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.     

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.     

Evidence for nifU and nifS participation in the biosynthesis of the iron-molybdenum cofactor of nitrogenase

The nifU and nifS genes encode the components of a cellular machinery dedicated to the assembly of [2Fe-2S] and [4Fe-4S] clusters required for growth under nitrogen-fixing conditions. The NifU and NifS proteins are involved in the production of active forms of the nitrogenase component proteins, NifH and NifDK. Although NifH contains a [4Fe-4S] cluster, the NifDK component carries two complex metalloclusters, the iron-molybdenum cofactor (FeMo-co) and the [8Fe-7S] P-cluster. FeMo-co, located at the active site of NifDK, is composed of 7 iron, 9 sulfur, 1 molybdenum, 1 homocitrate, and 1 unidentified light atom. To investigate whether NifUS are required for FeMo-co biosynthesis and to understand at what level(s) they might participate in this process, we analyzed the effect of nifU and nifS mutations on the formation of active NifB protein and on the accumulation of NifB-co, an isolatable intermediate of the FeMo-co biosynthetic pathway synthesized by the product of the nifB gene. The nifU and nifS genes were required to accumulate NifB-co in a nifN mutant background. This result clearly demonstrates the participation of NifUS in NifB-co synthesis and suggests a specific role of NifUS as the major provider of [Fe-S] clusters that serve as metabolic substrates for the biosynthesis of FeMo-co. Surprisingly, although nifB expression was attenuated in nifUS mutants, the assembly of the [Fe-S] clusters of NifB was compensated by other non-nif machinery for the assembly of [Fe-S] clusters, indicating that NifUS are not essential to synthesize active NifB.     

A proposed role for the Azotobacter vinelandii NfuA protein as an intermediate iron-sulfur cluster carrier

Iron-sulfur clusters ([Fe-S] clusters) are assembled on molecular scaffolds and subsequently used for maturation of proteins that require [Fe-S] clusters for their functions. Previous studies have shown that Azotobacter vinelandii produces at least two [Fe-S] cluster assembly scaffolds: NifU, required for the maturation of nitrogenase, and IscU, required for the general maturation of other [Fe-S] proteins. A. vinelandii also encodes a protein designated NfuA, which shares amino acid sequence similarity with the C-terminal region of NifU. The activity of aconitase, a [4Fe-4S] cluster-containing enzyme, is markedly diminished in a strain containing an inactivated nfuA gene. This inactivation also results in a null-growth phenotype when the strain is cultivated under elevated oxygen concentrations. NifU has a limited ability to serve the function of NfuA, as its expression at high levels corrects the defect of the nfuA-disrupted strain. Spectroscopic and analytical studies indicate that one [4Fe-4S] cluster can be assembled in vitro within a dimeric form of NfuA. The resultant [4Fe-4S] cluster-loaded form of NfuA is competent for rapid in vitro activation of apo-aconitase. Based on these results a model is proposed where NfuA could represent a class of intermediate [Fe-S] cluster carriers involved in [Fe-S] protein maturation.     

[4Fe-4S] cluster trafficking mediated by Arabidopsis mitochondrial ISCA and NFU proteins

Numerous iron-sulfur (Fe-S) proteins with diverse functions are present in the matrix and respiratory chain complexes of mitochondria. Although [4Fe-4S] clusters are the most common type of Fe-S cluster in mitochondria, the molecular mechanism of [4Fe-4S] cluster assembly and insertion into target proteins by the mitochondrial iron-sulfur cluster (ISC) maturation system is not well-understood. Here we report a detailed characterization of two late-acting Fe-S cluster-carrier proteins from Arabidopsis thaliana, NFU4 and NFU5. Yeast two-hybrid and bimolecular fluorescence complementation studies demonstrated interaction of both the NFU4 and NFU5 proteins with the ISCA class of Fe-S carrier proteins. Recombinant NFU4 and NFU5 were purified as apo-proteins after expression in Escherichia coli. In vitro Fe-S cluster reconstitution led to the insertion of one [4Fe-4S]²⁺ cluster per homodimer as determined by UV-visible absorption/CD, resonance Raman and EPR spectroscopy, and analytical studies. Cluster transfer reactions, monitored by UV-visible absorption and CD spectroscopy, showed that a [4Fe-4S]²⁺ cluster-bound ISCA1a/2 heterodimer is effective in transferring [4Fe-4S]²⁺ clusters to both NFU4 and NFU5 with negligible back reaction. In addition, [4Fe-4S]²⁺ cluster-bound ISCA1a/2, NFU4, and NFU5 were all found to be effective [4Fe-4S]²⁺ cluster donors for maturation of the mitochondrial apo-aconitase 2 as assessed by enzyme activity measurements. The results demonstrate rapid, unidirectional, and quantitative [4Fe-4S]²⁺ cluster transfer from ISCA1a/2 to NFU4 or NFU5 that further delineates their respective positions in the plant ISC machinery and their contributions to the maturation of client [4Fe-4S] cluster-containing proteins.     

NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU scaffold protein

NifU is a homodimeric modular protein comprising N- and C-terminal domains and a central domain with a redox-active [2Fe-2S](2+,+) cluster. It plays a crucial role as a scaffold protein for the assembly of the Fe-S clusters required for the maturation of nif-specific Fe-S proteins. In this work, the time course and products of in vitro NifS-mediated iron-sulfur cluster assembly on full-length NifU and truncated forms involving only the N-terminal domain or the central and C-terminal domains have been investigated using UV-vis absorption and M?ssbauer spectroscopies, coupled with analytical studies. The results demonstrate sequential assembly of labile [2Fe-2S](2+) and [4Fe-4S](2+) clusters in the U-type N-terminal scaffolding domain and the assembly of [4Fe-4S](2+) clusters in the Nfu-type C-terminal scaffolding domain. Both scaffolding domains of NifU are shown to be competent for in vitro maturation of nitrogenase component proteins, as evidenced by rapid transfer of [4Fe-4S](2+) clusters preassembled on either the N- or C-terminal domains to the apo nitrogenase Fe protein. Mutagenesis studies indicate that a conserved aspartate (Asp37) plays a critical role in mediating cluster transfer. The assembly and transfer of clusters on NifU are compared with results reported for U- and Nfu-type scaffold proteins, and the need for two functional Fe-S cluster scaffolding domains on NifU is discussed.     

The HBx protein from hepatitis B virus coordinates a redox-active Fe-S cluster

The viral protein HBx is the key regulatory factor of the hepatitis B virus (HBV) and the main etiology for HBV-associated liver diseases, such as cirrhosis and hepatocellular carcinoma. Historically, HBx has defied biochemical and structural characterization, deterring efforts to understand its molecular mechanisms. Here we show that soluble HBx fused to solubility tags copurifies with either a [2Fe-2S] or a [4Fe-4S] cluster, a feature that is shared among five HBV genotypes. We show that the O₂-stable [2Fe-2S] cluster form converts to an O₂-sensitive [4Fe-4S] state when reacted with chemical reductants, a transformation that is best described by a reductive coupling mechanism reminiscent of Fe-S cluster scaffold proteins. In addition, the Fe-S cluster conversions are partially reversible in successive reduction–oxidation cycles, with cluster loss mainly occurring during (re)oxidation. The considerably negative reduction potential of the [4Fe-4S]^2+/1+ couple (−520 mV) suggests that electron transfer may not be likely in the cell. Collectively, our findings identify HBx as an Fe-S protein with striking similarities to Fe-S scaffold proteins both in cluster type and reductive transformation. An Fe-S cluster in HBx offers new insights into its previously unknown molecular properties and sets the stage for deciphering the roles of HBx-associated iron (mis)regulation and reactive oxygen species in the context of liver tumorigenesis.     

Roles of ATP and NADPH in formation of the fe-s cluster of spinach ferredoxin

Ferredoxin (Fd) in higher plants is encoded by a nuclear gene, synthesized in the cytoplasm as a larger precursor, and imported into the chloroplast, where it is proteolytically processed, and assembled with the [2Fe-2S] cluster. The final step in the biosynthetic pathway of Fd can be analyzed by a reconstitution system composed of isolated chloroplasts and [³⁵S]cysteine, in which [³⁵S]sulfide and iron are incorporated into Fd to build up the ³⁵S-labeled Fe-S cluster. Although a lysed chloroplast system shows obligate requirements for ATP and NADPH, in vitro chemical reconstitution of the Fe-S cluster is generally thought to be energy-independent. The present study investigated whether ATP and NADPH in the chloroplast system of spinach (Spinacia oleracea) are involved in the supply of [³⁵S]sulfide or iron, or in Fe-S cluster formation itself. [³⁵S]Sulfide was liberated from [³⁵S] cysteine in an NADPH-dependent manner, whereas ATP was not necessary for this process. This desulfhydration of [³⁵S]cysteine occurred before the formation of the ³⁵S-labeled Fe-S cluster, and the amount of radioactivity in [³⁵S]sulfide was greater than that in ³⁵S-labeled holo-Fd by a factor of more than 20. Addition of nonradioactive sulfide (Na₂S) inhibited competitively formation of the ³⁵S-labeled Fe-S cluster along with the addition of nonradioactive cysteine, indicating that some of the inorganic sulfide released from cysteine is incorporated into the Fe-S cluster of Fd. ATP hydrolysis was not involved in the production of inorganic sulfide or in the supply of iron for assembly into the Fe-S cluster. However, ATP-dependent Fe-S cluster formation was observed even in the presence of sufficient amounts of [³⁵S]sulfide and iron. These results suggest a novel type of ATP-dependent in vivo Fe-S cluster formation that is distinct from in vitro chemical reconstitution. The implications of these results for the possible mechanisms of ATP-dependent Fe-S cluster formation are discussed.     

Fe-S Cluster Biogenesis in Isolated Mammalian Mitochondria: COORDINATED USE OF PERSULFIDE SULFUR AND IRON AND REQUIREMENTS FOR GTP,NADH, AND ATP*

Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [³⁵S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-³⁵S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the ³⁵S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia.     

Iron–sulfur protein maturation in Helicobacter pylori: identifying a Nfu‐type cluster carrier protein and its iron–sulfur protein targets

Helicobacter pylori is anomalous among non nitrogen‐fixing bacteria in containing an incomplete NIF system for Fe–S cluster assembly comprising two essential proteins, NifS (cysteine desulfurase) and NifU (scaffold protein). Although nifU deletion strains cannot be obtained via the conventional gene replacement, a NifU‐depleted strain was constructed and shown to be more sensitive to oxidative stress compared to wild‐type (WT) strains. The hp1492 gene, encoding a putative Nfu‐type Fe–S cluster carrier protein, was disrupted in three different H. pylori strains, indicating that it is not essential. However, Δnfu strains have growth deficiency, are more sensitive to oxidative stress and are unable to colonize mouse stomachs. Moreover, Δnfu strains have lower aconitase activity but higher hydrogenase activity than the WT. Recombinant Nfu was found to bind either one [2Fe–2S] or [4Fe–4S] cluster/dimer, based on analytical, UV–visible absorption/CD and resonance Raman studies. A bacterial two‐hybrid system was used to ascertain interactions between Nfu, NifS, NifU and each of 36 putative Fe–S‐containing target proteins. Nfu, NifS and NifU were found to interact with 15, 6 and 29 putative Fe–S proteins respectively. The results indicate that Nfu, NifS and NifU play a major role in the biosynthesis and/or delivery of Fe–S clusters in H. pylori.     

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.     

Controlled expression of nif and isc iron-sulfur protein maturation components reveals target specificity and limited functional replacement between the two systems

The nitrogen-fixing organism Azotobacter vinelandii contains at least two systems that catalyze formation of [Fe-S] clusters. One of these systems is encoded by nif genes, whose products supply [Fe-S] clusters required for maturation of nitrogenase. The other system is encoded by isc genes, whose products are required for maturation of [Fe-S] proteins that participate in general metabolic processes. The two systems are similar in that they include an enzyme for the mobilization of sulfur (NifS or IscS) and an assembly scaffold (NifU or IscU) upon which [Fe-S] clusters are formed. Normal cellular levels of the Nif system, which supplies [Fe-S] clusters for the maturation of nitrogenase, cannot also supply [Fe-S] clusters for the maturation of other cellular [Fe-S] proteins. Conversely, when produced at the normal physiological levels, the Isc system cannot supply [Fe-S] clusters for the maturation of nitrogenase. In the present work we found that such target specificity for IscU can be overcome by elevated production of NifU. We also found that NifU, when expressed at normal levels, is able to partially replace the function of IscU if cells are cultured under low-oxygen-availability conditions. In contrast to the situation with IscU, we could not establish conditions in which the function of IscS could be replaced by NifS. We also found that elevated expression of the Isc components, as a result of deletion of the regulatory iscR gene, improved the capacity for nitrogen-fixing growth of strains deficient in either NifU or NifS.     

IscA, an alternate scaffold for Fe-S cluster biosynthesis.

An IscA homologue within the nif regulon of Azotobacter vinelandii, designated (Nif)IscA, was expressed in Escherichia coli and purified to homogeneity. Purified (Nif)IscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form. Possible roles for (Nif)IscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorption, M?ssbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies. Although (Nif)IscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins. Rather, a role for (Nif)IscA as an alternate scaffold protein for Fe-S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile [4Fe-4S](2+) cluster per (Nif)IscA homodimer, via a transient [2Fe-2S](2+) cluster intermediate. The cluster assembly process was monitored temporally using UV-vis absorption and M?ssbauer spectroscopy, and the intermediate [2Fe-2S](2+)-containing species was additionally characterized by resonance Raman spectroscopy. The M?ssbauer and resonance Raman properties of the [2Fe-2S](2+) center are consistent with complete cysteinyl ligation. The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one [2Fe-2S](2+) or one [4Fe-4S](2+) per homodimer suggest that both cluster types are subunit bridging. In addition, (Nif)IscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur. The ability of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions. On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-directed cluster assembly on (Nif)IscA are proposed. It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly.     

Iron-sulfur cluster assembly: NifU-directed activation of the nitrogenase Fe protein

The NifU protein is a homodimer that is proposed to provide a molecular scaffold for the assembly of [Fe-S] clusters uniquely destined for the maturation of the nitrogenase catalytic components. There are three domains contained within NifU, with the N-terminal domain exhibiting a high degree of primary sequence similarity to a related family of [Fe-S] cluster biosynthetic scaffolds designated IscU. The C-terminal domain of NifU exhibits sequence similarity to a second family of proposed [Fe-S] cluster biosynthetic scaffolds designated Nfu. Genetic experiments described here involving amino acid substitutions within the N-terminal and C-terminal domains of NifU indicate that both domains can separately participate in nitrogenase-specific [Fe-S] cluster formation, although the N-terminal domain appears to have the dominant function. These in vivo experiments were supported by in vitro [Fe-S] cluster assembly and transfer experiments involving the activation of an apo-form of the nitrogenase Fe protein.     

Enzyme-mediated sulfide production for the reconstitution of [2Fe-2S] clusters into apo-biotin synthase of Escherichia coli. Sulfide transfer from cysteine to biotin.

We previously showed that biotin synthase in which the (Fe-S) cluster was labelled with 34S by reconstitution donates 34S to biotin [B. Tse Sum Bui, D. Florentin, F. Fournier, O. Ploux, A. Méjean & A. Marquet (1998) FEBS Lett. 440, 226-230]. We therefore proposed that the source of sulfur was very likely the (Fe-S) centre. This depletion of sulfur from the cluster during enzymatic reaction could explain the absence of turnover of the enzyme which means that to restore a catalytic activity, the clusters have to be regenerated. In this report, we show that the NifS protein from Azotobacter vinelandii and C-DES from Synechocystis as well as rhodanese from bovine liver can mobilize the sulfur, respectively, from cysteine and thiosulfate for the formation of a [2Fe-2S] cluster in the apoprotein of Escherichia coli biotin synthase. The reconstituted enzymes were as active as the native enzyme. When [35S]cysteine was used during the reconstitution experiments in the presence of NifS, labelled (Fe35S) biotin synthase was obtained. This enzyme produced [35S]biotin, confirming the results obtained with the 34S-reconstituted enzyme. NifS was also effective in mobilizing selenium from selenocystine to produce an (Fe-Se) cluster. However, though NifS could efficiently reconstitute holobiotin synthase from the apoform, starting from cysteine, these two effectors had no significant effect on the turnover of the enzyme in the in vitro assay.     

Iron-sulfur interconversions in the anaerobic ribonucleotide reductase from Escherichia coli

The anaerobic ribonucleotide reductase from Escherichia coli contains an iron-sulfur cluster which, in the reduced [4Fe-4S]⁺ form, serves to reduce S-adenosylmethionine and to generate a catalytically essential glycyl radical. The reaction of the reduced cluster with oxygen was studied by UV-visible, EPR, NMR, and Mössbauer spectroscopies. The [4Fe-4S]⁺ form is shown to be extremely sensitive to oxygen and converted to [4Fe-4S]²⁺, [3Fe-4S]^+/0, and to the stable [2Fe-2S]²⁺ form. It is remarkable that the oxidized protein retains full activity. This is probably due to the fact that during reduction, required for activity, the iron atoms, from 2Fe and 3Fe clusters, readily reassemble to generate an active [4Fe-4S] center. This property is discussed as a possible protective mechanism of the enzyme during transient exposure to air. Futhermore, the [2Fe-2S] form of the protein can be converted into a [3Fe-4S] form during chromatography on dATP-Sepharose, explaining why previous preparations of the enzyme were shown to contain large amounts of such a 3Fe cluster. This is the first report of a 2Fe to 3Fe cluster conversion.     

FeoC from Klebsiella pneumoniae Contains a [4Fe-4S] Cluster

Iron is essential for pathogen survival, virulence, and colonization. Feo is suggested to function as the ferrous iron (Fe²⁺) transporter. The enterobacterial Feo system is composed of 3 proteins: FeoB is the indispensable component and is a large membrane protein likely to function as a permease; FeoA is a small Src homology 3 (SH3) domain protein that interacts with FeoB; FeoC is a winged-helix protein containing 4 conserved Cys residues in a sequence suitable for harboring a putative iron-sulfur (Fe-S) cluster. The presence of an iron-sulfur cluster on FeoC has never been shown experimentally. We report that under anaerobic conditions, the recombinant Klebsiella pneumoniae FeoC (KpFeoC) exhibited hyperfine-shifted nuclear magnetic resonance (NMR) and a UV-visible (UV-Vis) absorbance spectrum characteristic of a paramagnetic center. The electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) results were consistent only with the [4Fe-4S] clusters. Substituting the cysteinyl sulfur with oxygen resulted in significantly reduced cluster stability, establishing the roles of these cysteines as the ligands for the Fe-S cluster. When exposed to oxygen, the [4Fe-4S] cluster degraded to [3Fe-4S] and eventually disappeared. We propose that KpFeoC may regulate the function of the Feo transporter through the oxygen- or iron-sensitive coordination of the Fe-S cluster.     

Bacterial ApbC can bind and effectively transfer iron-sulfur clusters

The metabolism of iron-sulfur ([Fe-S]) clusters requires a complex set of machinery that is still being defined. Mutants of Salmonella enterica lacking apbC have nutritional and biochemical properties indicative of defects in [Fe-S] cluster metabolism. ApbC is a 40.8 kDa homodimeric ATPase and as purified contains little iron and no acid-labile sulfide. An [Fe-S] cluster was reconstituted on ApbC, generating a protein that bound 2 mol of Fe and 2 mol of S (2-) per ApbC monomer and had a UV-visible absorption spectrum similar to known [4Fe-4S] cluster proteins. Holo-ApbC could rapidly and effectively activate Saccharomyces cerevisiae apo-isopropylmalate isolomerase (Leu1) in vitro, a process known to require the transfer of a [4Fe-4S] cluster. Maximum activation was achieved with 2 mol of ApbC per 1 mol of apo-Leu1. This article describes the first biochemical activity of ApbC in the context of [Fe-S] cluster metabolism. The data herein support a model in which ApbC coordinates an [4Fe-4S] cluster across its dimer interface and can transfer this cluster to an apoprotein acting as an [Fe-S] cluster scaffold protein, a function recently deduced for its eukaryotic homologues.