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Mitochondrial [2Fe-2S] cluster biosynthesis is driven by the coordinated activities of the Iron-Sulfur Cluster (ISC) pathway protein machinery. Within the ISC machinery, the protein that provides a structural scaffold on which [2Fe-2S] clusters are assembled is the ISCU protein in humans; this protein is referred to as the "Scaffold" protein. Truncation of the C-terminal portion of ISCU causes the fatal disease "ISCU Myopathy", which exhibits phenotypes of reduced Fe-S cluster assembly in cells. In this report, the yeast ISCU ortholog "Isu1" has been characterized to gain a better understanding of the role of the scaffold protein in relation to [2Fe-2S] assembly and ISCU Myopathy. Here we explored the biophysical characteristics of the C-terminal region of Isu1, the segment of the protein that is truncated on the human ortholog during the disease ISCU Myopathy. We characterized the role of this region in relation to iron binding, protein stability, assembly of the ISC multiprotein complex required to accomplish Fe-S cluster assembly, and finally on overall cell viability. We determined the Isu1 C-terminus is essential for the completion of the Fe-S cluster assembly but serves a function independent of protein iron binding.

Advances in genomic diagnostics hold promise for improved care of rare hematologic diseases. Here, we describe a novel targeted therapeutic approach for Ghosal hematodiaphyseal dysplasia, an autosomal recessive disease characterized by severe normocytic anemia and bone abnormalities due to loss-of-function mutations in thromboxane A synthase 1 (TBXAS1). TBXAS1 metabolizes prostaglandin H2 (PGH2), a cyclooxygenase (COX) product of arachidonic acid, into thromboxane A2. Loss-of-function mutations in TBXAS result in an increase in PGH2 availability for other PG synthases. The current treatment for Ghosal hematodiaphyseal dysplasia syndrome consists of corticosteroids. We hypothesize that nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit COX-1 and COX-2, could ameliorate the effects of TBXAS1 loss and improve hematologic function by reducing prostaglandin formation. We treated 2 patients with Ghosal hematodiaphyseal dysplasia syndrome, an adult and a child, with standard doses of NSAIDs (aspirin or ibuprofen). Both patients had rapid improvements concerning hematologic parameters and inflammatory markers without adverse events. Mass spectrometry analysis demonstrated that urinary PG metabolites were increased along with proinflammatory lipoxygenase (LOX) products 5-hydroxyeicosatetraenoic acid and leukotriene E4. Our data show that NSAIDs at standard doses surprisingly reduced both COX and LOX products, leading to the resolution of cytopenia, and should be considered for first-line treatment for Ghosal hematodiaphyseal dysplasia syndrome.

Saccharomyces cerevisiae Grx3 and Grx4 are multidomain monothiol glutaredoxins that are redundant with each other. They can be efficiently complemented by heterologous expression of their mammalian ortholog, PICOT, which has been linked to tumor development and embryogenesis. PICOT is now believed to act as a chaperone distributing Fe-S clusters, although the first link to iron metabolism was observed with its yeast counterparts. Like PICOT, yeast Grx3 and Grx4 reside in the cytosol and nucleus where they form unusual Fe-S clusters coordinated by two glutaredoxins with CGFS motifs and two molecules of glutathione. Depletion or deletion of Grx3/Grx4 leads to functional impairment of virtually all cellular iron-dependent processes and loss of cell viability, thus making these genes the most upstream components of the iron utilization system. Nevertheless, the Δgrx3/4 double mutant in the BY4741 genetic background is viable and exhibits slow but stable growth under hypoxic conditions. Upon exposure to air, growth of the double deletion strain ceases, and suppressor mutants appear. Adopting a high copy-number library screen approach, we discovered novel genetic interactions: overexpression of ESL1, ESL2, SOK1, SFP1 or BDF2 partially rescues growth and iron utilization defects of Δgrx3/4. This genetic escape from the requirement for Grx3/Grx4 has not been previously described. Our study shows that even a far-upstream component of the iron regulatory machinery (Grx3/4) can be bypassed, and cellular networks involving RIM101 pH sensing, cAMP signaling, mTOR nutritional signaling, or bromodomain acetylation, may confer the bypassing activities.

Rim2 is an unusual mitochondrial carrier protein capable of transporting both iron and pyrimidine nucleotides. Here we characterize two point mutations generated in the predicted substrate-binding site, finding that they yield disparate effects on iron and pyrimidine transport. The Rim2 (E248A) mutant was deficient in mitochondrial iron transport activity. By contrast, the Rim2 (K299A) mutant specifically abrogated pyrimidine nucleotide transport and exchange, while leaving iron transport activity largely unaffected. Strikingly, E248A preserved TTP/TTP homoexchange but interfered with TTP/TMP heteroexchange, perhaps because proton coupling was dependent on the E248 acidic residue. Rim2-dependent iron transport was unaffected by pyrimidine nucleotides. Rim2-dependent pyrimidine transport was competed by Zn2+ but not by Fe2+, Fe3+ or Cu2+. The iron and pyrimidine nucleotide transport processes displayed different salt requirements; pyrimidine transport was dependent on the salt content of the buffer whereas iron transport was salt independent. In mitochondria containing Rim2 (E248A), iron proteins were decreased, including aconitase (Fe-S), pyruvate dehydrogenase (lipoic acid containing) and cytochrome c (heme protein). Additionally, the rate of Fe-S cluster synthesis in isolated and intact mitochondria was decreased compared with the K299A mutant, consistent with the impairment of iron-dependent functions in that mutant. In summary, mitochondrial iron transport and pyrimidine transport by Rim2 occur separately and independently. Rim2 could be a bifunctional carrier protein.

Iron–sulfur clusters are essential cofactors of proteins. In eukaryotes, iron–sulfur cluster biogenesis requires a mitochondrial iron–sulfur cluster machinery (ISC) and a cytoplasmic iron–sulfur protein assembly machinery (CIA). Here we used mitochondria and cytoplasm isolated from yeast cells, and [(35)S]cysteine to detect cytoplasmic Fe–(35)S cluster assembly on a purified apoprotein substrate. We showed that mitochondria generate an intermediate, called (Fe–S)(int), needed for cytoplasmic iron–sulfur cluster assembly. The mitochondrial biosynthesis of (Fe–S)(int) required ISC components such as Nfs1 cysteine desulfurase, Isu1/2 scaffold, and Ssq1 chaperone. Mitochondria then exported (Fe–S)(int) via the Atm1 transporter in the inner membrane, and we detected (Fe–S)(int) in active form. When (Fe–S)(int) was added to cytoplasm, CIA utilized it for iron–sulfur cluster assembly without any further help from the mitochondria. We found that both iron and sulfur for cytoplasmic iron–sulfur cluster assembly originate from the mitochondria, revealing a surprising and novel mitochondrial role. Mitochondrial (Fe–S)(int) export was most efficient in the presence of cytoplasm containing an apoprotein substrate, suggesting that mitochondria respond to the cytoplasmic demand for iron–sulfur cluster synthesis. Of note, the (Fe–S)(int) is distinct from the sulfur intermediate called S(int), which is also made and exported by mitochondria but is instead used for cytoplasmic tRNA thiolation. In summary, our findings establish a direct and vital role of mitochondria in cytoplasmic iron–sulfur cluster assembly in yeast cells.

The cysteine desulfurase Nfs1/Isd11 uses the amino acid cysteine as the substrate and its activity is absolutely required for contributing persulfide sulfur to the essential process of iron-sulfur (Fe-S) cluster assembly in mitochondria. Here we describe a novel regulatory process involving phosphorylation of Nfs1 in mitochondria. Phosphorylation enhanced cysteine desulfurase activity, while dephosphorylation decreased its activity. Nfs1 phosphopeptides were identified, and the corresponding phosphosite mutants showed impaired persulfide formation. Nfs1 pull down from mitochondria recovered an associated kinase activity, and Yck2, a kinase present in the pull down, was able to phosphorylate Nfs1 in vitro and stimulate cysteine desulfurase activity. Yck2 exhibited an eclipsed distribution in the mitochondrial matrix, although other cellular localizations have been previously described. Mitochondria lacking the Yck2 protein kinase (∆yck2) showed less phosphorylating activity for Nfs1. Compared with wild-type mitochondria, ∆yck2 mitochondria revealed slower persulfide formation on Nfs1 consistent with a role of Yck2 in regulating mitochondrial cysteine desulfurase activity. We propose that Nfs1 phosphorylation may provide a means of rapid adaptation to increased metabolic demand for sulfur and Fe-S clusters within mitochondria.

Arbuscular mycorrhizal (AM) fungi can improve iron (Fe) acquisition of their host plants. Here, we report a characterization of two components of the high-affinity reductive Fe uptake system of Rhizophagus irregularis, the ferric reductase (RiFRE1) and the high affinity Fe permeases (RiFTR1-2). In the extraradical mycelia (ERM), Fe deficiency induced activation of a plasma membrane-localized ferric reductase, an enzyme that reduces Fe(III) sources to the more soluble Fe(II). Yeast mutant complementation assays showed that RiFRE1 encodes a functional ferric reductase and RiFTR1 an iron permease. In the heterologous system, RiFTR1 was expressed in the plasma membrane while RiFTR2 was expressed in the endomembranes. In the ERM, the highest expression levels of RiFTR1 were found in mycelia grown in media with 0.045 mM Fe, while RiFTR2 was upregulated under Fe-deficient conditions. RiFTR2 expression also increased in the intraradical mycelia (IRM) of maize plants grown without Fe. These data indicate that the Fe permease RiFTR1 plays a key role in Fe acquisition and that RiFTR2 is involved in Fe homeostasis under Fe-limiting conditions. RiFTR1 was highly expressed in the (IRM), which suggests that the maintenance of Fe homeostasis in the IRM might be essential for a successful symbiosis.

Mrs3 and Mrs4 are mitochondrial inner membrane proteins that deliver an unidentified cytosolic iron species into the matrix for use in iron-sulfur cluster (ISC) and heme biosynthesis. The Mrs3/4 double-deletion strain (ΔΔ) grew slowly in iron-deficient glycerol/ethanol medium but recovered to wild-type (WT) rates in iron-sufficient medium. ΔΔ cells grown under both iron-deficient and iron-sufficient respiring conditions acquired large amounts of iron relative to WT cells, indicating iron homeostatic dysregulation regardless of nutrient iron status. Biophysical spectroscopy (including Mössbauer, electron paramagnetic resonance, and electronic absorption) and bioanalytical methods (liquid chromatography with online inductively coupled plasma mass spectrometry detection) were used to characterize these phenotypes. Anaerobically isolated mitochondria contained a labile iron pool composed of a nonheme high-spin Fe(II) complex with primarily O and N donor ligands, called Fe(580). Fe(580) likely serves as feedstock for ISC and heme biosynthesis. Mitochondria from respiring ΔΔ cells grown under iron-deficient conditions were devoid of Fe(580), ISCs, and hemes; most iron was present as Fe(III) nanoparticles. O(2) likely penetrates the matrix of slow-growing poorly respiring iron-deficient ΔΔ cells and reacts with Fe(580) to form nanoparticles, thereby inhibiting ISC and heme biosynthesis. Mitochondria from iron-sufficient ΔΔ cells contained ISCs, hemes, and Fe(580) at concentrations comparable to those of WT mitochondria. The matrix of these mutant cells was probably sufficiently anaerobic to protect Fe(580) from degradation by O(2). An ~1100 Da manganese complex, an ~1200 Da zinc complex, and an ~5000 Da copper species were also present in ΔΔ and WT mitochondrial flow-through solutions. No lower-mass copper complex was evident.

Fe-S clusters are cofactors that participate in diverse and essential biological processes. Mitochondria contain a complete machinery for Fe-S cluster assembly. Cysteine desulfurase (Nfs1) is required generation of a form of activated sulfur and is essential for the initial Fe-S cluster assembly step. Using mass-spectometry we identified proteins that were copurified with Nfs1 using a pull-down strategy, including a novel protein kinase. Furthermore, we were able to identify phosphorylation sites on the Nfs1 protein. These data and analyses support the research article “Cysteine desulfurase is regulated by phosphorylation of Nfs1 in yeast mitochondria” by Rocha et al. (in press) [1].

FeS-clusters are utilized by numerous proteins within several biological pathways that are essential for life. In eukaryotes, the primary FeS-cluster production pathway is the mitochondrial iron-sulfur cluster (ISC) pathway. In Saccharomyces cerevisiae, de novo FeS-cluster formation is accomplished through coordinated assembly with the substrates iron and sulfur by the scaffold assembly protein "Isu1". Sulfur for cluster assembly is provided by cysteine desulfurase "Nfs1", a protein that works in union with its accessory protein "Isd11". Frataxin "Yfh1" helps direct cluster assembly by serving as a modulator of Nfs1 activity, by assisting in the delivery of sulfur and Fe(ii) to Isu1, or more likely through a combination of these and other possible roles. In vitro studies on the yeast ISC machinery have been limited, however, due to the inherent instability of recombinant Isu1. Isu1 is a molecule prone to degradation and aggregation. To circumvent Isu1 instability, we have replaced yeast Isu1 with the fly ortholog to stabilize our in vitro ISC assembly system and assist us in elucidating molecular details of the yeast ISC pathway. Our laboratory previously observed that recombinant frataxin from Drosophila melanogaster has remarkable stability compared to the yeast ortholog. Here we provide the first characterization of D. melanogaster Isu1 (fIscU) and demonstrate its ability to function within the yeast ISC machinery both in vivo and in vitro. Recombinant fIscU has physical properties similar to that of yeast Isu1. It functions as a stable dimer with similar Fe(ii) affinity and ability to form two 2Fe-2S clusters as the yeast dimer. The fIscU and yeast ISC proteins are compatible in vitro; addition of Yfh1 to Nfs1-Isd11 increases the rate of FeS-cluster formation on fIscU to a similar extent observed with Isu1. Finally, fIscU expressed in mitochondria of a yeast strain lacking Isu1 (and its paralog Isu2) is able to completely reverse the deletion phenotypes. These results demonstrate fIscU can functionally replace yeast Isu1 and it can serve as a powerful tool for exploring molecular details within the yeast ISC pathway.

Fe-S cluster assembly is an essential process for all cells. Impairment of Fe-S cluster assembly creates diseases in diverse and surprising ways. In one scenario, the loss of function of lipoic acid synthase, an enzyme with Fe-S cluster cofactor in mitochondria, impairs activity of various lipoamide-dependent enzymes with drastic consequences for metabolism. In a second scenario, the heme biosynthetic pathway in red cell precursors is specifically targeted, and iron homeostasis is perturbed, but lipoic acid synthesis is unaffected. In a third scenario, tRNA modifications arising from action of the cysteine desulfurase and/or Fe-S cluster proteins are lost, which may lead to impaired protein synthesis. These defects can then result in cancer, neurologic dysfunction or type 2 diabetes.

Vacuoles are acidic organelles that store FeIII polyphosphate, participate in iron homeostasis, and have been proposed to deliver iron to mitochondria for iron–sulfur cluster (ISC) and heme biosynthesis. Vma2Δ cells have dysfunctional V-ATPases, rendering their vacuoles nonacidic. These cells have mitochondria that are iron-dysregulated, suggesting disruption of a putative vacuole-to-mitochondria iron trafficking pathway. To investigate this potential pathway, we examined the iron content of a vma2Δ mutant derived from W303 cells using Mössbauer and EPR spectroscopies and liquid chromatography interfaced with inductively-coupled-plasma mass spectrometry. Relative to WT cells, vma2Δ cells contained WT concentrations of iron but nonheme FeII dominated the iron content of fermenting and respiring vma2Δ cells, indicating that the vacuolar FeIII ions present in WT cells had been reduced. However, vma2Δ cells synthesized WT levels of ISCs/hemes and had normal aconitase activity. The iron content of vma2Δ mitochondria was similar to WT, all suggesting that iron delivery to mitochondria was not disrupted. Chromatograms of cytosolic flow–through solutions exhibited iron species with apparent masses of 600 and 800 Da for WT and vma2∆, respectively. Mutant cells contained high copper concentrations and high concentrations of a species assigned to metallothionein, indicating copper dysregulation. vma2Δ cells from previously studied strain BY4741 exhibited iron-associated properties more consistent with prior studies, suggesting subtle strain differences. Vacuoles with functional V-ATPases appear unnecessary in W303 cells for iron to enter mitochondria and be used in ISC/heme biosynthesis; thus, there appears to be no direct or dedicated vacuole-to-mitochondria iron trafficking pathway. The vma2Δ phenotype may arise from alterations in trafficking of iron directly from cytosol to mitochondria.

Fe-S clusters are critically important cofactors implicated in numerous cellular processes, including respiration, amino acid biosynthesis, cofactor biosynthesis, tRNA modification, DNA repair and regulation of gene expression. In the accompanying manuscript, Tanaka et al. show that reengineering of the isoprenoid biosynthetic pathway in E. coli (to bypass the usage of essential Fe-S cluster proteins by inserting the mevalonate pathway) can offset the indispensability of the Fe-S cluster biosynthetic systems. They show that the resulting Δisc Δsuf double mutants supplemented with mevalonate can grow slowly without detectable Fe-S cluster proteins. This result is astounding and raises interesting questions about what is essential and what is dispensable in the compendium of Fe-S cluster protein functions in this cell.

Iron-sulfur (Fe-S) cluster containing proteins are utilized in almost every biochemical pathway. The unique redox and coordination chemistry associated with the cofactor allows these proteins to participate in a diverse set of reactions, including electron transfer, enzyme catalysis, DNA synthesis and signaling within several pathways. Due to the high reactivity of the metal, it is not surprising that biological Fe-S cluster assembly is tightly regulated within cells. In yeast, the major assembly pathway for Fe-S clusters is the mitochondrial ISC pathway. Yeast Fe-S cluster assembly is accomplished using the scaffold protein (Isu1) as the molecular foundation, with assistance from the cysteine desulfurase (Nfs1) to provide sulfur, the accessory protein (Isd11) to regulate Nfs1 activity, the yeast frataxin homologue (Yfh1) to regulate Nfs1 activity and participate in Isu1 Fe loading possibly as a chaperone, and the ferredoxin (Yah1) to provide reducing equivalents for assembly. In this report, we utilize calorimetric and spectroscopic methods to provide molecular insight into how wt-Isu1 from S. cerevisiae becomes loaded with iron. Isothermal Titration Calorimetry (ITC) and an iron competition binding assay were developed to characterize the energetics of protein Fe(II) binding. Differential Scanning Calorimetry (DSC) was used to identify thermodynamic characteristics of the protein in the apo state or under iron loaded conditions. Finally, X-ray Absorption Spectroscopy (XAS) was used to characterize the electronic and structural properties of Fe(II) bound to Isu1. Current data are compared to our previous characterization of the D37A Isu1 mutant, and these suggest that when Isu1 binds Fe(II) in a manner not perturbed by the D37A substitution, and that metal binding occurs at a site distinct from the cysteine rich active site in the protein.

In most cellular compartments cysteine residues are predominantly reduced. However, in the bacterial periplasm, the ER and the mitochondrial intermembrane space (IMS), sulfhydryl oxidases catalyze the formation of disulfide bonds. Nevertheless, many IMS proteins contain reduced cysteines that participate in binding metal- or heme-cofactors. In this study, we addressed the substrate specificity of the mitochondrial protein oxidation machinery. Dre2 is a cysteine-rich protein that is located in the cytosol. A large fraction of Dre2 bound to the cytosolic side of the outer membrane of mitochondria. Even when Dre2 is artificially targeted to the IMS, its cysteine residues remain in the reduced state. This indicates that protein oxidation in the IMS of mitochondria is not a consequence of the apparent oxidizing environment in this compartment but rather is substrate-specific and determined by the presence of Mia40-binding sites.

Summary In eukaryotes, mitochondria have been hypothesized to generate sulfur species required for tRNA thiolation in the cytosol, although no direct evidence thus far exists. Here we have detected these sulfur species, making use of our observation that isolated yeast cytosol alone is unable to thiolate tRNAs but can do so upon addition of mitochondria. Mitochondria were found to utilize the cysteine desulfurase Nfs1 to produce sulfur-containing species with masses ranging from 700 to 1,100 Da. Mitochondria exported these species via the Atm1 transporter in the inner membrane. Once exported to the cytosol, these sulfur species promoted cytosolic tRNA thiolation with no further requirement of mitochondria. Furthermore, we found that the Isu1/2 scaffolds but not the Ssq1 chaperone of the mitochondrial iron-sulfur cluster machinery were required for cytosolic tRNA thiolation, and thus the sulfur utilization pathway bifurcates at the Isu1/2 site for intra-organellar use in mitochondria or export to the cytosol.

Arbuscular mycorrhizal (AM) fungi can improve iron (Fe) acquisition of their host plants. Here, we report a characterization of two components of the high-affinity reductive Fe uptake system of Rhizophagus irregularis, the ferric reductase (RiFRE1) and the high affinity Fe permeases (RiFTR1-2). In the extraradical mycelia (ERM), Fe deficiency induced activation of a plasma membrane-localized ferric reductase, an enzyme that reduces Fe(III) sources to the more soluble Fe(II). Yeast mutant complementation assays showed that RiFRE1 encodes a functional ferric reductase and RiFTR1 an iron permease. In the heterologous system, RiFTR1 was expressed in the plasma membrane while RiFTR2 was expressed in the endomembranes. In the ERM, the highest expression levels of RiFTR1 were found in mycelia grown in media with 0.045 mM Fe, while RiFTR2 was upregulated under Fe-deficient conditions. RiFTR2 expression also increased in the intraradical mycelia (IRM) of maize plants grown without Fe. These data indicate that the Fe permease RiFTR1 plays a key role in Fe acquisition and that RiFTR2 is involved in Fe homeostasis under Fe-limiting conditions. RiFTR1 was highly expressed in the (IRM), which suggests that the maintenance of Fe homeostasis in the IRM might be essential for a successful symbiosis.

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 [(35)S]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-(35)S 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 (35)S-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.

Friedreich’s ataxia (FA) is an autosomal recessive disease caused by an intronic GAA triplet expansion in the FXN gene, leading to reduced expression of the mitochondrial protein frataxin. FA is estimated to affect 1 in 50 000 with a mean age of death in the fourth decade of life. There are no approved treatments for FA, although experimental approaches, which involve up-regulation or replacement of frataxin protein, are being tested. Frataxin is undetectable in serum or plasma, and whole blood cannot be used because it is present in long-lived erythrocytes. Therefore, an assay was developed for analyzing frataxin in platelets, which have a half-life of 10 days. The assay is based on stable isotope dilution immunopurification two-dimensional nano-ultra high performance liquid chromatography/parallel reaction monitoring/mass spectrometry. The lower limit of quantification was 0.078 pg frataxin/µg protein, and the assay had 100% sensitivity and specificity for discriminating between controls and FA cases. The mean levels of control and FA platelet frataxin were 9.4 ± 2.6 and 2.4 ± 0.6 pg/µg protein, respectively. The assay should make it possible to rigorously monitor the effects of therapeutic interventions on frataxin expression in this devastating disease.

Yeast Dre2 (anamorsin or CIAPIN1) is an essential component for cytosolic Fe/S cluster biosynthesis. The C-terminal domain contains eight evolutionarily conserved cysteine residues, and we previously demonstrated that the yeast Dre2 overexpressed in Escherichia coli contains one binuclear ([2Fe-2S]) cluster and one tetranuclear ([4Fe-4S]) cluster. In this study, we replaced each conserved cysteine with alanine and analyzed the effects by Electron Paramagnetic Resonance. Although the C311A mutant lacked both signals, our data clearly suggest that the [2Fe-2S] cluster is ligated to Cys252, Cys263, Cys266 and Cys268, whereas the [4Fe-4S] cluster is ligated to Cys311, Cys314, Cys322 and Cys325. By simulation analysis of the C263A and C322A data, we obtained the g-values for the [4Fe-4S] cluster (gx,y,z = 1.830, 1.947 and 2.018) and for the [2Fe-2S] cluster (gx,y,z =1.919, 1.962 and 2.001). We also observed spin-spin interaction between the two clusters, suggesting their close proximity. Chemically reconstituted Dre2 showed air sensitivity of the [4Fe-4S] cluster converting to a [2Fe-2S] cluster. Furthermore, using a yeast shuffle strain, we demonstrated for the first time that each of the Cys Fe-S cluster ligands with the exception of C252 is essential, indicating that both Dre2 clusters are needed for cell viability.

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 (1 mM) 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.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by GAA triplet expansions or point mutations in the FXN gene on chromosome 9q13. The gene product called frataxin, a mitochondrial protein that is severely reduced in FRDA patients, leads to mitochondrial iron accumulation, Fe-S cluster deficiency and oxidative damage. The tissue specificity of this mitochondrial disease is complex and poorly understood. While frataxin is ubiquitously expressed, the cellular phenotype is most severe in neurons and cardiomyocytes. Here, we conducted comprehensive proteomic, metabolic and functional studies to determine whether subclinical abnormalities exist in mitochondria of blood cells from FRDA patients. Frataxin protein levels were significantly decreased in platelets and peripheral blood mononuclear cells from FRDA patients. Furthermore, the most significant differences associated with frataxin deficiency in FRDA blood cell mitochondria were the decrease of two mitochondrial heat shock proteins. We did not observe profound changes in frataxin-targeted mitochondrial proteins or mitochondrial functions or an increase of apoptosis in peripheral blood cells, suggesting that functional defects in these mitochondria are not readily apparent under resting conditions in these cells.

In Saccharomyces cerevisiae, the mitochondrial inner membrane readily allows transport of cytosolic NAD(+), but not NADPH, to the matrix. Pos5p is the only known NADH kinase in the mitochondrial matrix. The enzyme phosphorylates NADH to NADPH and is the major source of NADPH in the matrix. The importance of mitochondrial NADPH for cellular physiology is underscored by the phenotypes of the Δpos5 mutant, characterized by oxidative stress sensitivity and iron-sulfur (Fe-S) cluster deficiency. Fe-S clusters are essential cofactors of proteins such as aconitase [4Fe-4S] and ferredoxin [2Fe-2S] in mitochondria. Intact mitochondria isolated from wild-type yeast can synthesize these clusters and insert them into the corresponding apoproteins. Here, we show that this process of Fe-S cluster biogenesis in wild-type mitochondria is greatly stimulated and kinetically favored by the addition of NAD(+) or NADH in a dose-dependent manner, probably via transport into mitochondria and subsequent conversion into NADPH. Unlike wild-type mitochondria, Δpos5 mitochondria cannot efficiently synthesize Fe-S clusters on endogenous aconitase or imported ferredoxin, although cluster biogenesis in isolated Δpos5 mitochondria is restored to a significant extent by a small amount of imported Pos5p. Interestingly, Fe-S cluster biogenesis in wild-type mitochondria is further enhanced by overexpression of Pos5p. The effects of Pos5p on Fe-S cluster generation in mitochondria indicate that one or more steps in the biosynthetic process require NADPH. The role of mitochondrial NADPH in Fe-S cluster biogenesis appears to be distinct from its function in anti-oxidant defense.

International audience; Friedreich ataxia is an inherited neurodegenerative disease caused by frataxin deficiency. Frataxin is a conserved mitochondrial protein that plays a role in FeS cluster assembly in mitochondria. FeS clusters are modular cofactors that perform essential functions throughout the cell. They are synthesized by a multistep and multisubunit mitochondrial machinery that includes the scaffold protein Isu for assembling a protein-bound FeS cluster intermediate. Frataxin interacts with Isu, iron, and the cysteine desulfurase Nfs1, which supplies sulfide, thus placing it at the center of mitochondrial FeS cluster biosynthesis.

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).

Recent data suggest that frataxin plays a key role in eukaryote cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (FeS) cluster biosynthesis. We have now identified a frataxin homologue ( T. vaginalis frataxin) from the human parasite Trichomonas vaginalis . Instead of mitochondria, this unicellular eukaryote possesses hydrogenosomes, peculiar organelles that produce hydrogen but nevertheless share common ancestry with mitochondria. T. vaginalis frataxin contains conserved residues implicated in iron binding, and in silico, it is predicted to form a typical α-β sandwich motif. The short N-terminal extension of T. vaginalis frataxin resembles presequences that target proteins to hydrogenosomes, a prediction confirmed by the results of overexpression of T. vaginalis frataxin in T. vaginalis . When expressed in the mitochondria of a frataxin-deficient Saccharomyces cerevisiae strain, T. vaginalis frataxin partially restored defects in heme and FeS cluster biosynthesis. Although components of heme synthesis or heme-containing proteins have not been found in T. vaginalis to date, T. vaginalis frataxin was also shown to interact with S. cerevisiae ferrochelatase by using a Biacore assay. The discovery of conserved iron-metabolizing pathways in mitochondria and hydrogenosomes provides additional evidence not only of their common evolutionary history, but also of the fundamental importance of this pathway for eukaryotes.

Friedreich ataxia has frequently been associated with an increased susceptibility to oxidative stress. We used the yeast (Saccharomyces cerevisiae) model of Friedreich ataxia to study the physiological consequences of a shift from anaerobiosis to aerobiosis. Cells lacking frataxin (Deltayfh1) showed no growth defect when cultured anaerobically. Under these conditions, a significant amount of aconitase was functional, with an intact 4 Fe/4 S cluster. When shifted to aerobic conditions, aconitase was rapidly degraded, and oxidatively modified proteins (carbonylated and HNE-modified proteins) accumulated in both the cytosol and the mitochondria. The ATP-dependent mitochondrial protease Pim1 (Lon) was strongly activated, although its expression level remained unchanged, and the cytosolic activity of the 20S proteasome was greatly decreased, compared to that in wild-type cells. Analysis of the purified proteasome revealed that the decrease in proteasome activity was likely due to both direct inactivation of the enzyme and inhibition by cytosolic oxidized proteins. These features indicate that the cells were subjected to major oxidative stress triggered by oxygen. Accumulation of oxidatively modified proteins, activation of Pim1, and proteasome inhibition did not directly depend on the amount of mitochondrial iron, because these phenotypes remained unchanged when the cells were grown under iron-limiting conditions, and these phenotypes were not observed in another mutant (Deltaggc1) which overaccumulates iron in its mitochondrial compartment. We conclude that oxygen is primarily involved in generating the deleterious phenotypes that are observed in frataxin-deficient yeast cells.

Fe-S clusters are critically important cofactors implicated in numerous cellular processes, including respiration, amino acid biosynthesis, cofactor biosynthesis, tRNA modification, DNA repair and regulation of gene expression. In the accompanying manuscript, Tanaka et al. show that reengineering of the isoprenoid biosynthetic pathway in E. coli (to bypass the usage of essential Fe-S cluster proteins by inserting the mevalonate pathway) can offset the indispensability of the Fe-S cluster biosynthetic systems. They show that the resulting Δisc Δsuf double mutants supplemented with mevalonate can grow slowly without detectable Fe-S cluster proteins. This result is astounding and raises interesting questions about what is essential and what is dispensable in the compendium of Fe-S cluster protein functions in this cell.

The reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) and other tetrazolium salts is widely used as an assay for bacterial, fungal and mammalian cell viability, but the genes encoding the reductase activities have not been defined. Here, it was shown that XTT and plasma membrane ferric reductase activities were 10–40-fold greater inCandida albicansthan inSaccharomyces cerevisiae. XTT reductase activity was induced fivefold inC. albicansgrown in low-iron conditions compared with iron-replete conditions, and for cells grown in unbuffered (pH 4.0–4.4) medium, XTT reductase activity was largely dependent onCaFRE10. XTT reductase activity ofC. albicansgrown in medium buffered to pH 6.8 was independent ofCaFRE10but, nonetheless, was upregulated in cells deprived of iron. Reduction of 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT), a membrane-permeable tetrazolium salt, occurred at an intracellular location and was independent ofCaFRE10. However, MTT activity was induced by iron deprivation inC. albicansbut not inS. cerevisiae.C. albicanspossessed multiple iron- and pH-regulated reductase activities capable of reducing tetrazolium salts, but, when grown in unbuffered medium,CaFRE10was required for XTT reductase activity.

Comparison and identification of mitochondrial matrix proteins from wild-type and cysteine desulfurase-defective (nfs1-14, carrying a hypomorphic allele of NFS1) yeast strains, using two-dimensional gel electrophoresis coupled to mass spectrometry analyses, revealed large changes in the amounts of various proteins. Protein spots that were specifically increased in the nfs1-14 mutant included subunits of lipoamide-containing enzyme complexes: Kgd2, Lat1, and Gcv3, subunits of the mitochondrial alpha-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and glycine cleavage system complexes, respectively. Moreover the increased protein spots corresponded to lipoamide-deficient forms in the nfs1-14 mutant. The increased proteins migrated as separate, cathode-shifted spots, consistent with gain of a lysine charge due to lack of lipoamide addition. Lack of lipoylation of these proteins was further validated using an antibody specific for lipoamide-containing proteins. In addition, this antibody revealed a fourth lipoamide-containing protein, probably corresponding to the E2 component of the branched-chain keto acid dehydrogenase complex. Like the lipoamide-containing forms of Kgd2, Lat1, and Gcv3, this protein also showed decreased lipoic acid reactivity in the nfs1-14 mutant. Cysteine desulfurases, such as yeast NFS1, are required for sulfur addition to iron-sulfur clusters and other sulfur-requiring processes. The results demonstrate that Nfs1 protein is required for the proper post-translational modification of the lipoamide-containing mitochondrial subproteome in yeast and pave the road toward a thorough understanding of its precise role in lipoic acid synthesis.

Frataxin is a conserved mitochondrial protein implicated in cellular iron metabolism. Deletion of the yeast frataxin homolog (YFH1) was combined with deletions of MRS3 and MRS4, mitochondrial carrier proteins implicated in iron homeostasis. As previously reported, the Deltayfh1 mutant accumulated iron in mitochondria, whereas the triple mutant (DeltaDeltaDelta) did not. When wild-type, Deltamrs3/4, Deltayfh1, and DeltaDeltaDelta strains were incubated anaerobically, all strains were devoid of heme and protected from iron and oxygen toxicity. The cultures were then shifted to air for a short time (4-5 h) or a longer time (15 h), and the evolving mutant phenotypes were analyzed (heme-dependent growth, total heme, cytochromes, heme proteins, and iron levels). A picture emerges from these data of defective heme formation in the mutants, with a markedly more severe defect in the DeltaDeltaDelta than in the individual Deltamrs3/4 or Deltayfh1 mutants (a "synthetic" defect in the genetic sense). The defect(s) in heme formation could be traced to lack of iron. Using a real time assay of heme biosynthesis, porphyrin precursor and iron were presented to permeabilized cells, and the appearance and disappearance of fluorescent porphyrins were followed. The Mrs3/4p carriers were required for rapid iron transport into mitochondria for heme synthesis, whereas there was also evidence for an alternative slower system. A different role for Yfh1p was observed under conditions of low mitochondrial iron and aerobic growth (revealed in the DeltaDeltaDelta), acting to protect bioavailable iron within mitochondria and to facilitate its use for heme synthesis.

The cellular structure of Trichomonas vaginalis substantially differs from other eukaryotes. Instead of mitochondria it possesses hydrogenosomes where key metabolic reactions are mediated by FeS proteins. The gene expression and the activity of FeS proteins is fully dependent on the iron availability. Although hydrogenosomes contain a high amount of iron, there is no information about hydrogenosomal iron homeostasis and FeS cluster formation. In mitochondria, frataxin plays an unclear role in iron metabolism. The deficiency of frataxin in human causes Friedreich's ataxia, which is associated with an increased level of mitochondrial iron. It has been suggested that frataxin is involved in various functions such as FeS cluster formation or iron storage. We cloned T. vaginalis frataxin homologue (TvFTX) coding for a protein of 121 amino acids. Although lacking transcription initiator element in 5′UTR region, the expression of tvftx was verified by a nuclear run-on assay. Moreover, the gene upregulation in iron deficiency was shown. The N-terminal part of TvFTX contains an extension similar to presequences targeting proteins to hydrogenosomes and mitochondria. To identify its cellular localization we transfected T. vaginalis with TvFTX fused to HA tag. The immunodetection of HA tag specifically labeled hydrogenosomes and the corresponding cellular fraction. The hydrogenosomal targeting sequence however does not support the translocation into yeast mitochondria. Thus for the complementation of yeast frataxin homologue (YFH1) a mitochondrial targeting sequence is required. When expressed in mitochondria of δYFH1 cells TvFTX complements the mutant growth defect and restores the activity of aconitase (iron-sulfur protein). Together with recent characterization of T. vaginalis cystein desulfurase these data indicate the conserved role of hydrogenosomes and mitochondria in FeS cluster assembly.

We cloned the CaYFH1 gene that encodes the yeast frataxin homologue in Candida albicans. CaYFH1 was expressed in Deltayfh1 Saccharomyces cerevisiae cells, where it compensated for all the phenotypes tested except for the lack of cytochromes. Double DeltaCayfh1/DeltaCayfh1 mutant had severe defective growth, accumulated iron in their mitochondria, lacked aconitase and succinate dehydrogenase activity and had defective respiration. The reductive, siderophore and haem uptake systems were constitutively induced and the cells excreted flavins, thus behaving like iron-deprived wild-type cells. Mutant cells accumulated reactive oxygen species and were hypersensitive to oxidative stress, but not to iron. Cytochromes were less abundant in mutants than in wild-type cells, but this did not result from defective haem synthesis. The low cytochrome concentration in mutant cells was comparable to that of iron-deprived wild-type cells. Mitochondrial iron was still available for haem synthesis in DeltaCayfh1/DeltaCayfh1 cells, in contrast to S. cerevisaeDeltayfh1 cells. CaYFH1 transcription was strongly induced by iron, which is consistent with a role of CaYfh1 in iron storage. Iron also regulated transcription of CaHEM14 (encoding protoporphyrinogen oxidase) but not that of CaHEM15 (encoding ferrochelatase). There are thus profound differences between S. cerevisiae and C. albicans in terms of haem synthesis, cytochrome turnover and the role of frataxin in these processes.

Mitochondria are the site of assembly of FeS centers of mitochondrial and cytosolic FeS proteins. Various microaerophilic or anaerobic unicellular eukaryotes lack typical mitochondria (“amitochondriate” protists). In some of these organisms, a metabolically different organelle, the hydrogenosome, is found, which is thought to derive from the same proteobacterial ancestor as mitochondria. Here, we show that hydrogenosomes of Trichomonas vaginalis , a human genitourinary parasite, contain a key enzyme of FeS center biosynthesis, cysteine desulfurase (TviscS-2), which is phylogenetically related to its mitochondrial homologs. Hydrogenosomes catalyze the enzymatic assembly and insertion of FeS centers into apoproteins, as shown by the reconstruction of the apoform of [2Fe-2S]ferredoxin and the incorporation of 35 S from labeled cysteine. Our results indicate that the biosynthesis of FeS proteins is performed by a homologous system in mitochondriate and amitochondriate eukaryotes and that this process is inherited from the proteobacterial ancestor of mitochondria.

TheYFH1 geneistheyeasthomologueofthehumanFRDAgene,whichencodesthefrataxinprotein.Saccharomyces cerevisiae cellslackingtheYFH1 geneshowedverylowcytochromecontent.InDyfh1strains,thelevelofferrochelatase(Hem15p)wasverylow,asaresultoftranscriptionalrepressionofHEM15.However,thelowamountofHem15pwasnotthecauseofhaemedeficiencyinDyfh1 cells.Ferrochelatase,amitochondrialprotein,abletomediateinsertionofironorzincintotheporphyrinprecursor,madeprimarilythezincprotoporphyrinproduct.ZincprotoporphyrininsteadofhaemeaccumulatedduringgrowthofDyfh1mutantcellsand,furthermore,preferentialformationofzincprotoporphyrinwasobservedinrealtime.Themethodforthesestudiesinvolveddirectpresentationofporphyrintomitochondriaandtoferrochelataseofpermeabilizedcellswithintactarchitecture,therebyspecificallytestingtheirondeliveryportionofthehaemebiosyntheticpathway.ThestudiesshowedthatDyfh1 mutantcellsaredefectiveinironusebyferrochelatase.Mo¨ssbauerspectroscopicanalysisshowedthatironwaspresentasamorphousnano-particlesofferricphosphateinDyfh1 mitochondria,whichcouldexplaintheunavailabilityofironforhaemesynthesis.Ahighfrequencyofsuppressormutationswasobserved,andthephenotypeofsuchmutantswascharacterizedbyrestorationofhaemesynthesisintheabsenceofYfh1p.Suppressorstrainsshowedanormalcytochromecontent,normalrespiration,butremaineddefectiveinFe–Sproteinsandstillaccumulatedironintomitochondriaalthoughtoalesserextent.Yfh1pandHem15pwereshowntointeractin vitro byBiacorestudies.OurresultssuggestthatYfh1mediatesironusebyferrochelatase.INTRODUCTIONThe YFH1 gene is the yeast homologue of the human FRDAgene, which encodes the frataxin protein. Mutations of FRDAassociated with decreased frataxin expression are responsiblefor Friedreich’s ataxia, the most common autosomal-recessiveneurodegenerative disease of Caucasians (1,2). Both genescode for mitochondrial proteins that are involved in ironhomeostasis and cellular respiration (3–8), but their preciseroles are unknown. Cardiac tissues from patients withFriedreich’s ataxia exhibit iron deposition, deficiencies in manyiron–sulphur cluster enzymes and reduced mitochondrial DNA(8,9). In addition, fibroblasts from these patients showhypersensitivity to oxidative stress that can be rescued bytreatment with iron chelators (10). A link to haeme biosynthesishas not been uncovered, and blood, bone marrow and red celldevelopment appear to be normal in patient with frataxindeficits. Frataxin is downregulated during erythroid develop-ment, suggesting that this protein is not involved in the high-volume iron trafficking that accompanies red cell production inthe bone marrow (11). However, activities of haeme enzymes inother tissues of Friedreich’s ataxia patients have not beenassessed, leaving open the possibility that tissue-specific haemedeficiencies may exist.Yeast cells lacking Yfh1p mirror many of the phenotypesobserved in disease tissues from patients with Friedreich’sataxia. These cells have defective respiration (3,5,12–14),unstable mitochondrial DNA and hypersensitivity to oxidative

Candida albicans, unlike Saccharomyces cerevisiae, was able to use extracellular haemin as an iron source. Haemin uptake kinetics by C. albicans cells showed two phases: a rapid phase of haemin binding (with a K(d) of about 0.2 microM) followed by a slower uptake phase. Both phases were strongly induced in iron-deficient cells compared to iron-rich cells. Haemin uptake did not depend on the previously characterized reductive iron uptake system and siderophore uptake system. CaHMX1, encoding a putative haem oxygenase, was shown to be required for iron assimilation from haemin. A double DeltaCahmx1 mutant was constructed. This mutant could not grow with haemin as the sole iron source, although haemin uptake was not affected. The three different iron uptake systems (reductive, siderophore and haemin) were regulated independently and in a complex manner. CaHMX1 expression was induced by iron deprivation, by haemin and by a shift of temperature from 30 to 37 degrees C. CaHMX1 expression was strongly deregulated in a Deltaefg1 mutant but not in a Deltatup1 mutant. C. albicans colonies forming on agar plates with haemin as the sole iron source showed a very unusual morphology. Colonies were made up of tubular structures that were organized into a complex network. The effect of haemin on filamentation was increased in the double DeltaCahmx1 mutant. This study provides the first experimental evidence that haem oxygenase is required for iron assimilation from haem by a pathogenic fungus.

High-affinity iron uptake by a ferrous permease in the opportunistic pathogen Candida albicans is required for virulence. Here this iron uptake system has been characterized by investigating three distinct activities: an externally directed surface ferric reductase, a membrane-associated PPD (p-phenylenediamine) oxidase and a cellular ferrous iron transport activity. Copper was required for the PPD oxidase and ferrous transport activities. In contrast, copper was not required for iron uptake from siderophores. Addition of iron to the growth medium repressed ferric reductase and ferrous transport, indicating homeostatic regulation. To identify the genes involved, orthologous mutants of Saccharomyces cerevisiae were transformed with a genomic library of C. albicans. CFL95, a gene with sequence similarity to ferric reductases, restored reductase activity to the orthologous S. cerevisiae mutant. CaFTR2 and CaFTR1, genes with homology to ferrous permeases, conferred ferrous transport activity to the orthologous S. cerevisiae mutant. However, neither a genomic library nor CaFET99, a multicopper oxidase homologue and candidate gene for the PPD oxidase, complemented the S. cerevisiae mutant, possibly because of problems with targeting or assembly. Transcripts for CFL95, CaFTR1 and CaFET99 were strongly repressed by iron, whereas the CaFTR2 transcript was induced by iron. Deletion of the TUP1 regulator perturbed the homeostatic control of reductive iron uptake. Incidentally, iron starvation was noted to induce flavin production and this was misregulated in the absence of TUP1 control. The opposite regulation of two iron permease genes and the role of TUP1 indicate that the process of iron acquisition by C. albicans may be more complex and potentially more adaptable than by S. cerevisiae.

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 [(35)S]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-(35)S 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 (35)S-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.

The yeast frataxin homolog (Yfh1p) participates in mitochondrial iron homeostasis. The phenotypic defects of the Delta yfh1 mutant include drastic accumulation of iron in mitochondria and slow growth. The Yfh1p precursor protein contains two N-terminal domains that are sequentially cleaved by the matrix processing peptidase on import into mitochondria, generating the mature protein. We have precisely mapped these two cleavage sites. Mutations blocking the first or the second cleavage of Yfh1p do not interfere with its in vitro import or with its ability to complement phenotypes of the Delta yfh1 mutant strain. Distinct roles have been ascertained for the two cleaved domains of Yfh1p. The first cleaved domain (domain I) is sufficient for in vitro mitochondrial import of a non-mitochondrial passenger protein. However, neither domain I nor other matrix-targeting signals alone can support efficient in vitro import of mature Yfh1p. The second cleaved domain (domain II) is required as a spacer between a targeting signal and mature Yfh1p. Likewise, when Yfh1p constructs lacking domain I or II are expressed in vivo, they fail to attain appreciable steady-state amounts in mitochondria and cannot complement phenotypes of the Delta yfh1 mutant.