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Fe-S Cluster Biogenesis in Isolated Mammalian Mitochondria

COORDINATED USE OF PERSULFIDE SULFUR AND IRON AND REQUIREMENTS FOR GTP, NADH, AND ATP*
  • Alok Pandey
    Footnotes
    Affiliations
    Department of Pharmacology and Physiology, New Jersey Medical School, Rutgers University, Newark, New Jersey 07101
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  • Jayashree Pain
    Footnotes
    Affiliations
    Department of Pharmacology and Physiology, New Jersey Medical School, Rutgers University, Newark, New Jersey 07101
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  • Arnab K. Ghosh
    Footnotes
    Affiliations
    Department of Pharmacology and Physiology, New Jersey Medical School, Rutgers University, Newark, New Jersey 07101
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  • Andrew Dancis
    Affiliations
    Department of Medicine, Division of Hematology-Oncology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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  • Debkumar Pain
    Correspondence
    To whom correspondence should be addressed: Dept. of Pharmacology and Physiology, New Jersey Medical School, Rutgers University, 185 S. Orange Ave., MSB I-669, Newark, NJ 07101-1709. Tel.: 973-972-3439; Fax: 973-972-7950
    Affiliations
    Department of Pharmacology and Physiology, New Jersey Medical School, Rutgers University, Newark, New Jersey 07101
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01GM107542 (to D. P.) and R37DK053953 (to A. D.). The work was also supported by a Friedreich's Ataxia Research Alliance grant.
    1 Both authors contributed equally to this work.
    2 Present address: Dept. of Surgery, Columbia University, New York, NY 10032.
Open AccessPublished:November 14, 2014DOI:https://doi.org/10.1074/jbc.M114.610402
      Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [35S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-35S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the 35S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia.Fe-S cluster assembly in mitochondria involves generation of an activated form of sulfur called persulfide.

      Results

      A novel experimental system tracks new Fe-S cluster synthesis in isolated mammalian mitochondria.

      Conclusion

      The use of persulfide sulfur and iron for Fe-S cluster biogenesis is tightly coordinated by processes requiring GTP, NADH, and ATP.

      Significance

      These cofactor targets can now be defined.

      Introduction

      Iron-sulfur (Fe-S) clusters are modular cofactors consisting of iron and inorganic sulfur. These cofactors are anchored in the polypeptide backbones of proteins, most often by liganding to sulfur atoms of cysteines (
      • Beinert H.
      • Holm R.H.
      • Münck E.
      Iron-sulfur clusters: Nature's modular, multipurpose structures.
      ). In cells, Fe-S cluster proteins are needed for many essential processes, including electron transfer, catalysis, iron regulation, DNA repair, ribosome biogenesis, and tRNA modifications (
      • Stemmler T.L.
      • Lesuisse E.
      • Pain D.
      • Dancis A.
      Frataxin and mitochondrial Fe-S cluster Biogenesis.
      ,
      • Rouault T.A.
      Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease.
      ,
      • Lill R.
      • Hoffmann B.
      • Molik S.
      • Pierik A.J.
      • Rietzschel N.
      • Stehling O.
      • Uzarska M.A.
      • Webert H.
      • Wilbrecht C.
      • Mühlenhoff U.
      The role of mitochondria in cellular iron-sulfur protein biogenesis and iron metabolism.
      ). The most common types of Fe-S clusters are [2Fe-2S] and [4Fe-4S], and several proteins with such clusters reside in mitochondria. Ferredoxins, which coordinate [2Fe-2S] clusters, acquire their clusters inside mitochondria but also play an essential role in Fe-S cluster biogenesis in mitochondria (
      • Lange H.
      • Kaut A.
      • Kispal G.
      • Lill R.
      A mitochondrial ferredoxin is essential for biogenesis of cellular iron-sulfur proteins.
      ). Aconitase, which requires a [4Fe-4S] cofactor for activity, is a key enzyme in the Krebs cycle in mitochondria, reversibly converting citrate to isocitrate (
      • Walden W.E.
      From bacteria to mitochondria: aconitase yields surprises.
      ). Fe-S cluster proteins also function in many other vital processes in mitochondria, including respiration, and the respiratory protein complexes I, II, and III coordinate numerous Fe-S clusters (
      • Lill R.
      Function and biogenesis of iron-sulphur proteins.
      ). However, both components (iron and sulfur) are toxic when unassembled or present in excess in mitochondria (
      • Pain D.
      • Dancis A.
      ). In fact, defects in Fe-S cluster synthesis in mitochondria are associated with a variety of human diseases, including neurodegenerative diseases such as Friedreich ataxia, myopathy such as ISCU myopathy, and inherited anemias such as sideroblastic anemia (
      • Rouault T.A.
      Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease.
      ,
      • Beilschmidt L.K.
      • Puccio H.M.
      Mammalian Fe-S cluster biogenesis and its implication in disease.
      ). Fe-S cluster assembly in mitochondria must therefore be both efficient and tightly regulated.
      Fe-S cluster biogenesis in mitochondria is highly complex. It is a multistep process involving many genes and proteins that contribute to the formation and regulation of the Fe-S cluster biosynthetic machinery. Lethal phenotypes are associated with disruption of most of these genes, consistent with the essential nature of the process. Our current knowledge of Fe-S cluster biogenesis in mitochondria is derived mostly from studies of proteins in Saccharomyces cerevisiae and their homologs in Escherichia coli (
      • Pain D.
      • Dancis A.
      ,
      • Johnson D.C.
      • Dean D.R.
      • Smith A.D.
      • Johnson M.K.
      Structure, function, and formation of biological iron-sulfur clusters.
      ,
      • Zheng L.
      • Cash V.L.
      • Flint D.H.
      • Dean D.R.
      Assembly of iron-sulfur clusters: identification of an iscSUA-jscBA-fdx gene cluster from Azotobacter vinelandii.
      ). The biosynthetic process can be broadly understood in terms of three phases. In the first phase, sulfur is mobilized from cysteine by an enzyme, cysteine desulfurase. In the second phase, sulfur is transferred from the enzyme to a scaffold protein and assembled with iron to form an Fe-S cluster intermediate. In the third phase, mediated by chaperones and glutaredoxins, the Fe-S cluster intermediate is transferred to apoproteins, forming holo and active proteins (
      • Stemmler T.L.
      • Lesuisse E.
      • Pain D.
      • Dancis A.
      Frataxin and mitochondrial Fe-S cluster Biogenesis.
      ,
      • Rouault T.A.
      Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease.
      ,
      • Lill R.
      • Hoffmann B.
      • Molik S.
      • Pierik A.J.
      • Rietzschel N.
      • Stehling O.
      • Uzarska M.A.
      • Webert H.
      • Wilbrecht C.
      • Mühlenhoff U.
      The role of mitochondria in cellular iron-sulfur protein biogenesis and iron metabolism.
      ).
      Studies of Fe-S cluster biogenesis in mammalian mitochondria are relatively recent and have focused primarily on the effects of in vivo depletion of a particular component of the mitochondrial Fe-S cluster machinery on Fe-S protein activities (
      • Shi Y.
      • Ghosh M.C.
      • Tong W.-H.
      • Rouault T.A.
      Human ISD11 is essential for both iron-sulfur cluster assembly and maintenance of normal cellular iron homeostasis.
      ,
      • Biederbick A.
      • Stehling O.
      • Rösser R.
      • Niggemeyer B.
      • Nakai Y.
      • Elsässer H.-P.
      • Lill R.
      Role of human mitochondrial Nfs1 in cytosolic iron-sulfur protein biogenesis and iron regulation.
      ,
      • Stehling O.
      • Elsässer H.P.
      • Brückel B.
      • Mühlenhoff U.
      • Lill R.
      Iron-sulfur protein maturation in human cells: evidence for a function of frataxin.
      ). However, this method has many limitations because of variable degrees of protein depletion and secondary effects occurring during the depletion time course (
      • Beilschmidt L.K.
      • Puccio H.M.
      Mammalian Fe-S cluster biogenesis and its implication in disease.
      ). Furthermore, in intact mammalian cells, it is not possible to manipulate critical molecules (e.g. nucleotides) to determine their roles in Fe-S cluster assembly without compromising other essential metabolic processes. Therefore, it has been difficult to dissect different steps of the mitochondrial Fe-S cluster biogenesis in vivo. Ideally, such studies must be performed with mitochondria isolated from mammalian cells. However, so far, there has been no experimental system available to study Fe-S cluster assembly in isolated mammalian mitochondria.
      Here we present the results of studies using isolated and metabolically active mammalian mitochondria supplemented with [35S]cysteine as the source of sulfur for Fe-S cluster synthesis. The experimental system resembles, in some respects, the system we developed for studying Fe-S cluster synthesis in yeast mitochondria (
      • Amutha B.
      • Gordon D.M.
      • Gu Y.
      • Lyver E.R.
      • Dancis A.
      • Pain D.
      GTP is required for iron-sulfur cluster biogenesis in mitochondria.
      ,
      • Amutha B.
      • Gordon D.M.
      • Dancis A.
      • Pain D.
      Nucleotide-dependent iron-sulfur cluster biogenesis of endogenous and imported apoproteins in isolated intact mitochondria.
      ,
      • Pain J.
      • Balamurali M.M.
      • Dancis A.
      • Pain D.
      Mitochondrial NADH kinase, Pos5p, is required for efficient iron-sulfur cluster biogenesis in Saccharomyces cerevisiae.
      ). We found that murine CAD
      The abbreviations used are: CAD
      Cath.A-derived
      αKG
      α-ketoglutarate
      EGS
      energy-regenerating system
      IP
      immunoprecipitation
      NMN
      nicotinamide mononucleotide
      SCS
      succinyl-CoA synthetase.
      mitochondria were able to generate an activated form of sulfur derived from cysteine and bound to the mitochondrial NFS1 cysteine desulfurase (NFS1-S-35SH). This activated persulfide sulfur was utilized during the synthesis of new Fe-35S clusters and inserted into endogenous apoaconitase or imported apoferredoxins. The overall process was strictly dependent on the availability of GTP, NADH, ATP, and iron. When GTP or NADH was lacking, the radiolabeled persulfide sulfur remained associated with NFS1, and Fe-S cluster assembly did not occur, even in the presence of added ATP and iron. Likewise, in iron-depleted mitochondria, the NFS1-bound persulfide sulfur was not released, even in the presence of added GTP, NADH, and ATP. In the presence of GTP, NADH, and iron but no ATP, the persulfide sulfur was released, but radiolabeled aconitase was not detected. Only in the presence of adequate levels of all four constituents (GTP, NADH, iron, and ATP) was the precursor-product relationship observed. That is, the radiolabeled NFS1 persulfide was decreased greatly, and radiolabeled aconitase was formed. The use of persulfide sulfur and iron for Fe-S cluster assembly was closely coordinated by processes requiring GTP, NADH, and ATP.

      DISCUSSION

      Fe-S cluster cofactors are essential for the function of numerous proteins. In eukaryotic cells, several Fe-S proteins, such as aconitase [4Fe-4S] and ferredoxin [2Fe-2S], are found in mitochondria, and the synthesis and assembly of these Fe-S clusters occur within the organelle. Numerous studies have been performed to investigate Fe-S cluster biogenesis in yeast mitochondria over the past two decades. By contrast, the biosynthetic process in mammalian mitochondria is receiving more attention only recently, and many aspects of the process remain poorly understood (
      • Beilschmidt L.K.
      • Puccio H.M.
      Mammalian Fe-S cluster biogenesis and its implication in disease.
      ). In fact, until now, no method has been available to directly study Fe-S cluster biogenesis in mitochondria isolated from any mammalian cells. Here, for the first time, we report new Fe-S cluster assembly by isolated and metabolically active mammalian mitochondria. An important feature of our assays is that they could be performed with intact mitochondria, thereby permitting manipulations of the organelle milieu. This novel system allowed us to identify some of the key regulatory aspects of Fe-S cluster biogenesis in mammalian mitochondria.
      We found that mitochondria isolated from murine CAD cells contain a complete machinery that can efficiently synthesize new Fe-S clusters and insert them into apoproteins. These mitochondria can handle Fe-S cluster assembly of protein substrates with different types of clusters. Specifically, assembly of [4Fe-4S] clusters on endogenous aconitase and [2Fe-2S] clusters on newly imported ferredoxins can occur simultaneously and in a similar manner (Figs. 6B and 7). Therefore, the process of Fe-S cluster biogenesis in isolated mammalian mitochondria faithfully recapitulates many aspects of the in vivo process taking place in cells. Importantly, our data show a regulatory relationship between nucleotide availability and Fe-S cluster biogenesis. For example, the overall process of Fe-S cluster assembly is greatly enhanced by the addition of ATP and GTP. Isolated mammalian mitochondria do not appear to contain adequate levels of endogenous ATP/GTP to support efficient Fe-S cluster biogenesis, thereby requiring addition of these nucleotides (Fig. 1A). The requirements for directly added ATP and/or GTP, however, can be bypassed when mitochondria are supplemented with αKG (Fig. 3A), thereby promoting metabolic generation of these nucleotides in the matrix, most likely by the ATP-specific (A-SCS) and GTP-specific (G-SCS) isoforms of SCS (Fig. 3B) (
      • Johnson J.D.
      • Mehus J.G.
      • Tews K.
      • Milavetz B.I.
      • Lambeth D.O.
      Genetic evidence for the expression of ATP- and GTP-specific succinyl-CoA synthetases in multicellular eucaryotes.
      ). Hydrolysis of both ATP and GTP is essential for [2Fe-2S] as well as [4Fe-4S] cluster biogenesis, and addition of ATP cannot circumvent the processes that require GTP hydrolysis and vice versa (Figs. 3, C and D, and 7A). These nucleotides are needed separately, possibly during different stages of Fe-S cluster biogenesis (see below).
      Some of the critical steps in Fe-S cluster biogenesis are thought to involve chemical reduction i.e. donation of electrons (see below). NADH and/or NADPH supplied to reductase enzymes in the matrix are the best candidates for providing the necessary reducing equivalents (
      • Lill R.
      Function and biogenesis of iron-sulphur proteins.
      ,
      • Pain J.
      • Balamurali M.M.
      • Dancis A.
      • Pain D.
      Mitochondrial NADH kinase, Pos5p, is required for efficient iron-sulfur cluster biogenesis in Saccharomyces cerevisiae.
      ). However, a role for NAD(P)H in Fe-S cluster assembly by mammalian mitochondria has so far not been directly demonstrated. Mitochondria as isolated appear to contain sufficient levels of NADH for Fe-S cluster biogenesis, and addition of NADH is unnecessary (Fig. 4A). However, if mitochondria are preincubated at 30 °C for 10 min, then endogenous NADH is likely to be depleted or consumed, perhaps by dehydrogenases in mitochondria, and, hence, mitochondria must be supplemented with NADH for Fe-S cluster synthesis to occur (Figs. 4B and 7B). Therefore, ATP, GTP, and NADH are required individually for Fe-S cluster assembly in mammalian mitochondria.
      Interestingly, isolated mammalian mitochondria contain a pool of stored iron that can be efficiently utilized for Fe-S cluster synthesis (Fig. 5A). When this endogenous pool of iron is depleted by treatment with a membrane-permeable chelator such as o-phenanthroline, Fe-S cluster assembly in isolated mitochondria does not occur. However, this is a reversible phenomenon because addition of ferrous iron salts restores the process (Fig. 5B). Added iron must be imported into the matrix to be utilized for Fe-S cluster synthesis. Therefore, isolated mammalian mitochondria are capable of using endogenous stored iron or newly imported iron for Fe-S cluster synthesis. Because of the stored pool of iron in mitochondria, we preferred 35S labeling, and not 55Fe labeling, of newly synthesized Fe-S clusters. Endogenous iron may compete with added 55Fe for incorporation into the clusters. Consequently, efficient 55Fe labeling may be achieved only after depletion of endogenous iron, requiring additional step(s). By contrast, the endogenous pool of unlabeled cysteine, if any, does not appear to drastically inhibit 35S labeling of new clusters.
      Cysteine desulfurases act on the substrate cysteine, removing sulfur and forming a covalent persulfide with the active site cysteine of the enzyme. Here we identified a bona fide persulfide intermediate on the NFS1 cysteine desulfurase that can be used productively for Fe-S cluster synthesis in intact mammalian mitochondria. When nucleotide/NADH-depleted mitochondria were incubated with [35S]cysteine, the radiolabeled persulfide covalently bound to NFS1 (NFS1-S-35SH) was detected by non-reducing SDS-PAGE (Fig. 8A, top panel). As expected, the NFS1-bound persulfide in mitochondria was sensitive to DTT treatment. More importantly, the NFS1-bound persulfide persisted under conditions that did not permit Fe-S cluster formation, such as lack of nucleotides, NADH, and/or iron availability (Fig. 9). Interestingly, the [35S]persulfide signal in nucleotide/NADH-depleted mitochondria was further enhanced with the addition of nucleotide analogs (ATPγS and/or GTPγS) (Fig. 8C). In the presence of increasing concentrations of these analogs, the residual ATP and/or GTP in these nucleotide/NADH-depleted mitochondria became increasingly unavailable for persulfide sulfur utilization, and, therefore, the persulfide remained more and more frozen on NFS1.
      Perhaps the most exciting and novel findings reported here involve the regulation of persulfide sulfur release from NFS1 and the highly coordinated utilization of persulfide sulfur and iron for Fe-S cluster synthesis. For example, when endogenous iron in mitochondria was depleted by o-phenanthroline pretreatment, the NFS1-bound persulfide was formed, but the persulfide sulfur was not released, even after the addition of GTP and NADH. Persulfide sulfur release was strictly dependent on the presence of GTP, NADH, and iron individually, and the process did not require ATP (Fig. 10). However, only in the presence of optimum levels of all four constituents together (GTP, NADH, iron, and ATP) was the NFS1-bound persulfide sulfur chased from the enzyme and subsequently utilized for Fe-S cluster assembly of aconitase (Fig. 9).
      Fe-S cluster biogenesis in mammalian mitochondria is a multistep process requiring many proteins. For brevity, the process can be divided into three key stages: stage 1, persulfide formation on NFS1; stage 2, formation of Fe-S cluster intermediates on the ISCU scaffold; and stage 3, transfer of cluster intermediates to apoproteins (Fig. 11). We propose that these distinct stages have different nucleotide requirements. In stage 1, the NFS1 cysteine desulfurase abstracts sulfur from the substrate cysteine, generating a persulfide on a conserved cysteine residue of the active site of the enzyme (NFS1-S-SH). This stage does not appear to require nucleotides, NADH, or iron (Fig. 9) (
      • Pandey A.
      • Golla R.
      • Yoon H.
      • Dancis A.
      • Pain D.
      Persulfide formation on mitochondrial cysteine desulfurase: enzyme activation by a eukaryote-specific interacting protein and Fe-S cluster synthesis.
      ,
      • Pandey A.
      • Gordon D.M.
      • Pain J.
      • Stemmler T.L.
      • Dancis A.
      • Pain D.
      Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly.
      ). However, the cysteine desulfurase activity is highly regulated by protein-protein interactions, and it seems that at least two separate conformational changes must occur in the enzyme for optimum activity. One change is mediated by frataxin interaction, exposing the substrate-binding sites and enhancing the binding of cysteine. A second change is mediated by an ISD11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation (
      • Pandey A.
      • Gordon D.M.
      • Pain J.
      • Stemmler T.L.
      • Dancis A.
      • Pain D.
      Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly.
      ). In stage 2, the persulfide sulfur is released and transferred from NFS1 to a scaffold protein, ISCU, and assembles with iron to form an Fe-S cluster intermediate (
      • Rouault T.A.
      Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease.
      ,
      • Lill R.
      • Hoffmann B.
      • Molik S.
      • Pierik A.J.
      • Rietzschel N.
      • Stehling O.
      • Uzarska M.A.
      • Webert H.
      • Wilbrecht C.
      • Mühlenhoff U.
      The role of mitochondria in cellular iron-sulfur protein biogenesis and iron metabolism.
      ). As described above, the release of persulfide sulfur occurs only in the presence of adequate levels of iron, NADH, and GTP (Fig. 10B). The source of iron has not been determined, but frataxin may play a role in iron donation for forming the ISCU intermediates (
      • Cook J.D.
      • Kondapalli K.C.
      • Rawat S.
      • Childs W.C.
      • Murugesan Y.
      • Dancis A.
      • Stemmler T.L.
      Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly.
      ). Importantly, this stage likely involves some critical reduction steps. For example, the sulfane sulfur (S0) of the NFS1-bound persulfide must be converted to sulfide (S2−) for incorporation into the nascent cluster. Likewise, iron must also be maintained in reduced form because only ferrous iron is useful for the assembly process. Furthermore, as in the case for bacterial IscU (
      • Chandramouli K.
      • Unciuleac M.C.
      • Naik S.
      • Dean D.R.
      • Huynh B.H.
      • Johnson M.K.
      Formation and properties of [4Fe-4S] clusters on the IscU scaffold protein.
      ), the reductive coupling of two distinct [2Fe-2S] clusters to a single [4Fe-4S] cluster may also occur on mammalian ISCU, requiring additional electrons. One or more of these steps may be mediated by reductases such as ferredoxin reductase, and NAD(P)H may be needed as electron supplier for the reductase. The enzyme provides electrons to ferredoxin(s) and then, probably, to other components of an Fe-S cluster assembly complex, thereby allowing cluster synthesis on ISCU. In human mitochondria, there are two different ferredoxins (FDX1 and FDX2), which are very similar. An earlier study suggested that only FDX2, and not FDX1, is required for Fe-S cluster assembly (
      • Sheftel A.D.
      • Stehling O.
      • Pierik A.J.
      • Elsässer H.-P.
      • Mühlenhoff U.
      • Webert H.
      • Hobler A.
      • Hannemann F.
      • Bernhardt R.
      • Lill R.
      Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis.
      ). However, a more recent study showed that both FDX1 and FDX2 are involved (
      • Shi Y.
      • Ghosh M.
      • Kovtunovych G.
      • Crooks D.R.
      • Rouault T.A.
      Both human ferredoxins 1 and 2 and ferredoxin reductase are important for iron-sulfur cluster biogenesis.
      ). In any case, the stimulatory effect of added NADH on Fe-S cluster biogenesis in our assays (Fig. 4B) is most likely due to its use for the ferredoxin reductase/ferredoxin redox chain. On one hand, the mammalian ferredoxin reductase prefers NADPH to NADH as electron supplier (
      • Lacour T.
      • Achstetter T.
      • Dumas B.
      Characterization of recombinant adrenodoxin reductase homologue (Arh1p) from yeast: implication in in vitro cytochrome P45011b monooxygenase system.
      ). On the other hand, the inner membrane of mitochondria is impermeable to cytosolic NADPH (
      • Pollak N.
      • Dölle C.
      • Ziegler M.
      The power to reduce: pyridine nucleotides: small molecules with a multitude of functions.
      ), consistent with the observation that directly added NADPH did not show any stimulation of cluster biogenesis in our assays with intact mitochondria (Fig. 4B). Therefore, NADPH must be made locally in the mitochondrial matrix. The observed stimulatory effects of NADH on cluster biogenesis of aconitase could be due to NADH, NADPH, or both. In the matrix, NADH may be converted to NADPH by a matrix-localized NAD(H) kinase. The NAD(H) kinases are the sole enzymes able to convert NAD(H) to NADP(H) using ATP as a phosphate donor. Such a kinase has been identified recently in mammalian mitochondria (
      • Ohashi K.
      • Kawai S.
      • Murata K.
      Identification and characterization of a human mitochondrial NAD kinase.
      ,
      • Zhang R.
      MNADK, a novel liver-enriched mitochondrion-localized NAD kinase.
      ), although a possible functional connection between the kinase and Fe-S cluster biogenesis remains to be determined. By contrast, the corresponding NADH kinase in the yeast mitochondrial matrix, called Pos5, has been characterized, and it seems that NADPH, generated by Pos5, plays an important role in Fe-S cluster biogenesis in yeast mitochondria (
      • Pain J.
      • Balamurali M.M.
      • Dancis A.
      • Pain D.
      Mitochondrial NADH kinase, Pos5p, is required for efficient iron-sulfur cluster biogenesis in Saccharomyces cerevisiae.
      ,
      • Outten C.E.
      • Culotta V.C.
      A novel NADH kinase is the mitochondrial source of NADPH in Saccharomyces cerevisiae.
      ).
      Figure thumbnail gr11
      FIGURE 11Model for nucleotide- and NADH-dependent Fe-S cluster assembly in mammalian mitochondria. Fe-S cluster biogenesis in mitochondria can be divided in three stages. Stage 1 involves persulfide formation on the cysteine desulfurase NFS1. This process does not require any nucleotides, NADH, or iron (
      • Pandey A.
      • Yoon H.
      • Lyver E.R.
      • Dancis A.
      • Pain D.
      Identification of a Nfs1p-bound persulfide intermediate in Fe-S cluster synthesis by intact mitochondria.
      ,
      • Pandey A.
      • Golla R.
      • Yoon H.
      • Dancis A.
      • Pain D.
      Persulfide formation on mitochondrial cysteine desulfurase: enzyme activation by a eukaryote-specific interacting protein and Fe-S cluster synthesis.
      ). Cysteine binds to NFS1, and frataxin plays a role by exposing cysteine binding sites (
      • Pandey A.
      • Gordon D.M.
      • Pain J.
      • Stemmler T.L.
      • Dancis A.
      • Pain D.
      Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly.
      ). ISD11 interacts with NFS1 and promotes persulfide formation (
      • Pandey A.
      • Gordon D.M.
      • Pain J.
      • Stemmler T.L.
      • Dancis A.
      • Pain D.
      Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly.
      ). In stage 2, iron and sulfur are combined, generating Fe-S cluster intermediates on the scaffold protein ISCU (
      • Stehling O.
      • Wilbrecht C.
      • Lill R.
      Mitochondrial iron-sulfur protein biogenesis and human disease.
      ). A GTPase and a reductase appear to be involved at this stage, requiring GTP and NADH, respectively. Frataxin may also play a role in this stage by providing iron or coordinating iron and sulfur donation to ISCU (
      • Cook J.D.
      • Kondapalli K.C.
      • Rawat S.
      • Childs W.C.
      • Murugesan Y.
      • Dancis A.
      • Stemmler T.L.
      Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly.
      ,
      • Colin F.
      • Martelli A.
      • Clémancey M.
      • Latour J.M.
      • Gambarelli S.
      • Zeppieri L.
      • Birck C.
      • Page A.
      • Puccio H.
      • Ollagnier de Choudens S.
      Mammalian frataxin controls sulfur production and iron entry during de novo Fe(4)S(4) cluster assembly.
      ). The GTPase involved has not yet been identified. The reductase is likely to be adrenodoxin reductase, which reduces ferredoxins FDX1 and FDX2 (
      • Sheftel A.D.
      • Stehling O.
      • Pierik A.J.
      • Elsässer H.-P.
      • Mühlenhoff U.
      • Webert H.
      • Hobler A.
      • Hannemann F.
      • Bernhardt R.
      • Lill R.
      Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis.
      ,
      • Shi Y.
      • Ghosh M.
      • Kovtunovych G.
      • Crooks D.R.
      • Rouault T.A.
      Both human ferredoxins 1 and 2 and ferredoxin reductase are important for iron-sulfur cluster biogenesis.
      ). The reducing equivalents for this electron transport chain may derive from NADPH (
      • Lacour T.
      • Achstetter T.
      • Dumas B.
      Characterization of recombinant adrenodoxin reductase homologue (Arh1p) from yeast: implication in in vitro cytochrome P45011b monooxygenase system.
      ), and, therefore an NAD(H) kinase may also be required inside mitochondria (
      • Pain J.
      • Balamurali M.M.
      • Dancis A.
      • Pain D.
      Mitochondrial NADH kinase, Pos5p, is required for efficient iron-sulfur cluster biogenesis in Saccharomyces cerevisiae.
      ,
      • Ohashi K.
      • Kawai S.
      • Murata K.
      Identification and characterization of a human mitochondrial NAD kinase.
      ). The substrate that receives electrons is unknown but could be the S0 and/or iron involved in Fe-S cluster intermediate formation (
      • Lill R.
      • Hoffmann B.
      • Molik S.
      • Pierik A.J.
      • Rietzschel N.
      • Stehling O.
      • Uzarska M.A.
      • Webert H.
      • Wilbrecht C.
      • Mühlenhoff U.
      The role of mitochondria in cellular iron-sulfur protein biogenesis and iron metabolism.
      ). In stage 3, Fe-S cluster intermediates are transferred from the scaffold protein to apoproteins such as aconitase or ferredoxins. Chaperones are involved at this stage, requiring ATP hydrolysis (
      • Schilke B.
      • Williams B.
      • Knieszner H.
      • Pukszta S.
      • D'Silva P.
      • Craig E.A.
      • Marszalek J.
      Evolution of mitochondrial chaperones utilized in Fe-S cluster biogenesis.
      ). The cochaperone HSC20 binds holo ISCU and initiates binding to the mitochondrial Hsp70, HSPA9 (
      • Maio N.
      • Singh A.
      • Uhrigshardt H.
      • Saxena N.
      • Tong W.H.
      • Rouault T.A.
      Cochaperone binding to LYR motifs confers specificity of iron sulfur cluster delivery.
      ). The Hsp70 reaction cycle requires ATP hydrolysis and mediates release and transfer of Fe-S cluster intermediates to glutaredoxins and apoproteins (
      • Stehling O.
      • Wilbrecht C.
      • Lill R.
      Mitochondrial iron-sulfur protein biogenesis and human disease.
      ). If mitochondrial levels of GTP, NADH or iron are inadequate, then the NFS1-bound persulfide remains trapped regardless of ATP levels. Only in the presence of sufficient levels of all four constituents (i.e. GTP, NADH, iron, and ATP) is the persulfide sulfur released from NFS1 and utilized for [2Fe-2S] and [4Fe-4S] cluster assembly. See text for details.
      In addition to NAD(P)H, GTP may also be required for efficient assembly of Fe-S cluster intermediates on ISCU. The NFS1-bound persulfide signal was enhanced in the presence of GTPγS or ATPγS individually (Fig. 8C), suggesting that the processes that require GTP hydrolysis or ATP hydrolysis for Fe-S cluster biogenesis lie downstream of stage 1. Interestingly, the maximum NFS1-bound persulfide signal was obtained in the presence of both GTPγS and ATPγS (Fig. 8C). Most likely, GTP hydrolysis and ATP hydrolysis occur at different stages and not during the same stage of Fe-S cluster assembly. This notion is supported by the observation that GTP, and not ATP, was specifically needed for the release of NFS1-bound persulfide sulfur (Fig. 10). We postulate that GTP plays an important role during synthesis of Fe-S cluster intermediates on ISCU in stage 2. This would be consistent with an earlier observation that, in yeast mitochondria, GTPγS inhibits assembly of Fe-S cluster intermediates on the scaffold protein (
      • Amutha B.
      • Gordon D.M.
      • Gu Y.
      • Lyver E.R.
      • Dancis A.
      • Pain D.
      GTP is required for iron-sulfur cluster biogenesis in mitochondria.
      ). At this stage, we can only speculate how a GTPase might be involved during stage 2. For example, the GTP hydrolysis mediated by a GTPase in the mitochondrial matrix might be required for trafficking of the persulfide sulfur from NFS1 to ISCU. Alternatively, the GTP hydrolysis might facilitate the insertion of ferrous ions into ISCU for the assembly of Fe-S cluster intermediates. In any case, the GTPase remains to be identified, and more work is needed to determine the precise role of GTP in Fe-S cluster assembly in mitochondria.
      In Stage 3, Hsp70 chaperones that are known to utilize ATP are involved. The cochaperone HSC20 (Jac1 in yeast) binds ISCU (Isu1 in yeast) and forms a complex with the generic mitochondrial Hsp70 chaperone, HSPA9 (Ssq1 in yeast) (
      • Maio N.
      • Singh A.
      • Uhrigshardt H.
      • Saxena N.
      • Tong W.H.
      • Rouault T.A.
      Cochaperone binding to LYR motifs confers specificity of iron sulfur cluster delivery.
      ). The cochaperone and the scaffold cooperatively stimulate the ATPase activity of the Hsp70 chaperone, which may be essential for the release of scaffold-bound cluster intermediates with concomitant transfer to the recipient apoprotein (
      • Schilke B.
      • Williams B.
      • Knieszner H.
      • Pukszta S.
      • D'Silva P.
      • Craig E.A.
      • Marszalek J.
      Evolution of mitochondrial chaperones utilized in Fe-S cluster biogenesis.
      ). Such a scenario may then explain the absolute requirement for ATP hydrolysis in our assays for Fe-S cluster biogenesis of aconitase and ferredoxins in isolated mammalian mitochondria (Figs. 3,C and D, and 7A). In addition to the transfer process (stage 3), other ATP-requiring steps in Fe-S cluster biogenesis are also possible, such as folding and/or maturation of the target substrates.
      Several human diseases are now known to be associated with deficiency of proteins involved in Fe-S cluster biogenesis in mitochondria. For example, frataxin is a key component of the Fe-S cluster assembly machinery, and deficiency of frataxin causes Friedreich ataxia (
      • Stemmler T.L.
      • Lesuisse E.
      • Pain D.
      • Dancis A.
      Frataxin and mitochondrial Fe-S cluster Biogenesis.
      ,
      • Campuzano V.
      • Montermini L.
      • Moltò M.D.
      • Pianese L.
      • Cossée M.
      • Cavalcanti F.
      • Monros E.
      • Rodius F.
      • Duclos F.
      • Monticelli A.
      • Zara F.
      • Cañizares J.
      • Koutnikova H.
      • Bidichandani S.I.
      • Gellera C.
      • Brice A.
      • Trouillas P.
      • De Michele G.
      • Filla A.
      • De Frutos R.
      • Palau F.
      • Patel P.I.
      • Di Donato S.
      • Mandel J.L.
      • Cocozza S.
      • Koenig M.
      • Pandolfo M.
      Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.
      ). This is an inherited neurodegenerative disease and is characterized by cardiomyopathy and the death of certain neuronal cells, particularly the dorsal root ganglia. Other cell types/tissues are unaffected. Another Fe-S disease, called ISCU myopathy, is caused by low levels of ISCU arising from a splicing mutation of ISCU (
      • Mochel F.
      • Knight M.A.
      • Tong W.H.
      • Hernandez D.
      • Ayyad K.
      • Taivassalo T.
      • Andersen P.M.
      • Singleton A.
      • Rouault T.A.
      • Fischbeck K.H.
      • Haller R.G.
      Splice mutation in the iron-sulfur cluster scaffold protein ISCU causes myopathy with exercise intolerance.
      ). Unlike Friedreich ataxia, the ISCU myopathy affects primarily skeletal muscles and only rarely the cardiac tissue. The reason for the tissue specificity of these diseases is not known, although tissue-specific splicing differences may contribute, and this has been a challenging issue in the field (
      • Rouault T.A.
      Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease.
      ,
      • Beilschmidt L.K.
      • Puccio H.M.
      Mammalian Fe-S cluster biogenesis and its implication in disease.
      ). Alternatively, different tissues may have different requirements for Fe-S clusters, and the corresponding mitochondria may need different levels of frataxin, ISCU, or other components of the cluster machinery. Likewise, nucleotide and/or NADH requirements and the corresponding regulatory steps could also be different from one tissue to the other. These issues can now be addressed, at least partly, by studying Fe-S cluster assembly in mitochondria isolated from different tissues.
      Recently, frataxin (Yfh1 in yeast) has been found to form a complex with NFS1 (Nfs1 in yeast), ISD11 (Isd11 in yeast), and ISCU (Isu1 in yeast) (
      • Colin F.
      • Martelli A.
      • Clémancey M.
      • Latour J.M.
      • Gambarelli S.
      • Zeppieri L.
      • Birck C.
      • Page A.
      • Puccio H.
      • Ollagnier de Choudens S.
      Mammalian frataxin controls sulfur production and iron entry during de novo Fe(4)S(4) cluster assembly.
      ,
      • Tsai C.-L.
      • Barondeau D.P.
      Human frataxin is an allosteric switch that activates the Fe-S cluster biosynthetic complex.
      ,
      • Schmucker S.
      • Martelli A.
      • Colin F.
      • Page A.
      • Wattenhofer-Donzé M.
      • Reutenauer L.
      • Puccio H.
      Mammalian frataxin: an essential function for cellular viability through an interaction with a preformed ISCU/NFS1/ISD11 iron-sulfur assembly complex.
      ,
      • Wang T.
      • Craig E.A.
      Binding of yeast frataxin to the scaffold for Fe-S cluster biogenesis, Isu.
      ). Using purified proteins, human frataxin has been found to stimulate the cysteine desulfurase activity of the complex (
      • Tsai C.-L.
      • Barondeau D.P.
      Human frataxin is an allosteric switch that activates the Fe-S cluster biosynthetic complex.
      ). In studies of the yeast counterparts using purified proteins and isolated mitochondria, Yfh1 directly interacted with Nfs1, exposing substrate-binding sites, most likely through a conformational change in the enzyme and enhancing the binding of cysteine (
      • Pandey A.
      • Gordon D.M.
      • Pain J.
      • Stemmler T.L.
      • Dancis A.
      • Pain D.
      Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly.
      ). This unique function of Yfh1 did not require Isu1 or Isd11. In addition, the nucleotides (ATP or GTP), NADH, or iron were not required for frataxin function. Frataxin can compensate for loss of Yfh1 in yeast, and, therefore, the human protein may function in mitochondria via a pathway similar to that in yeast (
      • Wilson R.B.
      • Roof D.M.
      Respiratory deficiency due to loss of mitochondrial DNA in yeast lacking the frataxin homologue.
      ,
      • Cavadini P.
      • Gellera C.
      • Patel P.I.
      • Isaya G.
      Human frataxin maintains mitochondrial iron homeostasis in Saccharomyces cerevisiae.
      ). Such a notion can now be directly tested using isolated mammalian mitochondria as described here. This is an important issue because lack of frataxin/Yfh1 mediated stimulatory effects on cysteine desulfurase activity may explain, at least in part, the deficiency of Fe-S clusters in mitochondria associated with frataxin/Yfh1 deficiency (
      • Yoon H.
      • Golla R.
      • Lesuisse E.
      • Pain J.
      • Donald J.E.
      • Lyver E.R.
      • Pain D.
      • Dancis A.
      Mutation in the Fe-S scaffold protein Isu bypasses frataxin deletion.
      ,
      • Yoon H.
      • Knight S.A.
      • Pandey A.
      • Pain J.
      • Zhang Y.
      • Pain D.
      • Dancis A.
      Frataxin-bypassing Isu1: characterization of the bypass activity in cells and mitochondria.
      ). The identification of the NFS1-bound persulfide and the tightly coordinated use of the persulfide sulfur with iron for Fe-S cluster synthesis in isolated mammalian mitochondria as described here will be useful in further elucidating the molecular basis of Friedreich ataxia.

      Acknowledgments

      We thank Dr. Silke Leimkühler for the pZM2 and pZM4 plasmids and Dr. Eldo Kuzhikandathil for the CAD cell line and advice regarding cell culture conditions.

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