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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
* 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.
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.
A novel experimental system tracks new Fe-S cluster synthesis in isolated mammalian mitochondria.
The use of persulfide sulfur and iron for Fe-S cluster biogenesis is tightly coordinated by processes requiring GTP, NADH, and ATP.
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 (
). 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 (
). 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 (
). 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 (
). 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 (
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 (
). 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 (
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.
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 (
). 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) (
). 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 (
). 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) (
). 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 (
). 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 (
). 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 (
), 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 (
). 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 (
), 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 (
), 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 (
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 (
). 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) (
). 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 (
). 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 (
). 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 (
). 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 (
). 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) (
). 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 (
). 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 (
). 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 (
). 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.
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.