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
IscA/SufA paralogs are the members of the iron-sulfur cluster assembly machinery in Escherichia coli. While deletion of either IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA results in a null-growth phenotype in minimal medium under aerobic growth conditions. Here we report that cell growth of the iscA/sufA double mutant (E. coli strain in which both iscA and sufA had been in-frame-deleted) can be partially restored by supplementing with BCAAs (branched-chain amino acids) and thiamin. We further demonstrate that deletion of IscA/SufA paralogs blocks the [4Fe-4S] cluster assembly in IlvD (dihydroxyacid dehydratase) of the BCAA biosynthesis pathway in E. coli cells under aerobic conditions and that addition of the iron-bound IscA/SufA efficiently promotes the [4Fe-4S] cluster assembly in IlvD and restores the enzyme activity in vitro, suggesting that IscA/SufA may act as an iron donor for the [4Fe-4S] cluster assembly under aerobic conditions. Additional studies reveal that IscA/SufA are also required for the [4Fe-4S] cluster assembly in protein ThiC of the thiamin biosynthesis pathway, aconitase B of the citrate acid cycle, and endonuclease III of the DNA base excision repair pathway in E. coli under aerobic conditions. Nevertheless, deletion of IscA/SufA does not significantly affect the [2Fe-2S] cluster assembly in the redox transcription factor SoxR, ferredoxin, and the siderophore-iron reductase FhuF. The results suggest that the biogenesis of the [4Fe-4S] clusters and the [2Fe-2S] clusters may have distinct pathways and that IscA/SufA paralogs are essential for the [4Fe-4S] cluster assembly, but are dispensable for the [2Fe-2S] cluster assembly in E. coli under aerobic conditions.
aconitase; branched-chain amino acids; dihydroxyacid dehydratase; iron-sulfur clusters; IscA/SufA paralogs; thiamin
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.
Escherichia coli [2Fe-2S]-ferredoxin
(Fdx) is encoded by the isc operon along with other
proteins involved in the ‘house-keeping’ mechanism of
iron–sulfur cluster biogenesis. Although it has been proposed
that Fdx supplies electrons to reduce sulfane sulfur (S0) produced by the cysteine desulfurase (IscS) to sulfide (S2–) as required for the assembly of Fe–S clusters on the scaffold
protein (IscU), direct experimental evidence for the role of Fdx has
been lacking. Here, we show that Fdx (in either oxidation state) interacts
directly with IscS. The interaction face on Fdx was found to include
residues close to its Fe–S cluster. In addition, C328 of IscS,
the residue known to pick up sulfur from the active site of IscS and
deliver it to the Cys residues of IscU, formed a disulfide bridge
with Fdx in the presence of an oxidizing agent. Electrons from reduced
Fdx were transferred to IscS only in the presence of l-cysteine,
but not to the C328S variant. We found that Fdx, IscU, and CyaY (the
bacterial frataxin) compete for overlapping binding sites on IscS.
This mutual exclusion explains the mechanism by which CyaY inhibits
Fe–S cluster biogenesis. These results (1) show that reduced
Fdx supplies one electron to the IscS complex as S0 is
produced by the enzymatic conversion of Cys to Ala and (2) explain
the role of Fdx as a member of the isc operon.
IscU is a scaffold protein that functions in iron-sulfur cluster assembly and transfer. Its critical importance has been recently underscored by the finding that a single intronic mutation in the human iscu gene is associated with a myopathy resulting from deficient succinate dehydrogenase and aconitase [Mochel, F., Knight, M. A., Tong, W. H., Hernandez, D., Ayyad, K., Taivassalo, T., Andersen, P. M., Singleton, A., Rouault, T. A., Fischbeck, K. H., and Haller, R. G. (2008) Am. J. Hum. Genet. 82, 652–660]. IscU functions through interactions with a chaperone protein HscA and a cochaperone protein HscB. To probe the molecular basis for these interactions, we have used NMR spectroscopy to investigate the solution structure of IscU from Escherichia coli and its interaction with HscB from the same organism. We found that wild-type apo-IscU in solution exists as two distinct conformations: one largely disordered and one largely ordered except for the metal binding residues. The two states interconvert on the millisecond time scale. The ordered conformation is stabilized by the addition of zinc or by the single site IscU mutation, D39A. We used apo-IscU(D39A) as a surrogate for the folded state of wild-type IscU and assigned its NMR spectrum. These assignments made it possible to identify the region of IscU with the largest structural differences in the two conformational states. Subsequently, by following the NMR signals of apo-IscU(D39A) upon addition of HscB, we identified the most perturbed regions as the two N-terminal β-strands and the C-terminal α-helix. On the basis of these results and analysis of IscU sequences from multiple species, we have identified the surface region of IscU that interacts with HscB. We conclude that the IscU:HscB complex exists as two (or more) distinct states that interconvert at a rate much faster than the dissociation of the complex and that HscB binds to and stabilizes the ordered state of apo-IscU.
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.
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.
Iron-sulphur cluster biogenesis requires coordinated delivery of iron and sulphur to scaffold proteins, followed by transfer of the assembled clusters from scaffold proteins to target proteins. This complex process is accomplished by a group of dedicated iron-sulphur cluster assembly proteins that are conserved from bacteria to humans. While sulphur in iron-sulphur clusters is provided by L-cysteine via cysteine desulfurase, the iron donor(s) for iron-sulphur cluster assembly remains largely elusive. Here we report that among the primary iron-sulphur cluster assembly proteins, IscA has a unique and strong binding activity for mononuclear iron in vitro and in vivo. Furthermore, the ferric iron centre tightly bound in IscA can be readily extruded by L-cysteine, followed by reduction to ferrous iron for iron-sulphur cluster biogenesis. Substitution of the highly conserved residue tyrosine 40 with phenylalanine (Y40F) in IscA results in a mutant protein that has a diminished iron binding affinity but retains the iron-sulphur cluster binding activity. Genetic complementation studies show that the IscA Y40F mutant is inactive in vivo, suggesting that the iron binding activity is essential for the function of IscA in iron-sulphur cluster biogenesis.
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.
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 clusters are one of the most ubiquitous redox centers in biology. Ironically, iron-sulfur clusters are highly sensitive to reactive oxygen species. Disruption of iron-sulfur clusters will not only change the activity of proteins that host iron-sulfur clusters, the iron released from the disrupted iron-sulfur clusters will further promote the production of deleterious hydroxyl free radicals via the Fenton reaction. Here, we report that ferritin A (FtnA), a major iron-storage protein in Escherichia coli, is able to scavenge the iron released from the disrupted iron-sulfur clusters and alleviates the production of hydroxyl free radicals. Furthermore, we find that the iron stored in ferritin A can be retrieved by an iron chaperon IscA for the re-assembly of the iron-sulfur cluster in a proposed scaffold IscU in the presence of the thioredoxin reductase system which emulates normal intracellular redox potential. The results suggest that E. coli ferritin A may act as an iron buffer to sequester the iron released from the disrupted iron-sulfur clusters under oxidative stress conditions and to facilitate the re-assembly of the disrupted iron-sulfur clusters under normal physiological conditions.
Ferritin A; hydroxyl free radicals; iron-sulfur clusters; IscA; IscU
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.
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
Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli
iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR in-frame deletion mutant (ΔiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF.
Bacterial pathogens use regulators that sense environmental cues to enhance their fitness. Here, we identify a transcriptional regulator in the human gut pathogen, Yersinia pseudotuberculosis, which controls a specialized secretion system essential for bacterial growth in mammalian tissues. This regulator was shown in other bacterial species to alter its activity in response to changes in iron concentration and oxidative stress, but has never been studied in Yersinia. Importantly, Y. pseudotuberculosis experiences large changes in iron bioavailability upon transit from the gut to deeper tissues and iron is a critical component in Yersinia virulence, as individuals with iron overload disorders have enhanced susceptibility to systemic Yersinia infections. Our work places this iron-modulated transcriptional regulator within the regulatory network that controls virulence gene expression in Y. pseudotuberculosis, identifying it as a potential new target for antimicrobial agents.
tRNA from Salmonella enterica serovar Typhimurium contains five thiolated nucleosides, 2-thiocytidine (s2C), 4-thiouridine (s4U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A). The levels of all of them are significantly reduced in cells with a mutated iscS gene, which encodes the cysteine desulfurase IscS, a member of the ISC machinery that is responsible for [Fe-S] cluster formation in proteins. A mutant (iscU52) was isolated that carried an amino acid substitution (S107T) in the IscU protein, which functions as a major scaffold in the formation of [Fe-S] clusters. In contrast to the iscS mutant, the iscU52 mutant showed reduced levels of only two of the thiolated nucleosides, ms2io6A (10-fold) and s2C (more than 2-fold). Deletions of the iscU, hscA, or fdx genes from the isc operon lead to a similar tRNA thiolation pattern to that seen for the iscU52 mutant. Unexpectedly, deletion of the iscA gene, coding for an alternative scaffold protein for the [Fe-S] clusters, showed a novel tRNA thiolation pattern, where the synthesis of only one thiolated nucleoside, ms2io6A, was decreased twofold. Based on our results, we suggest two principal distinct routes for thiolation of tRNA: (i) a direct sulfur transfer from IscS to the tRNA modifying enzymes ThiI and MnmA, which form s4U and the s2U moiety of (c)mnm5s2U, respectively; and (ii) an involvement of [Fe-S] proteins (an unidentified enzyme in the synthesis of s2C and MiaB in the synthesis of ms2io6A) in the transfer of sulfur to the tRNA.
The role of the Azotobacter vinelandii HscA/HscB co-chaperone system in ISC-mediated iron-sulfur cluster biogenesis has been investigated in vitro by using CD and EPR spectrometry to monitor the effect of HscA, HscB, MgATP, and MgADP on the time course of cluster transfer from [2Fe-2S]IscU to apo-Isc ferredoxin. CD spectra indicate that both HscB and HscA interact with [2Fe-2S] IscU and the rate of cluster transfer was stimulated more than 20-fold in the presence stoichiometric HscA and HscB and excess MgATP. No stimulation was observed in the absence of either HscB or MgATP and cluster transfer was found to be an ATP-dependent reaction based on concomitant phosphate production and the enhanced rates of cluster transfer in the presence of KCl which is known to stimulated HscA ATPase activity. The results demonstrate a role of the ISC HscA/HscB co-chaperone system in facilitating efficient [2Fe-2S] cluster transfer from the IscU scaffold protein to acceptor proteins and that [2Fe-2S] cluster transfer from IscU is an ATP-dependent process. The data are consistent with the proposed regulation of the HscA ATPase cycle by HscB and IscU (Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279, 53924-53931), and mechanistic proposals for coupling of the HscA ATPase cycle with cluster transfer from [2Fe-2S]IscU to apo-IscFdx are discussed.
Friedreich's ataxia is a neurodegenerative disorder caused by mutations in the frataxin gene that produces a predominantly mitochondrial protein whose primary function appears to be mitochondrial iron–sulfur cluster (ISC) biosynthesis. Previously we demonstrated that frataxin interacts with multiple components of the mammalian ISC assembly machinery. Here we demonstrate that frataxin interacts with the mammalian mitochondrial chaperone HSC20. We show that this interaction is iron-dependent. We also show that like frataxin, HSC20 interacts with multiple proteins involved in ISC biogenesis including the ISCU/Nfs1 ISC biogenesis complex and the GRP75 ISC chaperone. Furthermore, knockdown of HSC20 caused functional defects in activity of mitochondrial ISC-containing enzymes and also defects in ISC protein expression. Alterations up or down of frataxin expression caused compensatory changes in HSC20 expression inversely, as expected of two cooperating proteins operating in the same pathway and suggesting a potential therapeutic strategy for the disease. Knockdown of HSC20 altered cytosolic and mitochondrial iron pools and increased the expression of transferrin receptor 1 and iron regulatory protein 2 consistent with decreased iron bioavailability. These results indicate that HSC20 interacts with frataxin structurally and functionally and is important for ISC biogenesis and iron homeostasis in mammals. Furthermore, they suggest that HSC20 may act late in the ISC pathway as a chaperone in ISC delivery to apoproteins and that HSC20 should be included in multi-protein complex studies of mammalian ISC biogenesis.
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
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
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.
Reduced levels of frataxin, an essential protein of as yet unknown function, are responsible for causing the neurodegenerative pathology Friedreich's ataxia. Independent reports have linked frataxin to iron–sulphur cluster assembly through interactions with the two central components of this machinery: desulphurase Nfs1/IscS and the scaffold protein Isu/IscU. In this study, we use a combination of biophysical methods to define the structural bases of the interaction of CyaY (the bacterial orthologue of frataxin) with the IscS/IscU complex. We show that CyaY binds IscS as a monomer in a pocket between the active site and the IscS dimer interface. Recognition does not require iron and occurs through electrostatic interactions of complementary charged residues. Mutations at the complex interface affect the rates of enzymatic cluster formation. CyaY binding strengthens the affinity of the IscS/IscU complex. Our data suggest a new paradigm for understanding the role of frataxin as a regulator of IscS functions.
Frataxin is an essential protein that has been linked to iron–sulphur cluster assembly, and reduced levels are associated with Friedrich's ataxia. In this study, a combination of techniques is used to probe the interactions of the bacterial frataxin orthologue CyaY with the iron–sulphur cluster assembly machinery.
Biotin synthase (BioB) is an iron-sulfur enzyme that catalyzes the last step in biotin biosynthesis, the insertion of sulfur between the C6 and C9 carbons of dethiobiotin to complete the thiophane ring of biotin. Recent in vitro experiments suggest that the sulfur is derived from a [2Fe-2S]2+ cluster within BioB, and that the remnants of this cluster dissociate from the enzyme following each turnover. In order for BioB to catalyze multiple rounds of biotin synthesis, the [2Fe-2S]2+ cluster in BioB must be reassembled, a process that could be carried out in vivo by the ISC or SUF iron-sulfur cluster assembly systems. The bacterial ISC system includes HscA, an Hsp70-class molecular chaperone, whose yeast homolog has been shown to play an important but nonessential role in assembly of mitochondrial FeS clusters in S. cerevesiae. In the present work we show that in E. coli, HscA significantly improves the efficiency of the in vivo assembly of the [2Fe-2S]2+ cluster on BioB under conditions of low to moderate iron. In vitro, we show that HscA binds with increased affinity to BioB missing one or both FeS clusters, with a maximum of two HscA molecules per BioB dimer. BioB binds to HscA in an ATP/ADP-independent manner and a high affinity complex is also formed with a truncated form of HscA that lacks the nucleotide binding domain. Further, the BioB:HscA complex binds the FeS cluster scaffold protein IscU in a noncompetitive manner, generating a complex that contains all three proteins. We propose that HscA plays a role in facilitating the transfer of FeS clusters from IscU into the appropriate target apoproteins such as biotin synthase, perhaps by enhancing or prolonging the requisite protein:protein interaction.
Iron-sulfur (Fe/S) proteins are located in mitochondria, cytosol, and nucleus. Mitochondrial Fe/S proteins are matured by the iron-sulfur cluster (ISC) assembly machinery. Little is known about the formation of Fe/S proteins in the cytosol and nucleus. A function of mitochondria in cytosolic Fe/S protein maturation has been noted, but small amounts of some ISC components have been detected outside mitochondria. Here, we studied the highly conserved yeast proteins Isu1p and Isu2p, which provide a scaffold for Fe/S cluster synthesis. We asked whether the Isu proteins are needed for biosynthesis of cytosolic Fe/S clusters and in which subcellular compartment the Isu proteins are required. The Isu proteins were found to be essential for de novo biosynthesis of both mitochondrial and cytosolic Fe/S proteins. Several lines of evidence indicate that Isu1p and Isu2p have to be located inside mitochondria in order to perform their function in cytosolic Fe/S protein maturation. We were unable to mislocalize Isu1p to the cytosol due to the presence of multiple, independent mitochondrial targeting signals in this protein. Further, the bacterial homologue IscU and the human Isu proteins (partially) complemented the defects of yeast Isu protein-depleted cells in growth rate, Fe/S protein biogenesis, and iron homeostasis, yet only after targeting to mitochondria. Together, our data suggest that the Isu proteins need to be localized in mitochondria to fulfill their functional requirement in Fe/S protein maturation in the cytosol.
Iron-sulfur (Fe-S) clusters are ubiquitous cofactors that are crucial for many physiological processes in all organisms. In Escherichia coli, assembly of Fe-S clusters depends on the activity of the iron-sulfur cluster (ISC) assembly and sulfur mobilization (SUF) apparatus. However, the underlying molecular mechanisms and the mechanisms that control Fe-S cluster biogenesis and iron homeostasis are still poorly defined. In this study, we performed a global screen to identify the factors affecting Fe-S cluster biogenesis and iron homeostasis using the Keio collection, which is a library of 3,815 single-gene E. coli knockout mutants. The approach was based on radiolabeling of the cells with [2-14C]dihydrouracil, which entirely depends on the activity of an Fe-S enzyme, dihydropyrimidine dehydrogenase. We identified 49 genes affecting Fe-S cluster biogenesis and/or iron homeostasis, including 23 genes important only under microaerobic/anaerobic conditions. This study defines key proteins associated with Fe-S cluster biogenesis and iron homeostasis, which will aid further understanding of the cellular mechanisms that coordinate the processes. In addition, we applied the [2-14C]dihydrouracil-labeling method to analyze the role of amino acid residues of an Fe-S cluster assembly scaffold (IscU) as a model of the Fe-S cluster assembly apparatus. The analysis showed that Cys37, Cys63, His105, and Cys106 are essential for the function of IscU in vivo, demonstrating the potential of the method to investigate in vivo function of proteins involved in Fe-S cluster assembly.
The budding yeast Saccharomyces cerevisiae contains two homologues of bacterial IscA proteins, designated Isa1p and Isa2p. Bacterial IscA is a product of the isc (iron-sulfur cluster) operon and has been suggested to participate in Fe-S cluster formation or repair. To test the function of yeast Isa1p and Isa2p, single or combinatorial disruptions were introduced in ISA1 and ISA2. The resultant isaΔ mutants were viable but exhibited a dependency on lysine and glutamate for growth and a respiratory deficiency due to an accumulation of mutations in mitochondrial DNA. As with other yeast genes proposed to function in Fe-S cluster assembly, mitochondrial iron concentration was significantly elevated in the isa mutants, and the activities of the Fe-S cluster-containing enzymes aconitase and succinate dehydrogenase were dramatically reduced. An inspection of Isa-like proteins from bacteria to mammals revealed three invariant cysteine residues, which in the case of Isa1p and Isa2p are essential for function and may be involved in iron binding. As predicted, Isa1p is targeted to the mitochondrial matrix. However, Isa2p is present within the intermembrane space of the mitochondria. Our deletion analyses revealed that Isa2p harbors a bipartite N-terminal leader sequence containing a mitochondrial import signal linked to a second sequence that targets Isa2p to the intermembrane space. Both signals are needed for Isa2p function. A model for the nonredundant roles of Isa1p and Isa2p in delivering iron to sites of the Fe-S cluster assembly is discussed.