APO1 promotes the splicing of chloroplast group II introns and harbors a plant-specific zinc-dependent RNA binding domain

Arabidopsis thaliana APO1 is required for the accumulation of the chloroplast photosystem I and NADH dehydrogenase complexes and had been proposed to facilitate the incorporation of [4Fe-4S] clusters into these complexes. The identification of maize (Zea mays) APO1 in coimmunoprecipitates with a protein involved in chloroplast RNA splicing prompted us to investigate a role for APO1 in splicing. We show here that APO1 promotes the splicing of several chloroplast group II introns: in Arabidopsis apo1 mutants, ycf3-intron 2 remains completely unspliced, petD intron splicing is strongly reduced, and the splicing of several other introns is compromised. These splicing defects can account for the loss of photosynthetic complexes in apo1 mutants. Recombinant APO1 from both maize and Arabidopsis binds RNA with high affinity in vitro, demonstrating that DUF794, the domain of unknown function that makes up almost the entirety of APO1, is an RNA binding domain. We provide evidence that DUF794 harbors two motifs that resemble zinc fingers, that these bind zinc, and that they are essential for APO1 function. DUF794 is found in a plant-specific protein family whose members are all predicted to localize to mitochondria or chloroplasts. Thus, DUF794 adds a new example to the repertoire of plant-specific RNA binding domains that emerged as a product of nuclear-organellar coevolution.     

[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.     

In vitro activation of apo-aconitase using a [4Fe-4S] cluster-loaded form of the IscU [Fe-S] cluster scaffolding protein

Genetic experiments have established that IscU is involved in maturation of [Fe-S] proteins that require either [2Fe-2S] or [4Fe-4S] clusters for their biological activities. Biochemical studies have also shown that one [2Fe-2S] cluster can be assembled in vitro within each subunit of the IscU homodimer and that these clusters can be reductively coupled to form a single [4Fe-4S] cluster. In the present work, it is shown that the [4Fe-4S] cluster-loaded form of A. vinelandii IscU, but not the [2Fe-2S] cluster-loaded form, can be used for intact cluster transfer to an apo form of A. vinelandii aconitase A, a member of the monomeric dehydratase family of proteins that requires a [4Fe-4S] cluster for enzymatic activity. The rate of [4Fe-4S] cluster transfer from IscU to apo-aconitase A was not affected by the presence of the HscA/HscB co-chaperone system and MgATP. However, an altered form of a [4Fe-4S] cluster-containing IscU, having the highly conserved aspartate-39 residue substituted with alanine, is an effective inhibitor of wild-type [4Fe-4S] cluster-loaded IscU-directed activation of apo-aconitase A. In contrast, neither the clusterless form of IscU nor the [2Fe-2S] cluster-loaded form of IscU is an effective inhibitor of IscU-directed apo-aconitase A activation. These results are interpreted to indicate that the [2Fe-2S] and [4Fe-4S] cluster-loaded forms of IscU adopt different conformations that provide specificity with respect to the maturation of [2Fe-2S] and [4Fe-4S] centers in proteins.     

ISCA1 Orchestrates ISCA2 and NFU1 in the Maturation of Human Mitochondrial [4Fe-4S] Proteins

The late-acting steps of the pathway responsible for the maturation of mitochondrial [4Fe-4S] proteins are still elusive. Three proteins ISCA1, ISCA2 and NFU1 were shown to be implicated in the assembly of [4Fe-4S] clusters and their transfer into mitochondrial apo proteins. We present here a NMR-based study showing a detailed molecular model of the succession of events performed in a coordinated manner by ISCA1, ISCA2 and NFU1 to make [4Fe-4S] clusters available to mitochondrial apo proteins. We show that ISCA1 is the key player of the [4Fe-4S] protein maturation process because of its ability to interact with both NFU1 and ISCA2, which, instead do not interact each other. ISCA1 works as the promoter of the interaction between ISCA2 and NFU1 being able to determine the formation of a transient ISCA1-ISCA2-NFU1 ternary complex. We also show that ISCA1, thanks to its specific interaction with the C-terminal cluster-binding domain of NFU1, drives [4Fe-4S] cluster transfer from the site where the cluster is assembled on the ISCA1-ISCA2 complex to a cluster binding site formed by ISCA1 and NFU1 in the ternary ISCA1-ISCA2-NFU1 complex. Such mechanism guarantees that the [4Fe-4S] cluster can be safely moved from where it is assembled on the ISCA1-ISCA2 complex to NFU1, thereby resulting the [4Fe-4S] cluster available for the mitochondrial apo proteins specifically requiring NFU1 for their maturation.     

Arabidopsis cytosolic Nbp35 homodimer can assemble both [2Fe-2S] and [4Fe-4S] clusters in two distinct domains

Iron-sulfur proteins play physiologically important roles in a variety of metabolic processes in eukaryotes. In plants, iron-sulfur cluster biosynthesis is known to take place both in mitochondria and chloroplasts. However no components that mediate iron-sulfur cluster delivery in the plant cell cytosol have been identified so far. Here we report identification and characterization of a cytosolic Nbp35 homolog named AtNbp35 from Arabidopsis thaliana. AtNbp35-deficient Arabidopsis mutants were seedling lethal. Unlike the previously characterized yeast ScNbp35 which forms a heterotetramer with ScCfd1, AtNbp35 forms a homodimer in the cytosol and can harbor both [4Fe-4S] and [2Fe-2S] clusters on its amino- and carboxyl-terminal domains, respectively. Taken together, our data suggest that Nbp35 plays a pivotal role in iron-sulfur cluster assembly and delivery in the plant cell cytosol as a bifunctional molecular scaffold.     

Nfu2: a scaffold protein required for [4Fe-4S] and ferredoxin iron-sulphur cluster assembly in Arabidopsis chloroplasts

Nfu proteins are candidates to act as scaffold protein in vivo for iron-sulphur cluster biogenesis. In this work, Nfu2 protein function in the chloroplast was investigated in vivo using T-DNA insertion lines disrupted in AtNfu2 gene. Both alleles characterized presented the same dwarf phenotype due to photosynthetic and metabolic limitations. Nfu2 cDNA expression in nfu2.1 mutant rescued this phenotype. Photosynthesis study of these mutants revealed an altered photosystem I (PSI) activity together with a decrease in PSI amount confirmed by immunodetection experiments, and leading to an over reduction of the plastoquinol pool. Decrease of plastid 4Fe-4S sulphite reductase activity correlates with PSI amount decrease and supports an alteration of 4Fe-4S cluster biogenesis in nfu2 chloroplasts. The decrease of electron flow from the PSI is combined with a decrease in ferredoxin amount in nfu2 mutants. Our results are therefore in favour of a requirement of Nfu2 protein for 4Fe-4S and 2Fe-2S ferredoxin cluster assembly, conferring to this protein an important function for plant growth and photosynthesis as demonstrated by nfu2 mutant phenotype. As glutamate synthase and Rieske Fe-S proteins are not affected in nfu2 mutants, these data indicate that different pathways are involved in Fe-S biogenesis in Arabidopsis chloroplasts.     

Structural Plasticity of NFU1 Upon Interaction with Binding Partners: Insights into the Mitochondrial [4Fe-4S] Cluster Pathway

In humans, the biosynthesis and trafficking of mitochondrial [4Fe-4S]²⁺ clusters is a highly coordinated process that requires a complex protein machinery. In a mitochondrial pathway among various proposed to biosynthesize nascent [4Fe-4S]²⁺ clusters, two [2Fe-2S]²⁺ clusters are converted into a [4Fe-4S]²⁺ cluster on a ISCA1-ISCA2 complex. Along this pathway, this cluster is then mobilized from this complex to mitochondrial apo recipient proteins with the assistance of accessory proteins. NFU1 is the accessory protein that first receives the [4Fe-4S]²⁺ cluster from ISCA1-ISCA2 complex. A structural view of the protein–protein recognition events occurring along the [4Fe-4S]²⁺ cluster trafficking as well as how the globular N-terminal and C-terminal domains of NFU1 act in such process is, however, still elusive. Here, we applied small-angle X-ray scattering coupled with on-line size-exclusion chromatography and paramagnetic NMR to disclose structural snapshots of ISCA1-, ISCA2- and NFU1-containing apo complexes as well as the coordination of [4Fe-4S]²⁺ cluster bound to the ISCA1-NFU1 complex, which is the terminal stable species of the [4Fe-4S]²⁺ cluster transfer pathway involving ISCA1-, ISCA2- and NFU1 proteins. The structural modelling of ISCA1-ISCA2, ISCA1-ISCA2-NFU1 and ISCA1-NFU1 apo complexes, here reported, reveals that the structural plasticity of NFU1 domains is crucial to drive protein partner recognition and modulate [4Fe-4S]²⁺ cluster transfer from the cluster-assembly site in the ISCA1-ISCA2 complex to a cluster-binding site in the ISCA1-NFU1 complex. These structures allowed us to provide a first rational for the molecular function of the N-domain of NFU1, which can act as a modulator in the [4Fe-4S]²⁺ cluster transfer.     

Subunit D of RNA polymerase from Methanosarcina acetivorans contains two oxygen-labile [4Fe-4S] clusters: implications for oxidant-dependent regulation of transcription

Subunit D of multisubunit RNA polymerase from many species of archaea is predicted to bind one to two iron-sulfur (Fe-S) clusters, the function of which is unknown. A survey of encoded subunit D in the genomes of sequenced archaea revealed six distinct groups based on the number of complete or partial [4Fe-4S] cluster motifs within domain 3. Only subunit D from strictly anaerobic archaea, including all members of the Methanosarcinales, are predicted to bind two [4Fe-4S] clusters. We report herein the purification and characterization of Methanosarcina acetivorans subunit D in complex with subunit L. Expression of subunit D and subunit L in Escherichia coli resulted in the purification of a D-L heterodimer with only partial [4Fe-4S] cluster content. Reconstitution in vitro with iron and sulfide revealed that the M. acetivorans D-L heterodimer is capable of binding two redox-active [4Fe-4S] clusters. M. acetivorans subunit D deleted of domain 3 (DΔD3) was still capable of co-purifying with subunit L but was devoid of [4Fe-4S] clusters. Affinity purification of subunit D or subunit DΔD3 from M. acetivorans resulted in the co-purification of endogenous subunit L with each tagged subunit D. Overall, these results suggest that domain 3 of subunit D is required for [4Fe-4S] cluster binding, but the [4Fe-4S] clusters and domain 3 are not required for the formation of the D-L heterodimer. However, exposure of two [4Fe-4S] cluster-containing D-L heterodimer to oxygen resulted in loss of the [4Fe-4S] clusters and subsequent protein aggregation, indicating that the [4Fe-4S] clusters influence the stability of the D-L heterodimer and therefore have the potential to regulate the assembly and/or activity of RNA polymerase in an oxidant-dependent manner.     

The Arabidopsis chloroplastic NifU-like protein CnfU, which can act as an iron-sulfur cluster scaffold protein, is required for biogenesis of ferredoxin and photosystem I

The biosynthesis of iron-sulfur clusters is a highly regulated process involving several proteins. Among them, so-called scaffold proteins play pivotal roles in both the assembly and delivery of iron-sulfur clusters. Here, we report the identification of two chloroplast-localized NifU-like proteins, AtCnfU-V and AtCnfU-IVb, from Arabidopsis (Arabidopsis thaliana) with high sequence similarity to a cyanobacterial NifU-like protein that was proposed to serve as a molecular scaffold. AtCnfU-V is constitutively expressed in several tissues of Arabidopsis, whereas the expression of AtCnfU-IVb is prominent in the aerial parts. Mutant Arabidopsis lacking AtCnfU-V exhibited a dwarf phenotype with faint pale-green leaves and had drastically impaired photosystem I accumulation. Chloroplasts in the mutants also showed a decrease in both the amount of ferredoxin, a major electron carrier of the stroma that contains a [2Fe-2S] cluster, and in the in vitro activity of iron-sulfur cluster insertion into apo-ferredoxin. When expressed in Escherichia coli cells, AtCnfU-V formed a homodimer carrying a [2Fe-2S]-like cluster, and this cluster could be transferred to apo-ferredoxin in vitro to form holo-ferredoxin. We propose that AtCnfU has an important function as a molecular scaffold for iron-sulfur cluster biosynthesis in chloroplasts and thereby is required for biogenesis of ferredoxin and photosystem I.     

Biogenesis of iron-sulfur clusters in photosystem I: holo-NfuA from the cyanobacterium Synechococcus sp. PCC 7002 rapidly and efficiently transfers [4Fe-4S] clusters to apo-PsaC in vitro

The NfuA protein has been postulated to act as a scaffolding protein in the biogenesis of photosystem (PS) I and other iron-sulfur (Fe/S) proteins in cyanobacteria and chloroplasts. To determine the properties of NfuA, recombinant NfuA from Synechococcus sp. PCC 7002 was overproduced and purified. In vitro reconstituted NfuA contained oxygen- and EDTA-labile Fe/S cluster(s), which had EPR properties consistent with [4Fe-4S] clusters. After reconstitution with 57Fe2+, M?ssbauer studies of NfuA showed a broad quadrupole doublet that confirmed the presence of [4Fe-4S]2+ clusters. Native gel electrophoresis under anoxic conditions and chemical cross-linking showed that holo-NfuA forms dimers and tetramers harboring Fe/S cluster(s). Combined with iron and sulfide analyses, the results indicated that one [4Fe-4S] cluster was bound per NfuA dimer. Fe/S cluster transfer from holo-NfuA to apo-PsaC of PS I was studied by reconstitution of PS I complexes using P700-F(X) core complexes, PsaD, apo-PsaC, and holo-NfuA. Electron transfer measurements by time-resolved optical spectroscopy showed that holo-NfuA rapidly and efficiently transferred [4Fe-4S] clusters to PsaC in a reaction that required contact between the two proteins. The NfuA-reconstituted PS I complexes had typical charge recombination kinetics from [F(A)/F(B)](-) to P700+ and light-induced low-temperature EPR spectra. These results establish that cyanobacterial NfuA can act as a scaffolding protein for the insertion of [4Fe-4S] clusters into PsaC of PS I in vitro.     

IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU

Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes. The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide. In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography. The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and M?ssbauer investigations. The results show sequential cluster assembly with the initial IscU product containing one [2Fe-2S](2+) cluster per dimer converting first to a form containing two [2Fe-2S](2+) clusters per dimer and finally to a form that contains one [4Fe-4S](2+) cluster per dimer. Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2+) clusters in IscU are shown to have incomplete cysteinyl ligation. In addition, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins.     

Characterization of cluster N5 as a fast-relaxing [4Fe-4S] cluster in the Nqo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans

The NADH-quinone oxidoreductase from Paracoccus denitrificans consists of 14 subunits (Nqo1-14) and contains one FMN and eight iron-sulfur clusters. The Nqo3 subunit possesses fully conserved 11 Cys and 1 His in its N-terminal region and is considered to harbor three iron-sulfur clusters; however, only one binuclear (N1b) and one tetranuclear (N4) were previously identified. In this study, the Nqo3 subunit containing 1x[2Fe-2S] and 2x[4Fe-4S] clusters was expressed in Escherichia coli. The second [4Fe-4S](1+) cluster is detected by EPR spectroscopy below 6 K, exhibiting very fast spin relaxation. The resolved EPR spectrum of this cluster is broad and nearly axial. The subunit exhibits an absorption-type EPR signal around g approximately 5 region below 6 K, most likely arising from an S = 3/2 ground state of the fast-relaxing [4Fe-4S](1+) species. The substitution of the conserved His(106) with Cys specifically affected the fast-relaxing [4Fe-4S](1+) cluster, suggesting that this cluster is coordinated by His(106). In the cholate-treated NDH-1-enriched P. denitrificans membranes, we observed EPR signals arising from a [4Fe-4S] cluster below 6 K, exhibiting properties similar to those of cluster N5 detected in other complex I/NDH-1 and of the fast-relaxing [4Fe-4S](1+) cluster in the expressed Nqo3 subunit. Hence, we propose that the His-coordinated [4Fe-4S] cluster corresponds to cluster N5.     

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.     

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.     

Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe-2S] clusters

Glutaredoxins (Grxs) are small oxidoreductases that reduce disulphide bonds or protein-glutathione mixed disulphides. More than 30 distinct grx genes are expressed in higher plants, but little is currently known concerning their functional diversity. This study presents biochemical and spectroscopic evidence for incorporation of a [2Fe-2S] cluster in two heterologously expressed chloroplastic Grxs, GrxS14 and GrxS16, and in vitro cysteine desulphurase-mediated assembly of an identical [2Fe-2S] cluster in apo-GrxS14. These Grxs possess the same monothiol CGFS active site as yeast Grx5 and both were able to complement a yeast grx5 mutant defective in Fe-S cluster assembly. In vitro kinetic studies monitored by CD spectroscopy indicate that [2Fe-2S] clusters on GrxS14 are rapidly and quantitatively transferred to apo chloroplast ferredoxin. These data demonstrate that chloroplast CGFS Grxs have the potential to function as scaffold proteins for the assembly of [2Fe-2S] clusters that can be transferred intact to physiologically relevant acceptor proteins. Alternatively, they may function in the storage and/or delivery of preformed Fe-S clusters or in the regulation of the chloroplastic Fe-S cluster assembly machinery.     

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.     

Subunit location of the iron-sulfur clusters in fumarate reductase from Escherichia coli

The subunit location of the [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters in Escherichia coli fumarate reductase has been investigated by EPR studies of whole cells or whole cells extracts of a fumarate reductase deletion mutant with plasmid amplified expression of discrete fumarate reductase subunits or groups of subunits. The results indicate that both the [2Fe-2S] and [3Fe-4S] clusters are located entirely in the iron-sulfur protein subunit. Information concerning the specific cysteine residues that ligate these clusters has been obtained by investigating the EPR characteristics of cells of the deletion mutant amplified with a plasmid coding for the flavoprotein subunit and a truncated iron-sulfur protein subunit. While the results are not definitive with respect to the location of the [4Fe-4S] cluster, they are most readily interpreted in terms of this cluster being entirely in the flavoprotein subunit or bridging between the two catalytic domain subunits. These new results are discussed in light of the amino acid sequences of the two subunits and the sequences of structurally well characterized iron-sulfur proteins containing [2Fe-2S], [3Fe-4S], and [4Fe-4S] centers.     

Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins

Most eukaryotes contain iron-sulfur cluster (ISC) assembly proteins related to Saccharomyces cerevisiae Isa1 and Isa2. We show here that Isa1 but not Isa2 can be functionally replaced by the bacterial relatives IscA, SufA, and ErpA. The specific function of these "A-type" ISC proteins within the framework of mitochondrial and bacterial Fe/S protein biogenesis is still unresolved. In a comprehensive in vivo analysis, we show that S. cerevisiae Isa1 and Isa2 form a complex that is required for maturation of mitochondrial [4Fe-4S] proteins, including aconitase and homoaconitase. In contrast, Isa1-Isa2 were dispensable for the generation of mitochondrial [2Fe-2S] proteins and cytosolic [4Fe-4S] proteins. Targeting of bacterial [2Fe-2S] and [4Fe-4S] ferredoxins to yeast mitochondria further supported this specificity. Isa1 and Isa2 proteins are shown to bind iron in vivo, yet the Isa1-Isa2-bound iron was not needed as a donor for de novo assembly of the [2Fe-2S] cluster on the general Fe/S scaffold proteins Isu1-Isu2. Upon depletion of the ISC assembly factor Iba57, which specifically interacts with Isa1 and Isa2, or in the absence of the major mitochondrial [4Fe-4S] protein aconitase, iron accumulated on the Isa proteins. These results suggest that the iron bound to the Isa proteins is required for the de novo synthesis of [4Fe-4S] clusters in mitochondria and for their insertion into apoproteins in a reaction mediated by Iba57. Taken together, these findings define Isa1, Isa2, and Iba57 as a specialized, late-acting ISC assembly subsystem that is specifically dedicated to the maturation of mitochondrial [4Fe-4S] proteins.