, Iba57, physically interacts with Isa1 and Isa2 in yeast. To test the cellular role of human ISCA1, ISCA2, and IBA57, HeLa cells were depleted for any of these proteins by RNA interference technology. Depleted cells contained massively swollen and enlarged mitochondria that were virtually devoid of cristae membranes, demonstrating the importance of these proteins for mitochondrial biogenesis. The activities of mitochondrial [4Fe-4S] proteins, including aconitase, respiratory complex I, and lipoic acid synthase, were diminished following depletion of the three proteins. In contrast, the mitochondrial [2Fe-2S] enzyme ferrochelatase and cellular heme content were unaffected. We further provide evidence against a localization and direct Fe/S protein maturation function of ISCA1 and ISCA2 in the cytosol. Taken together, our data suggest that ISCA1, ISCA2, and IBA57 are specifically involved in the maturation of mitochondrial [4Fe-4S] proteins functioning late in the ISC assembly pathway.
A system for the controlled expression of genes in Azotobacter vinelandii by using genomic fusions to the sucrose catabolic regulon was developed. This system was used for the functional analysis of the A. vinelandii isc genes, whose products are involved in the maturation of [Fe-S] proteins. For this analysis, the scrX gene, contained within the sucrose catabolic regulon, was replaced by the contiguous A. vinelandii iscS, iscU, iscA, hscB, hscA, fdx, and iscX genes, resulting in duplicate genomic copies of these genes: one whose expression is directed by the normal isc regulatory elements (Pisc) and the other whose expression is directed by the scrX promoter (PscrX). Functional analysis of [Fe-S] protein maturation components was achieved by placing a mutation within a particular Pisc-controlled gene with subsequent repression of the corresponding PscrX-controlled component by growth on glucose as the carbon source. This experimental strategy was used to show that IscS, IscU, HscBA, and Fdx are essential in A. vinelandii and that their depletion results in a deficiency in the maturation of aconitase, an enzyme that requires a [4Fe-4S] cluster for its catalytic activity. Depletion of IscA results in a null growth phenotype only when cells are cultured under conditions of elevated oxygen, marking the first null phenotype associated with the loss of a bacterial IscA-type protein. Furthermore, the null growth phenotype of cells depleted of HscBA could be partially reversed by culturing cells under conditions of low oxygen. Conserved amino acid residues within IscS, IscU, and IscA that are essential for their respective functions and/or whose replacement results in a partial or complete dominant-negative growth phenotype were also identified using this system.
Crystal structures reveal how distinct sites on the cysteine desulfurase IscS bind two different sulfur-acceptor proteins, IscU and TusA, to transfer sulfur atoms for iron-sulfur cluster biosynthesis and tRNA thiolation.
The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer via persulfide to a range of acceptor proteins involved in Fe-S cluster assembly, tRNA modifications, and sulfur-containing cofactor biosynthesis. Several IscS-interacting partners including IscU, a scaffold for Fe-S cluster assembly; TusA, the first member of a sulfur relay leading to sulfur incorporation into the wobble uridine of several tRNAs; ThiI, involved in tRNA modification and thiamine biosynthesis; and rhodanese RhdA are sulfur acceptors. Other proteins, such as CyaY/frataxin and IscX, also bind to IscS, but their functional roles are not directly related to sulfur transfer. We have determined the crystal structures of IscS-IscU and IscS-TusA complexes providing the first insight into their different modes of binding and the mechanism of sulfur transfer. Exhaustive mutational analysis of the IscS surface allowed us to map the binding sites of various partner proteins and to determine the functional and biochemical role of selected IscS and TusA residues. IscS interacts with its partners through an extensive surface area centered on the active site Cys328. The structures indicate that the acceptor proteins approach Cys328 from different directions and suggest that the conformational plasticity of a long loop containing this cysteine is essential for the ability of IscS to transfer sulfur to multiple acceptor proteins. The sulfur acceptors can only bind to IscS one at a time, while frataxin and IscX can form a ternary complex with IscU and IscS. Our data support the role of frataxin as an iron donor for IscU to form the Fe-S clusters.
Sulfur is incorporated into the backbone of almost all proteins in the form of the amino acids cysteine and methionine. In some proteins, sulfur is also present as iron–sulfur clusters, sulfur-containing vitamins, and cofactors. What's more, sulfur is important in the structure of tRNAs, which are crucial for translation of the genetic code from messenger RNA for protein synthesis. The biosynthetic pathways for assembly of these sulfur-containing molecules are generally well known, but the molecular details of how sulfur is delivered from protein to protein are less well understood. In bacteria, one of three pathways for sulfur delivery is the isc (iron-sulfur clusters) system. First, an enzyme called IscS extracts sulfur atoms from cysteine. This versatile enzyme can then interact with several proteins to deliver sulfur to various pathways that make iron–sulfur clusters or transfer sulfur to cofactors and tRNAs. This study describes in atomic detail precisely how IscS binds in a specific and yet distinct way to two different proteins: IscU (a scaffold protein for iron–sulfur cluster formation) and TusA (which delivers sulfur for tRNA modification). Furthermore, by introducing mutations into IscS, we have identified the region on the surface of this protein that is involved in binding its target proteins. These findings provide a molecular view of the protein–protein interactions involved in sulfur transfer and advance our understanding of how sulfur is delivered from one protein to another during biosynthesis of iron–sulfur clusters.
Maturation of iron-sulfur proteins is achieved by the SUF machinery in a wide number of Eubacteria and Archaea as well as eukaryotic chloroplasts. This machinery is encoded in E. coli by the sufABCDSE operon, where three Suf components, SufB, SufC, and SufD, form a complex and appear to provide an intermediary site for the iron-sulfur cluster assembly. Here we report the quaternary structure of SufC2-SufD2 complex in which SufC is bound to the C-terminal domain of SufD. Comparison with the monomeric structure of SufC revealed conformational change of the active site residues: SufC becomes competent for ATP-binding and hydrolysis upon association with SufD. The two SufC subunits were spatially separated in the SufC2-SufD2 complex, whereas cross-linking experiments in solution have indicated that two SufC molecules associate with each other in the presence of Mg2+ and ATP. Such dimer formation of SufC may lead to a gross structural change of the SufC2-SufD2 complex. Furthermore, genetic analysis of SufD revealed an essential histidine residue buried inside the dimer interface, suggesting that conformational change may expose this crucial residue. These findings together with biochemical characterization of the SufB-SufC-SufD complex have led us to propose a model for the iron-sulfur cluster biosynthesis in the complex.
ATP-binding cassette ATPase; crystallography; iron-sulfur protein maturation; scaffold; SUF machinery
A regulatory network of Sinorhizobium meliloti genes involved in adaptation to iron-limiting conditions and the involvement of the rhizobial iron regulator gene (rirA) were analyzed by mutation and microarray analyses. A constructed S. meliloti rirA mutant exhibited growth defects and enhanced H2O2 sensitivity in the presence of iron, but symbiotic nitrogen fixation was not affected. To identify iron-responsive and RirA-regulated S. meliloti genes, a transcriptome approach using whole-genome microarrays was used. Altogether, 45 genes were found to be jointly derepressed by mutation of rirA and under different iron-limited conditions. As expected, a number of genes involved in iron transport (e.g., hmuPSTU, shmR, rhbABCDEF, rhtX, and rhtA) and also genes with predicted functions in energy metabolism (e.g., fixN3, fixP3, and qxtAB) and exopolysaccharide production (e.g., exoY and exoN) were found in this group of genes. In addition, the iron deficiency response of S. meliloti also involved rirA-independent expression changes, including repression of the S. meliloti flagellar regulon. Finally, the RirA modulon also includes genes that are not iron responsive, including a gene cluster putatively involved in Fe-S cluster formation (sufA, sufS, sufD, sufC, and sufB).