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.
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) 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
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
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
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.
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
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 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.
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 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.
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
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.
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.
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.
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
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.
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
Cloning, expression, purification, crystallization and data collection are reported for a member of the SufE family of proteins involved in the biosynthesis of Fe–S clusters in prokaryotes. Diffraction data were collected to 1.9 Å resolution and an interpretable electron-density map has been obtained by molecular replacement.
Xanthomonas axonopodis pv. citri (Xac) SufE (XAC2355) is a member of a family of bacterial proteins that are conserved in several pathogens and phytopathogens. The Escherichia coli suf operon is involved in iron–sulfur cluster biosynthesis under iron-limitation and stress conditions. It has recently been demonstrated that SufE and SufS form a novel two-component cysteine desulfarase in which SufS catalyses the conversion of l-cysteine to l-alanine, forming a protein-bound persulfide intermediate. The S atom is then transferred to SufE, from which it is subsequently transferred to target molecules or reduced to sulfide in solution. Here, the cloning, expression, crystallization and phase determination of Xac SufE crystals are described. Recombinant SufE was crystallized in space group P212121 and diffracted to 1.9 Å resolution at a synchrotron source. The unit-cell parameters are a = 45.837, b = 58.507, c = 98.951 Å, α = β = γ = 90°. The calculated Matthews coefficient indicated the presence of two molecules in the asymmetric unit. Phasing was performed by molecular-replacement using E. coli SufE as a model (PDB code 1mzg) and an interpretable map was obtained.
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.
Escherichia coli fhuF mutants, a sufS::MudI mutant, and a sufD::MudI mutant were found to have the same phenotype: the inability to use ferrioxamine B as an iron source in a plate assay. In addition, the sufS and sufD genes were shown to be regulated by the iron-dependent Fur repressor. Sequence analysis revealed that the sufS open reading frame corresponds to orf f406. The protein SufS belongs to the family of NifS-like proteins, which supply sulfur for [Fe-S] centers. The protein FhuF contains a [2Fe-2S] center. A mutation in the upstream sufD gene (orf f423) caused the same phenotype. The T7 expression system and a His tag allow the isolation in good yield of the FhuF protein from a wild-type strain. In contrast, overproduction of the protein in a ΔsufD strain failed. Radioactive labeling of N-His-FhuF with [35S]methionine showed that the protein was unstable in the ΔsufD mutant.
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.
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 metabolic consequences of two insertions, iscR1::MudJ and iscA2::MudJ, in the isc gene cluster of Salmonella enterica serovar Typhimurium were studied. Each of these insertions had polar effects and caused a nutritional requirement for the thiazole moiety of thiamine. Data showed that IscS was required for the synthesis of nicotinic acid and the thiazole moiety of thiamine and that one or more additional isc gene products were required for a distinct step in the thiazole biosynthetic pathway. Strains with isc lesions had reduced succinate dehydrogenase and aconitase activities. Furthermore, isc mutants accumulated increased levels of pyruvate in the growth medium in response to exogenously added iron (FeCl3), and this response required a functional ferric uptake regulator, Fur.
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.