Stage-specific requirement for Isa1 and Isa2 proteins in the mitochondrion of Trypanosoma brucei and heterologous rescue by human and Blastocystis orthologues

IscA/Isa proteins function as alternative scaffolds for the assembly of Fe-S clusters and/or provide iron for their assembly in prokaryotes and eukaryotes. Isa are usually non-essential and in most organisms are confined to the mitochondrion. We have studied the function of TbIsa1 and TbIsa2 in Trypanosoma brucei, where the requirement for both of them to sustain cell growth depends on the life cycle stage. The TbIsa proteins are abundant in the procyclic form, which contains an active organelle. Both proteins are indispensable for growth, as they are required for the assembly of Fe-S clusters in mitochondrial aconitase, fumarase and succinate dehydrogenase. Reactive oxygen species but not iron accumulate in the procyclic mitochondrion upon ablation of the TbIsa proteins, but their depletion does not influence the assembly of Fe-S clusters in cytosolic proteins. In the bloodstream form, which has a downregulated mitochondrion, the TbIsa proteins are non-essential. The Isa2 orthologue of the anaerobic protist Blastocystis partially rescued the growth and enzymatic activities of TbIsa1/2 knock-down. Rescues of single knock-downs as well as heterologous rescues with human Isa orthologues partially recovered the activities of aconitase and fumarase. These results show that the Isa1 and Isa2 proteins of diverse eukaryotes have overlapping functions.     

CpNifS-dependent iron-sulfur cluster biogenesis in chloroplasts

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

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

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

Biosynthesis of iron-sulphur clusters is a complex and highly conserved process

Iron-sulphur ([Fe-S]) clusters are simple inorganic prosthetic groups that are contained in a variety of proteins having functions related to electron transfer, gene regulation, environmental sensing and substrate activation. In spite of their simple structures, biological [Fe-S] clusters are not formed spontaneously. Rather, a consortium of highly conserved proteins is required for both the formation of [Fe-S] clusters and their insertion into various protein partners. Among the [Fe-S] cluster biosynthetic proteins are included a pyridoxal phosphate-dependent enzyme (NifS) that is involved in the activation of sulphur from l-cysteine, and a molecular scaffold protein (NifU) upon which [Fe-S] cluster precursors are formed. The formation or transfer of [Fe-S] clusters appears to require an electron-transfer step. Another complexity is that molecular chaperones homologous to DnaJ and DnaK are involved in some aspect of the maturation of [Fe-S]-cluster-containing proteins. It appears that the basic biochemical features of [Fe-S] cluster formation are strongly conserved in Nature, since organisms from all three life Kingdoms contain the same consortium of homologous proteins required for [Fe-S] cluster formation that were discovered in the eubacteria.     

铁硫簇的生物合成

铁硫簇在细胞的生物学过程中起着重要的作用,可参与电子传递、代谢控制和基因调节等过程。研究显示铁硫簇具有多样性,它的合成依赖于ISC和SUF系统,固氮酶中还需要NIF系统的参与。ISC系统由iscSUA-hscBA-fdx基因串编码,合成的是一类“管家”蛋白,适于在正常条件下表达。SUF系统由基因串sufABCDSE编码,常在恶劣环境如氧化应激和铁饥饿条件下表达。NIF系统由nifSU基因编码,适于固氮酶(厌氧条件下起作用)铁硫簇的合成。     

Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors composed of iron and inorganic sulfur. They are required for the function of proteins involved in a wide range of activities, including electron transport in respiratory chain complexes, regulatory sensing, photosynthesis and DNA repair. The proteins involved in the biogenesis of Fe-S clusters are evolutionarily conserved from bacteria to humans, and many insights into the process of Fe-S cluster biogenesis have come from studies of model organisms, including bacteria, fungi and plants. It is now clear that several rare and seemingly dissimilar human diseases are attributable to defects in the basic process of Fe-S cluster biogenesis. Although these diseases –which include Friedreich’s ataxia (FRDA), ISCU myopathy, a rare form of sideroblastic anemia, an encephalomyopathy caused by dysfunction of respiratory chain complex I and multiple mitochondrial dysfunctions syndrome – affect different tissues, a feature common to many of them is that mitochondrial iron overload develops as a secondary consequence of a defect in Fe-S cluster biogenesis. This Commentary outlines the basic steps of Fe-S cluster biogenesis as they have been defined in model organisms. In addition, it draws attention to refinements of the process that might be specific to the subcellular compartmentalization of Fe-S cluster biogenesis proteins in some eukaryotes, including mammals. Finally, it outlines several important unresolved questions in the field that, once addressed, should offer important clues into how mitochondrial iron homeostasis is regulated, and how dysfunction in Fe-S cluster biogenesis can contribute to disease.     

Global Identification of Genes Affecting Iron-Sulfur Cluster Biogenesis and Iron Homeostasis

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.     

Iron-sulfur cluster biosynthesis in bacteria: Mechanisms of cluster assembly and transfer

Iron-sulfur [Fe-S] clusters are ubiquitous ancient prosthetic groups that are required to sustain fundamental life processes. Formation of intracellular [Fe-S] clusters does not occur spontaneously but requires a complex biosynthetic machinery. Different types of [Fe-S] cluster assembly systems have been discovered. All of them have in common the requirement of a cysteine desulfurase and the participation of [Fe-S] scaffold proteins. The purpose of this review is to discuss various aspects of the molecular mechanisms of [Fe-S] cluster assembly in living organisms: (i) mechanism of sulfur donor enzymes, namely the cysteine desulfurases; (ii) mechanism by which clusters are preassembled on scaffold proteins and (iii) mechanism of [Fe-S] cluster transfer from scaffold to target proteins.     

Functions of mitochondrial ISCU and cytosolic ISCU in mammalian iron-sulfur cluster biogenesis and iron homeostasis

Iron-sulfur (Fe-S) clusters are required for the functions of mitochondrial aconitase, mammalian iron regulatory protein 1, and many other proteins in multiple subcellular compartments. Recent studies in Saccharomyces cerevisiae indicated that Fe-S cluster biogenesis also has an important role in mitochondrial iron homeostasis. Here we report the functional analysis of the mitochondrial and cytosolic isoforms of the human Fe-S cluster scaffold protein, ISCU. Suppression of human ISCU by RNAi not only inactivated mitochondrial and cytosolic aconitases in a compartment-specific manner but also inappropriately activated the iron regulatory proteins and disrupted intracellular iron homeostasis. Furthermore, endogenous ISCU levels were suppressed by iron deprivation. These results provide evidence for a coordinated response to iron deficiency that includes activation of iron uptake, redistribution of intracellular iron, and decreased utilization of iron in 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.     

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

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

Iron-regulated assembly of the cytosolic iron–sulfur cluster biogenesis machinery

The cytosolic iron–sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron–sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.     

A-type carrier protein ErpA is essential for formation of an active formate-nitrate respiratory pathway in Escherichia coli K-12

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.     

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.     

Characteristics of the aerobic respiratory chains of the microaerophiles Campylobacter jejuni and Helicobacter pylori

The respiratory chain enzymes of microaerophilic bacteria should play a major role in their adaptation to growth at low oxygen tensions. The genes encoding the putative NADH:quinone reductases (NDH-1), the ubiquinol:cytochrome c oxidoreductases (bc1 complex) and the terminal oxidases of the microaerophiles Campylobacter jejuni and Helicobacter pylori were analysed to identify structural elements that may be required for their unique energy metabolism. The gene clusters encoding NDH-1 in both C. jejuni and H. pylori lacked nuoE and nuoF, and in their place were genes encoding two unknown proteins. The NuoG subunit in these microaerophilic bacteria appeared to have an additional Fe-S cluster that is not present in NDH-1 from other organisms; but C. jejuni and H. pylori differed from each other in a cysteine-rich segment in this subunit, which is present in some but not all NDH-1. Both organisms lacked genes orthologous to those encoding NDH-2. The subunits of the bc1 complex of both bacteria were similar, and the Rieske Fe-S and cytochrome b subunits had significant similarity to those of Paracoccus denitrificans and Rhodobacter capsulatus, well-studied bacterial bc1 complexes. The composition of the terminal oxidases of C. jejuni and H. pylori was different; both bacteria had cytochrome cbb3 oxidases, but C. jejuni also contained a bd-type quinol oxidase. The primary structures of the major subunits of the cbb3-type (terminal) oxidase of C. jejuni and H. pylori indicated that they form a separate group within the cbb3 protein family. The implications of the results for the function of the enzymes and their adaptation to microaerophilic growth are discussed.     

Delivery of iron-sulfur clusters to the hydrogen-oxidizing [NiFe]-hydrogenases in Escherichia coli requires the A-type carrier proteins ErpA and IscA

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.     

Characterization of the NifU and NifS Fe-S cluster formation proteins essential for viability in Helicobacter pylori

The Fe-S cluster formation proteins NifU and NifS are essential for viability in the ulcer causing human pathogen Helicobacter pylori. Obtaining viable H. pylori mutants upon mutagenesis of the genes encoding NifU and NifS was unsuccessful even by growing the potential transformants under many different conditions including low O(2) atmosphere and supplementation with both ferric and ferrous iron. When a second copy of nifU was introduced into the chromosome at a unrelated site, creating a mero-diploid strain for nifU, this second copy of the gene could be disrupted at high frequency. This indicates that the procedures used for transformation were capable of nifU mutagenesis, so that the failure to recover mutants is solely due to the requirement of nifU for H. pylori viability. H. pylori NifU and NifS were expressed in Escherichia coli and purified to near homogeneity, and the proteins were characterized. Purified NifU is a red protein that contains approximately 1.5 atoms of iron per monomer. This iron was determined to be in the form of a redox-active [2Fe-2S](2+,+) cluster by characteristic UV-visible, EPR, and MCD spectra. The primary structure of NifU also contains the three conserved cysteine residues which are involved in providing the scaffold for the assembly of a transient Fe-S cluster for insertion into apoprotein. Purified NifS has a yellow color and UV-visible spectra characteristic of a pyridoxal phosphate containing enzyme. NifS is a cysteine desulfurase, releasing sulfur or sulfide (depending on the reducing environment) from L-cysteine, in agreement with its proposed role as a sulfur donor to Fe-S clusters. The results here indicate that the NifU type of Fe-S cluster formation proteins is not specific for maturation of the nitrogenase proteins and, as H. pylori lacks other Fe-S cluster assembly proteins, that the H. pylori NifS and NifU are responsible for the assembly of many (non-nitrogenase) Fe-S clusters.     

Cobalt stress in Escherichia coli. The effect on the iron-sulfur proteins

Cobalt is toxic for cells, but mechanisms of this toxicity are largely unknown. The biochemical and genetic experiments reported here demonstrate that iron-sulfur proteins are greatly affected in cobalt-treated Escherichia coli cells. Exposure of a wild-type strain to intracellular cobalt results in the inactivation of three selected iron-sulfur enzymes, the tRNA methylthio-transferase, aconitase, and ferrichrome reductase. Consistently, mutant strains lacking the [Fe-S] cluster assembly SUF machinery are hypersensitive to cobalt. Last, expression of iron uptake genes is increased in cells treated with cobalt. In vitro studies demonstrated that cobalt does not react directly with fully assembled [Fe-S] clusters. In contrast, it reacts with labile ones present in scaffold proteins (IscU, SufA) involved in iron-sulfur cluster biosynthesis. We propose a model wherein cobalt competes out iron during synthesis of [Fe-S] clusters in metabolically essential proteins.     

Hyperproduction of recombinant ferredoxins in escherichia coli by coexpression of the ORF1-ORF2-iscS-iscU-iscA-hscB-hs cA-fdx-ORF3 gene cluster.

Fe-S proteins acquire Fe-S clusters by an unknown post-translational mechanism. To study the in vivo synthesis of the Fe-S clusters, we constructed an experimental system to monitor the expressed ferredoxin (Fd) as a reporter of protein-bound Fe-S clusters assembled in Escherichia coli. Overexpression of five Fds in a T7 polymerase-based system led to the formation of soluble apoFds and mature holoFds, indicating that assembly of the Fe-S cluster into apoFd polypeptides is a rate-limiting step. We examined the coexpression of the E. coli ORF1-ORF2-iscS-iscU-iscA-hscB-hsc A-fdx-ORF3 gene cluster, which has recently been suggested to be involved in the formation or repair of Fe-S protein [Zheng, L., Cash, V.L., Flint, D.H., and Dean, D.R. (1998) J. Biol. Chem. 273, 13264-13272], with reporter Fds using compatible plasmids. The production of all five reporter holoFds examined was dramatically increased by the coexpression of the gene cluster, and apparent specificity to the polypeptides or to the type of Fe-S clusters was not observed. The increase in holoFd production was observed under the coexpression conditions in all culture media examined, with either 2 x YT medium or Terrific broth, and with or without supplemental cysteine or iron. These results indicate that the proteins encoded by the gene cluster are involved in the assembly of the Fe-S clusters in a wide variety of Fe-S proteins.     

Drosophila frataxin: an iron chaperone during cellular Fe-S cluster bioassembly

Frataxin, a mitochondrial protein that is directly involved in regulating cellular iron homeostasis, has been suggested to serve as an iron chaperone during cellular Fe-S cluster biosynthesis. In humans, decreased amounts or impaired function of frataxin causes the autosomal recessive neurodegenerative disorder Friedreich's ataxia. Cellular production of Fe-S clusters is accomplished by the Fe cofactor assembly platform enzymes Isu (eukaryotes) and IscU (prokaryotes). In this report, we have characterized the overall stability and iron binding properties of the Drosophila frataxin homologue (Dfh). Dfh is highly folded with secondary structural elements consistent with the structurally characterized frataxin orthologs. While the melting temperature ( T M approximately 59 degrees C) and chemical stability ([urea] 50% approximately 2.4 M) of Drosophila frataxin, measured using circular dichroism (CD) and fluorescence spectroscopy, closely match values determined for the human ortholog, pure Dfh is more stable against autodegradation than both the human and yeast proteins. The ferrous iron binding affinity ( K d approximately 6.0 microM) and optimal metal to protein stoichiometry (1:1) for Dfh have been measured using isothermal titration calorimetry (ITC). Under anaerobic conditions with salt present, holo-Dfh is a stable iron-loaded protein monomer. Frataxin prevents reactive oxygen species-induced oxidative damage to DNA when presented with both Fe(II) and H 2O 2. Ferrous iron bound to Dfh is high-spin and held in a partially symmetric Fe-(O/N) 6 coordination environment, as determined by X-ray absorption spectroscopy (XAS). Extended X-ray absorption fine structure (EXAFS) simulations indicate the average Fe-O/N bond length in Dfh is 2.13 A, consistent with a ligand geometry constructed by water and carboxylate oxygens most likely supplied in part by surface-exposed conserved acidic residues located on helix 1 and strand 1 in the structurally characterized frataxin orthologs. The iron-dependent binding affinity ( K d approximately 0.21 microM) and optimal holo-Dfh to Isu monomer stoichiometry (1:1) have also been determined using ITC. Finally, frataxin mediates the delivery of Fe(II) to Isu, promoting Fe-S cluster assembly in vitro. The Dfh-assisted assembly of Fe-S clusters occurs with an observed kinetic rate constant ( k obs) of 0.096 min (-1).