Interchangeability and distinct properties of bacterial Fe-S cluster assembly systems: functional replacement of the isc and suf operons in Escherichia coli with the nifSU-like operon from Helicobacter pylori

The assembly of iron-sulfur (Fe-S) clusters, a key step in the post-translational maturation of Fe-S proteins, is mediated by a complex apparatus. In E. coli, this process involves two independent systems called ISC and SUF encoded by the iscSUA-hscBA-fdx gene cluster and sufABCDSE operon, respectively. Another system, termed NIF (nifSU), is required for the maturation of nitrogenase in nitrogen-fixing bacteria. We have developed a novel genetic system to gain further insight into these multi-component systems, and to determine how ISC, SUF and NIF might differ in their roles in Fe-S assembly. We have constructed an E. coli mutant lacking both the isc and suf operons, and this strain can only survive in the presence of a complementing plasmid. Using the plasmid replacement technique, we examined the isc and suf operons, and identified the genes essential for the function. Additionally, we have found that nifSU-like genes cloned from Helicobacter pylori are functionally exchangeable with the isc and suf operons. Thus, the NIF-like system participates in the maturation of a wide variety of Fe-S proteins. An increased ability of NIF to complement isc and suf loss was seen under anaerobic conditions. This may explain why the NIF system is only found in a limited number of bacterial species, and most other organisms prefer the ISC and/or SUF systems. While the differences between ISC and SUF were small with respect to the complementing activity, the SUF system appears to be more advantageous for bacterial growth in the presence of hydrogen peroxide.     

A third bacterial system for the assembly of iron-sulfur clusters with homologs in archaea and plastids

The assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery. In several proteobacteria, this process involves ISC (Fe-S cluster assembly) machinery composed of at least six components also conserved in mitochondria from lower to higher eukaryotes. In nitrogen-fixing bacteria, another system, termed NIF (nitrogen fixation), is required for the maturation of nitrogenase. Here we report the identification of a third system, designated the SUF machinery, the components of which are encoded in Escherichia coli by an unassigned operon, sufABCDSE. We have analyzed spontaneous pseudorevertants isolated from a mutant strain lacking all the components of the ISC machinery. The suppressor mutations in the revertants have been localized to the regulatory region of the suf operon; overexpression of this operon restores the growth phenotypes and activity of Fe-S proteins in mutant cells lacking ISC. Disruption of the suf operon alone does not cause any major defects, but synthetic lethality was observed when both the isc and suf operons were inactivated. These results indicate that proteins encoded by the suf operon participate in the ISC-independent minor pathway for the assembly of Fe-S clusters. The genes homologous to sufBC are present in a wide range of bacteria, Archaea, and plastids, suggesting that this type of system is almost ubiquitous in nature.     

Effect of RyhB small RNA on global iron use in Escherichia coli

RyhB is a noncoding RNA regulated by the Fur repressor. It has previously been shown to cause the rapid degradation of a number of mRNAs that encode proteins that utilize iron. Here we examine the effect of ectopic RyhB production on global gene expression by microarray analysis. Many of the previously identified targets were found, as well as other mRNAs encoding iron-binding proteins, bringing the total number of regulated operons to at least 18, encoding 56 genes. The two major operons involved in Fe-S cluster assembly showed different behavior; the isc operon appears to be a direct target of RyhB action, while the suf operon does not. This is consistent with previous findings suggesting that the suf genes but not the isc genes are important for Fe-S cluster synthesis under iron-limiting conditions, presumably for essential iron-binding proteins. In addition, we observed repression of Fur-regulated genes upon RyhB expression, interpreted as due to intracellular iron sparing resulting from reduced synthesis of iron-binding proteins. Our results demonstrate the broad effects of a single noncoding RNA on iron homeostasis.     

Lack of YggX results in chronic oxidative stress and uncovers subtle defects in Fe-S cluster metabolism in Salmonella enterica

As components involved in Fe-S cluster metabolism are described, the challenge becomes defining the integrated process that occurs in vivo based on the individual functions characterized in vitro. Strains lacking yggX have been used here to mimic chronic oxidative stress and uncover subtle defects in Fe-S cluster metabolism. We describe the in vivo similarities and differences between isc mutants, which have a known function in cluster assembly, and mutants disrupted in four additional loci, gshA, apbC, apbE, and rseC. The latter mutants share similarities with isc mutants: (i) a sensitivity to oxidative stress, (ii) a thiamine auxotrophy in the absence of the YggX protein, and (iii) decreased activities of Fe-S proteins, including aconitase, succinate dehydrogenase, and MiaB. However, they differ from isc mutants by displaying a phenotypic dependence on metals and a distinct defect in the SoxRS response to superoxides. Results presented herein support the proposed role of YggX in iron trafficking and protection against oxidative stress, describe additional phenotypes of isc mutants, and suggest a working model in which the ApbC, ApbE, and RseC proteins and glutathione participate in Fe-S cluster repair.     

Lack of the ApbC or ApbE protein results in a defect in Fe-S cluster metabolism in Salmonella enterica serovar Typhimurium

The isc genes function in the assembly of Fe-S clusters and are conserved in many prokaryotic and eukaryotic organisms. In most bacteria studied, the isc operon can be deleted without loss of cell viability, indicating that additional systems for Fe-S cluster assembly must exist. Several laboratories have described nutritional and biochemical defects resulting from mutations in the isc operon. Here we demonstrate that null mutations in two genes of unknown function, apbC and apbE, result in similar cellular deficiencies. Exogenous ferric chloride suppressed these deficiencies in the apbC and apbE mutants, distinguishing them from previously described isc mutants. The deficiencies caused by the apbC and isc mutations were additive, which is consistent with Isc and ApbC's having redundant functions or with Isc and ApbC's functioning in different areas of Fe-S cluster metabolism (e.g., Fe-S cluster assembly and Fe-S cluster repair). Both the ApbC and ApbE proteins are similar in sequence to proteins that function in metal cofactor assembly. Like the enzymes with sequence similarity to ApbC, purified ApbC protein was able to hydrolyze ATP. The data herein are consistent with the hypothesis that the ApbC and ApbE proteins function in Fe-S cluster metabolism in vivo.     

SufA from Erwinia chrysanthemi. Characterization of a scaffold protein required for iron-sulfur cluster assembly

SufA is a component of the recently discovered suf operon, which has been shown to play an important function in bacteria during iron-sulfur cluster biosynthesis and resistance to oxidative stress. The SufA protein from Erwinia chrysanthemi, a Gram-negative plant pathogen, has been purified to homogeneity and characterized. It is a homodimer with the ability to assemble rather labile [2Fe-2S] and [4Fe-4S] clusters as shown by M?ssbauer spectroscopy. These clusters can be transferred to apoproteins such as ferredoxin or biotin synthase during a reaction that is not inhibited by bathophenanthroline, an iron chelator. Cluster assembly in these proteins is much more efficient when iron and sulfur are provided by holoSufA than by free iron sulfate and sodium sulfide. We propose the function of SufA is that of a scaffold protein for [Fe-S] cluster assembly and compare it to IscA, a member of the isc operon also involved in cluster biosynthesis in both prokaryotes and eukaryotes. Mechanistic and physiological implications of these results are also discussed.     

Genetic analysis of the isc operon in Escherichia coli involved in the biogenesis of cellular iron-sulfur proteins.

The iron-sulfur (Fe-S) cluster, the nonheme-iron cofactor essential for the activity of many proteins, is incorporated into target proteins with the aid of complex machinery. In bacteria, several proteins encoded by the iscRSUA-hscBA-fdx-ORF3 cluster (isc operon) have been proposed to execute crucial tasks in the assembly of Fe-S clusters. To elucidate the in vivo function, we have undertaken a systematic mutational analysis of the genes in the Escherichia coli isc operon. In all functional tests, i.e. growth rate, nutritional requirements and activities of Fe-S enzymes, the inactivation of the iscS gene elicited the most drastic alteration. Strains with mutations in the iscU, hscB, hscA, and fdx genes also exhibited conspicuous phenotypical consequences almost identical to one another. The effect of the inactivation of iscA was small but appreciable on Fe-S enzymes. In contrast, mutants with inactivated iscR or ORF3 showed virtually no differences from wild-type cells. The requirement of iscSUA-hscBA-fdx for the assembly of Fe-S clusters was further confirmed by complementation experiments using a mutant strain in which the entire isc operon was deleted. Our findings support the conclusion that IscS, via cysteine desulfurase activity, provides the sulfur that is subsequently incorporated into Fe-S clusters by assembler machinery comprising of the iscUA-hscBA-fdx gene products. The results presented here indicate crucial roles for IscU, HscB, HscA, and Fdx as central components of the assembler machinery and also provide evidence for interactions among them.     

The SufE sulfur-acceptor protein contains a conserved core structure that mediates interdomain interactions in a variety of redox protein complexes

The isc and suf operons in Escherichia coli represent alternative genetic systems optimized to mediate the essential metabolic process of iron-sulfur cluster (Fe-S) assembly under basal or oxidative-stress conditions, respectively. Some of the proteins in these two operons share strong sequence homology, e.g. the cysteine desulfurases IscS and SufS, and presumably play the same role in the oxygen-sensitive assembly process. However, other proteins in these operons share no significant homology and occur in a mutually exclusive manner in Fe-S assembly operons in other organisms (e.g. IscU and SufE). These latter proteins presumably play distinct roles adapted to the different assembly mechanisms used by the two systems. IscU has three invariant cysteine residues that function as a template for Fe-S assembly while accepting a sulfur atom from IscS. SufE, in contrast, does not function as an Fe-S assembly template but has been suggested to function as a shuttle protein that uses a persulfide linkage to a single invariant cysteine residue to transfer a sulfur atom from SufS to an alternative Fe-S assembly template. Here, we present and analyze the 2.0A crystal structure of E.coli SufE. The structure shows that the persulfide-forming cysteine occurs at the tip of a loop with elevated B-factors, where its side-chain is buried from solvent exposure in a hydrophobic cavity located beneath a highly conserved surface. Despite the lack of sequence homology, the core of SufE shows strong structural similarity to IscU, and the sulfur-acceptor site in SufE coincides with the location of the cysteine residues mediating Fe-S cluster assembly in IscU. Thus, a conserved core structure is implicated in mediating the interactions of both SufE and IscU with the mutually homologous cysteine desulfurase enzymes present in their respective operons. A similar core structure is observed in a domain found in a variety of Fe-S cluster containing flavoenzymes including xanthine dehydrogenase, where it also mediates interdomain interactions. Therefore, the core fold of SufE/IscU has been adapted to mediate interdomain interactions in diverse redox protein systems in the course of evolution.     

Iron-sulfur cluster assembly: characterization of IscA and evidence for a specific and functional complex with ferredoxin

The synthesis of iron-sulfur clusters in Escherichia coli is believed to require a complex protein machinery encoded by the isc (iron-sulfur cluster) operon. The product of one member of this operon, IscA, has been overexpressed, purified, and characterized. It can assemble an air-sensitive [2Fe-2S] cluster as shown by UV-visible and resonance Raman spectroscopy. The metal form but not the apoform of IscA binds ferredoxin, another member of the isc operon, selectively, allowing transfer of iron and sulfide from IscA to ferredoxin and formation of the [2Fe-2S] holoferredoxin. These results thus suggest that IscA is involved in ferredoxin cluster assembly and activation. This is an important function because a functional ferredoxin is required for maturation of other cellular Fe-S proteins.     

An intestinal parasitic protist, Entamoeba histolytica, possesses a non-redundant nitrogen fixation-like system for iron-sulfur cluster assembly under anaerobic conditions

We have characterized the iron-sulfur (Fe-S) cluster formation in an anaerobic amitochondrial protozoan parasite, Entamoeba histolytica, in which Fe-S proteins play an important role in energy metabolism and electron transfer. A genomewide search showed that E. histolytica apparently possesses a simplified and non-redundant NIF (nitrogen fixation)-like system for the Fe-S cluster formation, composed of only a catalytic component, NifS, and a scaffold component, NifU. Amino acid alignment and phylogenetic analyses revealed that both amebic NifS and NifU (EhNifS and EhNifU, respectively) showed a close kinship to orthologs from epsilon-proteobacteria, suggesting that both of these genes were likely transferred by lateral gene transfer from an ancestor of epsilon-proteobacteria to E. histolytica. The EhNifS protein expressed in E. coli was present as a homodimer, showing cysteine desulfurase activity with a very basic optimum pH compared with NifS from other organisms. Eh-NifU protein existed as a tetramer and contained one stable [2Fe-2S]2+ cluster per monomer, revealed by spectroscopic and iron analyses. Fractionation of the whole parasite lysate by anion exchange chromatography revealed three major cysteine desulfurase activities, one of which corresponded to the EhNifS protein, verified by immunoblot analysis using the specific EhNifS antibody; the other two peaks corresponded to methionine gamma-lyase and cysteine synthase. Finally, ectopic expression of the EhNifS and EhNifU genes successfully complemented, under anaerobic but not aerobic conditions, the growth defect of an Escherichia coli strain, in which both the isc and suf operons were deleted, suggesting that EhNifS and EhNifU are necessary and sufficient for Fe-S clusters of non-nitrogenase Fe-S proteins to form under anaerobic conditions. This is the first demonstration of the presence and biological significance of the NIF-like system in eukaryotes.     

Role of Saccharomyces cerevisiae ISA1 and ISA2 in iron homeostasis

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 isaDelta 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.     

Hydrogen peroxide inactivates the Escherichia coli Isc iron-sulphur assembly system, and OxyR induces the Suf system to compensate

Environmental H(2) O(2) creates several injuries in Escherichia coli, including the oxidative conversion of dehydratase [4Fe-4S] clusters to an inactive [3Fe-4S] form. To protect itself, H(2) O(2) -stressed E. coli activates the OxyR regulon. This regulon includes the suf operon, which encodes an alternative to the housekeeping Isc iron-sulphur 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 H(2) O(2) , we imposed chronic low-grade stress and found that suf mutants could not maintain the activity of isopropylmalate isomerase, a key iron-sulphur 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, submicromolar H(2) O(2) 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 H(2) O(2) -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.     

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

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

Effect of iron-sulfur cluster assembly proteins on the expression of Escherichia coli lipoic acid synthase

Lipoic Acid Synthase (LipA) can accommodate a [4Fe-4S] cluster that is thought to be essential for the insertion of sulfur into an octanoyl substrate during the biosynthesis of lipoic acid. With the objective of improving soluble holo-LipA expression, a series of multi-cistronic plasmids were constructed carrying lipA in combination with one of the three systems: groE/SL, trxA, or the isc operon. Co-expression of lipA with the isc operon approximately trebled the isolated yield of soluble LipA and resulted in efficient assembly of the Fe-S cluster. This strategy may be helpful in the soluble expression of a wide range of Fe-S cluster-dependent proteins.     

Repair of oxidized iron-sulfur clusters in Escherichia coli

The [4Fe-4S]2+ clusters of dehydratases are rapidly damaged by univalent oxidants, including hydrogen peroxide, superoxide, and peroxynitrite. The loss of an electron destabilizes the cluster, causing it to release its catalytic iron atom and converting the cluster initially to an inactive [3Fe-4S]1+ form. Continued exposure to oxidants in vitro leads to further iron release. Experiments have shown that these clusters are repaired in vivo. We sought to determine whether repair is mediated by either the Isc or Suf cluster-assembly systems that have been identified in Escherichia coli. We found that all the proteins encoded by the isc operon were critical for de novo assembly, but most of these were unnecessary for cluster repair. IscS, a cysteine desulfurase, appeared to be an exception: although iscS mutants repaired damaged clusters, they did so substantially more slowly than did wild-type cells. Because sulfur mobilization should be required only if clusters degrade beyond the [3Fe-4S]1+ state, we used whole cell EPR to visualize the fate of oxidized enzymes in vivo. Fumarase A was overproduced. Brief exposure of cells to hydrogen peroxide resulted in the appearance of the characteristic [3Fe-4S]1+ signal of the oxidized enzyme. When hydrogen peroxide was then scavenged, the enzyme activity reappeared within minutes, in concert with the disappearance of the EPR signal. Thus it is unclear why IscS is required for efficient repair. The iscS mutants grew poorly, allowing the possibility that metabolic defects indirectly slow the repair process. Our data did indicate that damaged clusters decompose beyond the [3Fe-4S]1+ state in vivo when stress is prolonged. Under the conditions of our experiments, mutants that lacked other repair candidates--Suf proteins, glutathione, and NADPH: ferredoxin reductase--all repaired clusters at normal rates. We conclude that the mechanism of cluster repair is distinct from that of de novo assembly and that this is true because mild oxidative stress does not degrade clusters in vivo to the point of presenting an apoenzyme to the de novo cluster-assembly systems.