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.     

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).     

Acidic residues of yeast frataxin have an essential role in Fe-S cluster assembly

Friedreich ataxia is caused by decreased levels of frataxin, a mitochondrial acidic protein that is assumed to act as chaperone in the assembly of Fe-S clusters on the scaffold Isu protein. Frataxin has the in vitro capacity to form iron-loaded multimers, which also suggests an iron storage function. It has been reported that alanine substitution of residues in an acidic ridge of yeast frataxin (Yfh1) elicits loss of iron binding in vitro but has no effect on Fe-S cluster synthesis in vivo. Here, we show that a marked change in the electrostatic properties of a specific region of Yfh1 surface - by substituting two or four acidic residues by lysine or alanine, respectively - impairs Fe-S cluster assembly, weakens the interaction between Yfh1 and Isu1, and increases oxidative damage. Therefore, the acidic ridge is essential for the Yfh1 function and is likely to be involved in iron-mediated protein-protein interactions.     

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.     

A novel eukaryotic factor for cytosolic Fe-S cluster assembly

Iron regulatory protein 1 (IRP1) is regulated through the assembly/disassembly of a [4Fe-4S] cluster, which interconverts IRP1 with cytosolic aconitase. A genetic screen to isolate Saccharomyces cerevisiae strains bearing mutations in genes required for the conversion of IRP1 to c-aconitase led to the identification of a previously uncharacterized, essential gene, which we call CFD1 (cytosolic Fe-S cluster deficient). CFD1 encodes a highly conserved, putative P-loop ATPase. A non-lethal mutation of CFD1 (cfd1-1) reduced c-aconitase specific activity in IRP1-transformed yeast by >90%, although IRP1 in these cells could be readily converted to c-aconitase in vitro upon incubation with iron alone. IRP1-transformed cfd1-1 yeast lacked EPR-detectable Fe-S clusters in c-aconitase, pointing to a defect in Fe-S cluster assembly. The specific activity of another cytosolic Fe-S protein, Leu1p, was also inhibited by >90% in cfd1-1 yeast, whereas activity of mitochondrial Fe-S proteins was not inhibited. Consistent with a cytosolic site of activity, Cfd1p was localized in the cytoplasm. To our knowledge, Cfd1p is the first cytoplasmic Fe-S cluster assembly factor described in eukaryotes.     

Evolution of mitochondrial chaperones utilized in Fe-S cluster biogenesis

Biogenesis of Fe-S clusters is an essential process [1]. In both Escherichia coli and Saccharomyces cerevisiae, insertion of clusters into an apoprotein requires interaction between a scaffold protein on which clusters are assembled and a molecular chaperone system--an unusually specialized mitochondrial Hsp70 (mtHsp70) and its J protein cochaperone [2]. It is generally assumed that mitochondria inherited their Fe-S cluster assembly machinery from prokaryotes via the endosymbiosis of a bacterium that led to formation of mitochondria. Indeed, phylogenetic analyses demonstrated that the S. cerevisiae J protein, Jac1, and the scaffold, Isu, are orthologous to their bacterial counterparts [3, 4]. However, our analyses indicate that the specialized mtHsp70, Ssq1, is only present in a subset of fungi; most eukaryotes have a single mtHsp70, Ssc1. We propose that an Hsp70 having a role limited to Fe-S cluster biogenesis arose twice during evolution. In the fungal lineage, the gene encoding multifunctional mtHsp70, Ssc1, was duplicated, giving rise to specialized Ssq1. Therefore, Ssq1 is not orthologous to the specialized Hsp70 from E. coli (HscA), but shares a striking level of convergence at the biochemical level. Thus, in the vast majority of eukaryotes, Jac1 and Isu function with the single, multifunctional mtHsp70 in Fe-S cluster biogenesis.     

Direct interaction of coenzyme M with the active-site Fe-S cluster of heterodisulfide reductase

Heterodisulfide reductase (HDR) catalyzes the formation of coenzyme M (CoM-SH) and coenzyme B (CoB-SH) by the reversible reduction of the heterodisulfide, CoM-S-S-CoB. This reaction recycles the two thiol coenzymes involved in the final step of microbial methanogenesis. Electron paramagnetic resonance (EPR) and variable-temperature magnetic circular dichroism spectroscopic experiments on oxidized HDR incubated with CoM-SH revealed a S=1/2 [4Fe-4S]3) cluster, the EPR spectrum of which is broadened in the presence of CoM-33SH [Duin, E.C., Madadi-Kahkesh, S., Hedderich, R., Clay, M.D. and Johnson, M.K. (2002) Heterodisulfide reductase from Methanothermobacter marburgensis contains an active-site [4Fe-4S] cluster that is directly involved in mediating heterodisulfide reduction. FEBS Lett. 512, 263-268; Duin, E.C., Bauer, C., Jaun, B. and Hedderich, R. (2003) Coenzyme M binds to a [4Fe-4S] cluster in the active site of heterodisulfide reductase as deduced from EPR studies with the [33S]coenzyme M-treated enzyme. FEBS Lett. 538, 81-84]. These results provide indirect evidence that the disulfide binds to the iron-sulfur cluster during reduction. We report here direct structural evidence for this interaction from Se X-ray absorption spectroscopic investigation of HDR treated with the selenium analog of coenzyme M (CoM-SeH). Se K edge extended X-ray absorption fine structure confirms a direct interaction of the Se in CoM-SeH-treated HDR with an Fe atom of the Fe-S cluster at an Fe-Se distance of 2.4A.     

Biogenesis of Fe-S cluster by the bacterial Suf system: SufS and SufE form a new type of cysteine desulfurase

Biosynthesis of iron-sulfur clusters (Fe-S) depends on multiprotein systems. Recently, we described the SUF system of Escherichia coli and Erwinia chrysanthemi as being important for Fe-S biogenesis under stressful conditions. The SUF system is made of six proteins: SufC is an atypical cytoplasmic ABC-ATPase, which forms a complex with SufB and SufD; SufA plays the role of a scaffold protein for assembly of iron-sulfur clusters and delivery to target proteins; SufS is a cysteine desulfurase which mobilizes the sulfur atom from cysteine and provides it to the cluster; SufE has no associated function yet. Here we demonstrate that: (i) SufE and SufS are both cystosolic as all members of the SUF system; (ii) SufE is a homodimeric protein; (iii) SufE forms a complex with SufS as shown by the yeast two-hybrid system and by affinity chromatography; (iv) binding of SufE to SufS is responsible for a 50-fold stimulation of the cysteine desulfurase activity of SufS. This is the first example of a two-component cysteine desulfurase enzyme.     

The asymmetric IscA homodimer with an exposed [2Fe-2S] cluster suggests the structural basis of the Fe-S cluster biosynthetic scaffold

It has been shown that the so-called scaffold proteins are vital in Fe-S cluster biosynthesis by providing an intermediate site for the assembly of Fe-S clusters. However, since no structural information on such scaffold proteins with bound Fe-S cluster intermediates is available, the structural basis of the core of Fe-S cluster biosynthesis remains poorly understood. Here we report the first Fe-S cluster-bound crystal structure of a scaffold protein, IscA, from Thermosynechococcus elongatus, which carries three strictly conserved cysteine residues. Surprisingly, one partially exposed [2Fe-2S] cluster is coordinated by two conformationally distinct IscA protomers, termed alpha and beta, with asymmetric cysteinyl ligation by Cys37, Cys101, Cys103 from alpha and Cys103 from beta. In the crystal, two alphabeta dimers form an unusual domain-swapped tetramer via central domains of beta protomers. Together with additional biochemical data supporting its physiologically relevant configuration, we propose that the unique asymmetric Fe-S cluster coordination and the resulting distinct conformational stabilities of the two IscA protomers are central to the function of IscA-type Fe-S cluster biosynthetic scaffold.     

Compensation for a defective interaction of the hsp70 ssq1 with the mitochondrial Fe-S cluster scaffold isu

Ssq1, a specialized yeast mitochondrial Hsp70, plays a critical role in the biogenesis of proteins containing Fe-S clusters through its interaction with Isu, the scaffold on which clusters are built. Two substitutions within the Ssq1 substrate binding cleft, both of which severely reduced affinity for Isu, had very different effects in vivo. Cells expressing Ssq1(F462S), which had no detectable affinity for Isu, are indistinguishable from Deltassq1 cells, underscoring the importance of the Ssq1-Isu1 interaction in vivo. In contrast, cells expressing Ssq1(V472F), whose affinity for Isu is at least 10-fold lower than that of wild-type Ssq1, had only moderately reduced Fe-S enzyme activities and increased iron levels and grew similarly to wild-type cells. Consistent with the reduced affinity for Isu, the ATPase activity of Ssq1(V472F) was stimulated less well than that of Ssq1 upon addition of Isu and Jac1, the J-protein partner of Ssq1. However, higher concentrations of Jac1 or Isu1, which form a stable complex, could compensate for this defect in stimulation of Ssq1(V472F). Expression of Isu1 was up-regulated 10-fold in ssq1(V472F) compared with wild-type cells, suggesting that formation of a Jac1-Isu1 complex can overcome a lowered affinity of Ssq1 for Isu in vivo as well as in vitro.     

CpSufE activates the cysteine desulfurase CpNifS for chloroplastic Fe-S cluster formation

CpNifS, a cysteine desulfurase required to supply sulfur for ironsulfur cluster biogenesis in Arabidopsis thaliana chloroplasts, belongs to a class of NifS-like enzymes with low endogenous cysteine desulfurase activity. Its bacterial homologue SufS is stimulated by SufE. Here we characterize the Arabidopsis chloroplast protein CpSufE, which has an N-terminal SufE-like domain and a C-terminal BolA-like domain unique to higher plants. CpSufE is targeted to the chloroplast stroma, indicated by green fluorescent protein localization and immunoblot experiments. Like CpNifS, CpSufE is expressed in all major tissues, with higher expression in green parts. Its expression is light-dependent and regulated at the mRNA level. The addition of purified recombinant CpSufE increased the Vmax for the cysteine desulfurase activity of CpNifS over 40-fold and decreased the KM toward cysteine from 0.1 to 0.043 mm. In contrast, CpSufE addition decreased the affinity of CpNifS for selenocysteine, as indicated by an increase in the KM from 2.9 to 4.17 mm, and decreased the Vmax for selenocysteine lyase activity by 30%. CpSufE forms dynamic complexes with CpNifS, indicated by gel filtration, native PAGE, and affinity chromatography experiments. A mutant of CpSufE in which the single cysteine was changed to serine was not active in stimulating CpNifS, although it did compete with WT CpSufE. The iron-sulfur cluster reconstitution activity of the CpNifS-CpSufE complex toward apoferredoxin was 20-fold higher than that of CpNifS alone. We conclude that CpNifS and CpSufE together form a cysteine desulfurase required for iron-sulfur cluster formation in chloroplasts.     

Iron chaperones for mitochondrial Fe-S cluster biosynthesis and ferritin iron storage

Protein controlled iron homeostasis is essential for maintaining appropriate levels and availability of metal within cells. Recently, two iron chaperones have been discovered that direct metal within two unique pathways: (1) mitochondrial iron-sulfur (Fe-S) cluster assembly and (2) within the ferritin iron storage system. Although structural and functional details describing how these iron chaperones operate are emerging, both share similar iron binding affinities and metal-ligand site structures that enable them to bind and release Fe2+ to specific protein partners. Molecular details related to iron binding and delivery by these chaperones will be explored within this review.     

Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

Alpha-toxin-induced phosphorylation of PDK1 via the tyrosine kinase A (TrkA) receptor signaling pathway plays an important role in the activation of rabbit neutrophils. The relation between the toxin and TrkA, however, remains poorly understood. Here, we show that the toxin-induced phosphorylation of TrkA is closely related to the induction of neurite-outgrowth in PC12 cells. The toxin induced neurite-outgrowth and phosphorylation of TrkA in the cells in a dose-dependent manner. K252a, a TrkA inhibitor, and shRNA for TrkA inhibited the toxin-induced neurite-outgrowth, and phosphorylation of TrkA and ERK1/2. PD98059, an inhibitor of the ERK1/2 cascade, inhibited phosphorylation of ERK1/2 and the neurite-outgrowth induced by alpha-toxin. The wild-type toxin induced the formation of diacylglycerol, and neurite-outgrowth, but H148G, a variant toxin which binds to cell membranes and has lost the enzymatic activity did not. We demonstrated that the phosphorylation of TrkA through the phospholipid metabolism induced by the toxin synergistically play a key role in neurite-outgrowth.     

Identification of a Nfs1p-bound persulfide intermediate in Fe-S cluster synthesis by intact mitochondria

Cysteine desulfurases generate a covalent persulfide intermediate from cysteine, and this activated form of sulfur is essential for the synthesis of iron-sulfur (Fe-S) clusters. In yeast mitochondria, there is a complete machinery for Fe-S cluster synthesis, including a cysteine desulfurase, Nfs1p. Here we show that following supplementation of isolated mitochondria with [(35)S]cysteine, a radiolabeled persulfide could be detected on Nfs1p. The persulfide persisted under conditions that did not permit Fe-S cluster formation, such as nucleotide and/or iron depletion of mitochondria. By contrast, under permissive conditions, the radiolabeled Nfs1p persulfide was greatly reduced and radiolabeled aconitase was formed, indicating transfer of persulfide to downstream Fe-S cluster recipients. Nfs1p in mitochondria was found to be relatively more resistant to inactivation by N-ethylmaleimide (NEM) as compared with a prokaryotic cysteine desulfurase. Mitochondria treated with NEM (1mM) formed the persulfide on Nfs1p but failed to generate Fe-S clusters on aconitase, likely due to inactivation of downstream recipient(s) of the Nfs1p persulfide. Thus the Nfs1p-bound persulfide as described here represents a precursor en route to Fe-S cluster synthesis in mitochondria.     

Arabidopsis AtIscA-I is affected by deficiency of Fe-S cluster biosynthetic scaffold AtCnfU-V

IscA has been proposed to be a scaffold protein of the iron-sulfur cluster biosynthetic machinery. We have identified the IscA homolog to be localized to plastids, termed AtIscA-I, in Arabidopsis thaliana. The AtIscA-I protein was apparently constitutively expressed in all tissues analyzed in Arabidopsis. The AtIscA-I protein exists in the stroma as a soluble protein which tends to form a homo-dimer and can host a [2Fe-2S]-like cluster. Complete loss of the protein from plastids did not cause any significant defect either in normal plant growth or in biogenesis of major iron-sulfur proteins, indicating this protein is not essential or redundant for these functions. In contrast, loss of one of the three plastid-localized CnfU scaffold proteins, AtCnfU-V, caused significant reduction in the level of AtIscA-I. These data suggest that efficient biogenesis of AtIscA-I scaffold requires function of another essential scaffold protein CnfU.     

Iron-responsive degradation of iron-regulatory protein 1 does not require the Fe-S cluster

The generally accepted role of iron-regulatory protein 1 (IRP1) in orchestrating the fate of iron-regulated mRNAs depends on the interconversion of its cytosolic aconitase and RNA-binding forms through assembly/disassembly of its Fe-S cluster, without altering protein abundance. Here, we show that IRP1 protein abundance can be iron-regulated. Modulation of IRP1 abundance by iron did not require assembly of the Fe-S cluster, since a mutant with all cluster-ligating cysteines mutated to serine underwent iron-induced protein degradation. Phosphorylation of IRP1 at S138 favored the RNA-binding form and promoted iron-dependent degradation. However, phosphorylation at S138 was not required for degradation. Further, degradation of an S138 phosphomimetic mutant was not blocked by mutation of cluster-ligating cysteines. These findings were confirmed in mouse models with genetic defects in cytosolic Fe-S cluster assembly/disassembly. IRP1 RNA-binding activity was primarily regulated by IRP1 degradation in these animals. Our results reveal a mechanism for regulating IRP1 action relevant to the control of iron homeostasis during cell proliferation, inflammation, and in response to diseases altering cytosolic Fe-S cluster assembly or disassembly.     

NapF is a cytoplasmic iron-sulfur protein required for Fe-S cluster assembly in the periplasmic nitrate reductase

The periplasmic nitrate reductase (Nap) is wide-spread in proteobacteria. NapA, the nitrate reductase catalytic subunit, contains a Mo-bisMGD cofactor and one [4Fe-4S] cluster. The nap gene clusters in many bacteria, including Rhodobacter sphaeroides DSM158, contain an napF gene, disruption of which drastically decreases both in vitro and in vivo nitrate reductase activities. In spite its importance in the Nap system, NapF has never been characterized biochemically, and its role remains unknown. The NapF protein has four polycysteine clusters that suggest that it is an iron-sulfur-containing protein. In the present study, a His(6)-tagged NapF protein was overproduced in Escherichia coli and purified anaerobically. The purified NapF protein was used to obtain polyclonal antibodies raised in rabbit, and cellular fractionation of R. sphaeroides followed by immunoprobing with anti-NapF antibodies revealed that the native NapF protein is located in the cytoplasm. This contrasts with the periplasmic location of the mature NapA. However, NapA could not be detected in an isogenic napF(-) strain of R. sphaeroides. The His(6)-tagged NapF protein displayed spectral properties indicative of Fe-S clusters, but these features were rapidly lost, suggesting cluster lability. However, reconstitution of the Fe-S centers into the apo-NapF protein was achieved in the presence of Azotobacter vinelandii cysteine desulfurase (NifS), and this allowed the recovery of nitrate reductase activity in NapA protein that had previously been treated with 2,2'-dipyridyl to remove the [4Fe-4S] cluster. This activity was not recovered in the absence of NapF. Taking into account the cytoplasmic localization of NapF, the presence of labile Fe-S clusters in the protein, the napF(-) strain phenotype, and the NapF-dependent reactivation of the 2,2'-dipyridyl-treated NapA, we propose a role for NapF in assembling the [4Fe-4S] center of the catalytic subunit NapA.     

Binding of yeast frataxin to the scaffold for Fe-S cluster biogenesis, Isu

Friedreich ataxia is caused by reduced activity of frataxin, a conserved iron-binding protein of the mitochondrial matrix, thought to supply iron for formation of Fe-S clusters on the scaffold protein Isu. Frataxin binds Isu in an iron-dependent manner in vitro. However, the biological relevance of this interaction and whether in vivo the interaction between frataxin and Isu is mediated by adaptor proteins is a matter of debate. Here, we report that alterations of conserved, surface-exposed residues of yeast frataxin, which have deleterious effects on cell growth, impair Fe-S cluster biogenesis and interaction with Isu while altering neither iron binding nor oligomerization. Our results support the idea that the surface of the beta-sheet, adjacent to the acidic, iron binding ridge, is important for interaction of Yfh1 with the Fe-S cluster scaffold and point to a critical role for frataxin in Fe-S cluster biogenesis.