Iron–sulfur cluster biogenesis and trafficking in mitochondriaPublished, Papers in Press, June 14, 2017, DOI 10.1074/jbc.R117.787101

X Joseph J. Braymer‡ and X Roland Lill‡§1

From the ‡Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch-Strasse 6, 35032 Marburg and§LOEWE Zentrum für Synthetische Mikrobiologie SynMikro, Hans-Meerwein-Strasse, 35043 Marburg, Germany

Edited by F. Peter Guengerich

The biogenesis of iron–sulfur (Fe/S) proteins in eukaryotes isa multistage, multicompartment process that is essential for abroad range of cellular functions, including genome mainte-nance, protein translation, energy conversion, and the antiviralresponse. Genetic and cell biological studies over almost 2decades have revealed some 30 proteins involved in the synthe-sis of cellular [2Fe-2S] and [4Fe-4S] clusters and their incorpo-ration into numerous apoproteins. Mechanistic aspects of Fe/Sprotein biogenesis continue to be elucidated by biochemical andultrastructural investigations. Here, we review recent develop-ments in the pursuit of constructing a comprehensive model ofFe/S protein assembly in the mitochondrion.

Ubiquitous iron–sulfur clusters and their synthesis: anoverview

Iron–sulfur (Fe/S)2 clusters are inorganic cofactors that areessential for the proper functioning of virtually all biologicalcells (1). The chemical versatility of these clusters is utilized infundamental life processes such as energy production, meta-bolic conversions, DNA maintenance, gene expression regula-tion, protein translation, and the antiviral response (Fig. 1)(2– 4). In eukaryotes, Fe/S proteins are found in or associatedwith the mitochondrion, endoplasmic reticulum, cytosol, andthe nucleus. These cofactors participate in electron transferreactions, Lewis acid catalysis, transfer of sulfur atoms, or facil-itating structural roles (5, 6). Although the activities of someFe/S proteins are dispensable for cell survival under certainconditions, for example fungal Fe/S enzymes in the metabolismof amino acids, others such as Fe/S proteins involved in DNAmaintenance or protein translation are essential for cell viabil-ity (Fig. 1). The growing number of diseases that implicate Fe/Sproteins or their assembly factors illustrates the essentiality of

the various functions of these protein cofactors (7–9). The phe-notypes associated with these “Fe/S diseases” and the in vivowork in model systems such as Saccharomyces cerevisiae andhuman cell culture have led to a mechanistic model of eukary-otic Fe/S protein biogenesis, in which the sequence of eventsrequired for proper synthesis and trafficking of Fe/S clustershas been elucidated (2). This model has been invaluable todiagnosing new mitochondrial disorders, and its continuedadvancement will enhance the ability for early diagnosis (10 –13). The focus of this review will be on the latest developmentsof the functional and mechanistic aspects that have advancedthe model of mitochondrial Fe/S protein biogenesis.

Cells typically maintain a strict balance of iron and sulfide ionconcentrations due to their damaging redox reactions whenpresent in excess (14, 15). The cell also uses a complex biosyn-thetic system to ensure that the sometimes redox-sensitive andlabile Fe/S clusters are assembled correctly, trafficked to spe-cific target apoproteins, and remain protected during these pro-cesses. This cellular control is exemplified by the 18 known“Fe/S cluster assembly” (ISC) proteins involved in the properbiogenesis and trafficking of clusters in mitochondria and the11 known “cytosolic Fe/S protein assembly” (CIA) proteinsresponsible for synthesis, trafficking, and insertion of clustersin the cytosol and nucleus (Figs. 1 and 2) (2, 16). Mitochondriaor the evolutionarily derived hydrogenosomes and mitosomesappear to be essential for biogenesis of all cellular Fe/S proteins(7, 17, 18). The only notable exception to this rule may be anewly characterized eukaryotic organism, which appears to bedevoid of mitochondria (19).

Mitochondrial Fe/S protein assembly can be divided intofour steps (Figs. 1 and 2). In the first step, de novo [2Fe-2S]cluster synthesis is orchestrated on a scaffold protein (Isu1) by aset of essential ISC proteins (Fig. 2). In the second step, a chap-erone/co-chaperone system facilitates the release of the newlysynthesized [2Fe-2S] cluster from the scaffold and its binding toa downstream ISC transfer protein (Grx5). The [2Fe-2S] clusterthen is inserted into [2Fe-2S] target proteins, trafficked to thelate-acting ISC machinery for [4Fe-4S] cluster synthesis, orused for synthesis and mitochondrial export of a yet unknownsulfur-containing species (X-S in Fig. 1) to be utilized by theCIA system. In the third step, conversion of the [2Fe-2S] into a[4Fe-4S] cluster requires a second hub of cluster synthesis. Thefourth step involves the specific insertion of the newly gener-ated [4Fe-4S] clusters into apoproteins by dedicated ISC target-ing factors (Fig. 2).

In keeping with the evolutionary origin of the mitochon-drion, the eukaryotic ISC machinery is believed to have been

This work was supported by the European Union’s Horizon 2020 research andinnovation programme under the Marie Sklodowska-Curie agreementGrant 659325 (to J. J. B.) and generous financial support (to R. L.) from theDeutsche Forschungsgemeinschaft (Koselleck Grant, LI 415/5, SFB 987,SPP 1710, and SPP 1927), LOEWE program of State Hessen, and German-Israeli Foundation (GIF). This is the fourth article in the Thematic Minire-view series “Metals in Biology 2017: Iron transport, storage, and the rami-fications.” The authors declare that they have no conflicts of interest withthe contents of this article. The content is solely the responsibility of theauthors and does not necessarily represent the official views of theNational Institutes of Health.

1 To whom correspondence should be addressed. Tel.: 49-6421-286-6449;Fax: 49-6421-286-6414; E-mail: lill@staff.uni-marburg.de.

2 The abbreviations used are: Fe/S, iron–sulfur; ISC, iron–sulfur cluster assem-bly; CIA, cytosolic iron–sulfur protein assembly; LYRM, leucine-tyrosine-arginine motif; PLP, pyridoxal 5�-phosphate; PDB, Protein Data Bank.

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Figure 1. Simplified overview of the assembly of eukaryotic Fe/S proteins and their cellular functions. The process begins in the mitochondrion withcomponents of the early ISC machinery synthesizing a [2Fe-2S] cluster on a scaffold protein. This requires iron, cysteine, and electrons as the basic components.Following the de novo assembly reaction, the cluster is released from the scaffold and bound to a transfer protein, from where the transiently associated[2Fe-2S] cluster is trafficked to [2Fe-2S] targets or the late ISC machinery for [4Fe-4S] cluster synthesis. Additionally, an unknown sulfur-containing component,X-S, is generated (shown exported from the mitochondrion) and delivered to the CIA system for maturation of cytosolic and nuclear Fe/S proteins. Mitochon-drial [4Fe-4S] clusters are finally trafficked to apoproteins by ISC targeting factors. Prominent examples of eukaryotic Fe/S proteins are listed with theircorresponding cellular roles.

Figure 2. Cartoon model of the mitochondrial Fe/S protein assembly process. A cascade of ISC proteins is required for the de novo synthesis of [2Fe-2S] and[4Fe-4S] clusters and their proper trafficking to target apoproteins in mitochondria. Initially, a [2Fe-2S] cluster is synthesized by the early ISC machinery,composed of the Isu1 scaffold protein requiring sulfide from the cysteine desulfurase complex Nfs1-Isd11-Acp1, electrons from the transfer chain NADPH-Arh1and the ferredoxin Yah1, and the regulator and/or iron donor Yfh1. The Isu1-bound [2Fe-2S] cluster is then delivered to the monothiol glutaredoxin Grx5, areaction accomplished by the Hsp70 chaperone Ssq1 with the help of the J-type co-chaperone Jac1. This reaction is dependent on ATP hydrolysis by Ssq1. Theexchange factor Mge1 facilitates the exchange of ADP for ATP. The resulting bridging [2Fe-2S] cluster on a Grx5 dimer is inserted directly into [2Fe-2S] recipientapoproteins or trafficked to the late ISC machinery for [4Fe-4S] cluster biogenesis. The early ISC machinery, including the chaperones and Grx5, is alsoresponsible for generating the component X-S for transport of sulfur out of the mitochondria to the CIA machinery for cytosolic-nuclear Fe/S protein biogen-esis. The late ISC machinery consists of the yet structurally and functionally uncharacterized Isa1-Isa2-Iba57 complex and is needed for the generation of[4Fe-4S] clusters. Trafficking and insertion of the [4Fe-4S] clusters into target Fe/S proteins are facilitated by specific ISC targeting factors, such as Nfu1, thecomplex I-specific Ind1, and the Bol proteins. Dashed arrows indicate steps that remain poorly elucidated on the biochemical level.

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inherited from an alphaproteobacterium (18), and hence manyof the mitochondrial and bacterial ISC proteins are highly sim-ilar in both structure and function (20, 21). In fact, the func-tional investigation of the mitochondrial ISC system has largelybenefited from the progress made in studying the related bac-terial system and vice versa (6, 20, 22). Nevertheless, evolutionhas slightly extended the function of the eukaryotic ISCmachinery in various ways, and it has modified the regulation ofthe biosynthetic process (23, 24). Because of its genetic tracta-bility and rapid cell growth, S. cerevisiae has proven to be anoptimal model organism for studying the essential process, yethuman cell culture has also been used successfully for func-tional studies on the ISC system. Here, we primarily use thecorresponding yeast nomenclature to describe the highly con-served protein machinery and biochemical reactions (24). Keyto recent progress in the untangling of the molecular mecha-nisms involved has been the development of in vitro reconsti-tution assays that employ spectroscopic techniques to followFe/S cluster synthesis (25–28), labeling methods (29), or the useof isolated mitochondria (30). Structural and biophysical workon Fe/S cluster biogenesis factors from bacteria to humans hasstimulated and complemented these assays, providing molecu-lar details into the ISC proteins and the modes of transient Fe/Scluster coordination by these factors.

De novo synthesis of a [2Fe-2S] cluster on themitochondrial scaffold Isu1

Central to the de novo synthesis of [2Fe-2S] clusters is thescaffold protein Isu1 where the cluster is initially assembledwith the assistance from other ISC proteins (Figs. 2 and 3) (31).A key ISC protein is the cysteine desulfurase Nfs1, along with itspartner proteins Isd11 and Acp1 (32). The Nfs1-Isd11-Acp1complex is responsible for the generation of transient per-sulfides (–SSH) on the active-site cysteine of Nfs1. Two co-crystal structures of the bacterial desulfurase IscS and thescaffold IscU have depicted that the ISC scaffold proteinbinds near a flexible loop of the desulfurase containing theactive-site cysteine (Cys loop, Fig. 3) (33, 34). The structuresalso indicate that Nfs1 acts as a homodimer providing twoindependent sites for Isu1 binding and hence two possibleactive sites for [2Fe-2S] cluster synthesis. Mutations of bothS. cerevisiae Nfs1 and Isu1 at their putative binding inter-faces show decreased interaction, supporting this mode ofassociation for the scaffold protein (35). The small proteinIsd11 contributes to the stability and possibly the regulationof the desulfurase enzyme (36 – 40). Isd11 (mammalianLYRM4) belongs to the leucine–tyrosine–arginine-motif(LYRM) family of proteins, members of which bind to respi-ratory complexes I–III and V or their assembly intermedi-

Figure 3. Structural insights of the early ISC machinery for [2Fe-2S] cluster synthesis. Structural and functional studies of the early ISC machineryhave suggested a six-membered multimeric protein complex composed of two copies of each of the following proteins. The cysteine desulfurase Nfs1(modeled from IscS-IscU PDB code 4EB7) (100), which binds its partner proteins Isd11 and Acp1 at an unknown location; the scaffold Isu1 (modeled fromIscS-IscU PDB code 3LVL); the ferredoxin Yah1 (PDB code 2MJE), and Yfh1 (PDB code 3FQL). All three proteins are expected to bind close to the Cys loop(red) of Nfs1. Note that the second set of Yah1, Yfh1, and Isd11-Acp1 components are not shown for clarity. Paramount to the synthesis of a [2Fe-2S]cluster is the scaffold protein Isu1 containing a possible cluster-binding site of three cysteines and a histidine (lower inset). The exact coordination of the[2Fe-2S] cluster is unclear throughout the biosynthetic process (designated by X). Nfs1 delivers two persulfides (–SSH) from the PLP active site (PLPrepresented as spheres) via the Cys loop (lower inset, red) to the active site of Isu1. Electrons are shuttled via the ferredoxin Yah1. Interaction of reducedYah1 with Isu1 is proposed to be facilitated by �-helix 3 (red). How 2 eq of ferrous iron gain access to the active site of Fe/S cluster synthesis remainsunclear. Allosteric regulation of sulfur transfer from Nfs1 to Isu1 is conferred by Yfh1. Isd11-Acp1 stabilizes the 200-kDa complex with Nfs1, yet theirprecise function is unknown. The Isd11-Acp1 structure may be similar to that of an LYR protein-Acp1 dimer in respiratory complex I (upper inset). Thesubunit B14 from Bos taurus containing an LYR motif (red) interacts with SDAP-� (Acp1), which covalently binds a 4�-phosphopantetheine moiety via aconserved serine (orange) (PDB code 5LDW) (41, 43).

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ates (41– 43). Although S. cerevisiae has no complex I, that ofthe yeast Yarrowia lipolytica contains the LYRM6 protein,which is essential for the catalytic activity of this respiratorycomplex. LYRM6 and the LYR family members subunit B14and B22 of mammalian complex I anchor the mitochondrialacyl-carrier protein (mammalian SDAP-�/�, Y. lipolyticaACPM1) to the complex, and the 3D structure of this dimerhas been resolved (43) (Fig. 3). A similar interaction modecan now be expected for Isd11 attaching Acp1 to the Fe/Scluster biogenesis complex in S. cerevisiae (32, 44). The dualrole of Acp1 in Fe/S protein biogenesis and mitochondrialfatty acid metabolism, including lipoic acid synthesis, mayprovide a regulatory device linking the biogenesis of respira-tory functions and metabolic activities. Critical to the func-tion of Acp1 in complex I, fatty acid synthesis, and the ISCcomplex is a 4�-phosphopantetheine moiety covalentlybound via an invariant serine residue and carrying a fatty acylchain (Fig. 3) (45). This raises the question of how Acp1coordinates all these diverse mitochondrial functions. Theinteraction surface of the Isd11-Acp1 subcomplex and theNfs1 dimer remains elusive; however, mutations in �-helix 1,�-helix 3, and the C-terminal region of Isd11 have beenshown to compromise interactions with the desulfurase (40).

Allosteric regulation of persulfide transfer from Nfs1 to thescaffold protein Isu1 is proposed to be conferred by Yfh1(human frataxin) (29, 46). The Nfs1 desulfurase reaction takesplace on a pyridoxal 5�-phosphate (PLP) cofactor that is situ-ated at the dimer interface (Fig. 3), where free L-cysteine formsa Schiff base with PLP that is primed for sulfur release (47). TheCys loop can bind near the PLP site, which may also allosteri-cally control access to the PLP site (48). The active-site cysteineof Nfs1 strips a sulfur atom, in the form of a persulfide, from theSchiff base and shuttles it to the active site of the scaffold pro-tein Isu1 (34, 47). Based on small-angle X-ray scattering withEscherichia coli ISC proteins and mutational studies inS. cerevisiae, monomeric Yfh1 (or bacterial CyaY) binds to apocket between Nfs1 and Isu1 (Fig. 3) (49, 50). This localizesYfh1 in the vicinity of both the flexible active-site Cys loop ofNfs1 and the cluster-binding site of Isu1 suggestive of its allos-teric regulatory role in persulfide transfer. A proposed secondfunction of Yfh1 is in iron supply, which will be discussedbelow.

The presence of a ferredoxin in the ISC operon in pro-karyotes and the requirement of Yah1 in vivo in yeast had sug-gested that an electron source was required for Fe/S proteinbiogenesis (31, 51). The dependence of this process on the [2Fe-2S] ferredoxin Yah1 or on human FDX2 (52, 53) in the early ISCmachinery has now been recapitulated in vitro by reconstitu-tion assays in both prokaryotic and eukaryotic systems (25, 54).Previous assays were independent of Yah1 because of the pres-ence of the artificial reductant dithiothreitol (DTT), which canreplace the ferredoxins as an electron source, and thus changethe mechanism to a non-physiological variation. The use ofDTT in reactions involving Fe/S cluster synthesis and traffick-ing must therefore be critically evaluated due to the ability ofDTT to chemically promote the release of sulfide from theNfs1-bound persulfide and thus chemically rather than bio-chemically support Fe/S cluster formation (25, 28, 54). In the

mitochondrion, the ferredoxin reductase Arh1 is reduced byNADPH, and then shuttles its electrons to Yah1, which usesthem for the construction of the [2Fe-2S]2� cluster on Isu1(Figs. 2 and 3). An interaction between Isu1 and Yah1 wasobserved in S. cerevisiae mitochondria and further confirmedin vitro by chemical cross-linking, gel filtration, NMR, andmicroscale thermophoresis (25). In contrast to Yfh1, reducedYah1 interacts strongly only with Isu1 and not significantly withthe desulfurase as in prokaryotes (Fig. 3) (25, 54, 55). Asexpected from a reductive role of Yah1, only its reduced formhad a high affinity for Isu1 suggesting that electron transferloosens the interaction and facilitates dissociation of the oxi-dized Yah1 from Isu1. At what point the electrons are trans-ferred to the scaffold protein by Yah1 remains unclear; how-ever, NMR studies suggest that a hydrophilic patch in �-helix-3of Yah1 could mediate the transfer (Fig. 3) (25).

Although it is generally accepted that the newly synthesized[2Fe-2S] cluster is constructed on the scaffold protein Isu1, themechanism of cluster synthesis remains vague (Fig. 3). Theacceptor sites of the two persulfides needed for [2Fe-2S] clusterformation on the scaffold protein have yet to be determined invivo. In vitro studies have indicated that Isu1 can indeed bepersulfurated (29, 46, 56, 57), but experiments in isolated mito-chondria have suggested that iron must be present before apersulfide can be transferred (30). How iron gains access to theactive site remains enigmatic. Apo-Isu1 does not appear to bindiron at the active site with significant affinity (58), suggestingthat iron may be actively delivered to the ISC complex. Yfh1and its human homologue frataxin can bind ferrous iron viaacidic residues on the N-terminal �-helix, but concrete in vivoevidence of Yfh1 supplying iron is still lacking (15, 59). Interest-ingly, frataxin-independent cluster synthesis using an Isu1mutant protein may rule against a specific iron delivery func-tion of frataxin (60). More detailed structural information mayhelp resolve this still open question.

How the [2Fe-2S] cluster is coordinated on Isu1 during andshortly after its synthesis remains unclear (Fig. 3). Crystal struc-tures with ISC proteins from Archaeoglobus fulgidus have sug-gested an intermediate, where the [2Fe-2S] cluster binds to Cysresidues contributed from both IscS and IscU (34). Althoughthis arrangement is highly attractive as a synthesis intermedi-ate, it has later been noted that the IscS used in this studylikely does not function as a bona fide desulfurase because itlacks both the PLP-coordinating Lys residue and desulfuraseactivity (61). In vitro reconstitution of Fe/S cluster synthesison Isu1 yielded a bridging [2Fe-2S] cluster on an Isu1 dimeras a final product, but this arrangement remains to be con-firmed in vivo (25).

Overall, the current model for the multimeric early-actingISC complex includes six proteins in stoichiometric amounts,Isu1, Nfs1–Isd11–Acp1, Yfh1, and Yah1, and is required for theconstruction of the [2Fe-2S] cluster (25, 32). Other studies havesuggested different oligomeric states of the biosyntheticmachinery, yet it remains unclear how the active site of Isu1would remain available for cluster construction and traffickingin these large complexes (62, 63).

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Chaperone-facilitated [2Fe-2S] cluster release from Isu1and trafficking by Grx5

A second multimeric protein complex is responsible for facil-itating the release of the Isu1-bound [2Fe-2S] cluster for down-stream trafficking. The specialized Hsp70 chaperone Ssq1 andits co-chaperone Jac1 work together in targeting the Isu1 scaf-fold for specific release of the bound [2Fe-2S] cluster to themonothiol glutaredoxin Grx5 (Fig. 2). Initially, Jac1 recognizesthe cluster-bound Isu1 and directs it to Ssq1 (64). Jac1 eitherfacilitates the dissociation of cluster-bound Isu1 from the early-acting ISC complex (35) or alternatively Jac1 binds to an Isu1holo-dimer that has already dissociated from the biosyntheticmachinery after Fe/S cluster synthesis (25, 65, 66). In eithercase, the dynamic chaperone complex is used to specificallytraffic the [2Fe-2S] cluster to Grx5 or possibly directly to [2Fe-2S] target apoproteins (Fig. 2) (67– 69). Grx5 is the only known[2Fe-2S] cluster-binding protein able to directly receive clus-ters from Isu1 by physically interacting with Ssq1 at a non-substrate-binding site (70). Notably, the suggested role of thechaperone system in [2Fe-2S] cluster trafficking has beenreconstituted in vitro only in the bacterial ISC system, wherethe chaperones HscA and HscB stimulated up to 700-fold the[2Fe-2S] cluster transfer from the IscU scaffold to bacterialGrxD (27).

As surmised from the biochemical assays, ATP hydrolysis onSsq1 induces a conformational change in the peptide-bindingdomain of Ssq1 that leads to its stable binding to the LPPVmotif of Isu1 (Fig. 2) (27, 65, 67). In turn, that reaction isbelieved to labilize the [2Fe-2S] cluster on Isu1 providing afacile relay to Grx5. Once the cluster release step is com-plete, the nucleotide exchange factor Mge1 can recycle Ssq1by assisting ADP dissociation and allowing the rebinding ofATP (70). These concerted, regulated steps of the chaperone

cycle could prevent unwanted [2Fe-2S] cluster release to thesolvent, small molecules, or adventitious metal-bindingsites. In support of this view, depletion of the chaperonesystem or of Grx5 in vivo shows an increase in Fe/S clusterbinding on Isu1 (31). S. cerevisiae strains lacking Ssq1 areviable, yet JAC1 deletions are lethal indicating its indispens-ability for Fe/S cluster biogenesis (71). This difference for thetwo chaperones is explained by the presence of the second,more general Hsp70 isoform Ssc1 in S. cerevisiae partiallytaking over the function of Ssq1 (72).

The role of Grx5 as an Fe/S cluster transfer protein is sup-ported by the crystal structure of the human homologueGLRX5 with a bridging [2Fe-2S] cluster (73). The definition forbacterial GrxD as a cluster “carrier protein” cannot be adoptedfor mitochondria to avoid confusion with “mitochondrial car-rier proteins” involved in inner membrane transport of metab-olites (74). The [2Fe-2S] cluster is coordinated between twoGrx5 monomers using the active-site cysteine of Grx5 and thecysteine of a non-covalently Grx5-bound glutathione molecule(Fig. 4, A and B). In S. cerevisiae, the inability to immunopre-cipitate Grx5 with a radiolabeled 55Fe/S cluster suggests thismoiety to be bound in a labile fashion, a property that would bebeneficial for trafficking the cluster to further downstream tar-gets. Cluster binding was, however, detectable by 55Fe radiola-beling, when Schizosaccharomyces pombe or human Grx5 ho-mologues were expressed ectopically in yeast indicating thatthese foreign proteins bind the cluster more stably (70). Sur-prisingly, deletion of S. cerevisiae GRX5 is not lethal, suggestingthat the protein’s function can be bypassed to some extent,despite its currently accepted central role in Fe/S cluster traf-ficking in mitochondria (Fig. 2). Furthermore, Grx5 function ishardly needed under anaerobic conditions suggesting that itstrafficking role is particularly required under ambient or high

Figure 4. ISC transfer proteins and targeting factors assisting [2Fe-2S] and [4Fe-4S] cluster trafficking. A, crystal structure of the human Grx5 homologueGLRX5 (PDB code 2WUL) as a homodimer. The different protomers are different shades of gray with the GSH carbon atoms colored in magenta (blue, nitrogen;red, oxygen; orange, iron; yellow, sulfur). B, [2Fe-2S] cluster coordination in the Grx5 homodimer involves a cysteine from each protomer and each GSHmolecule, with coordination bonds shown by dotted lines. C–E, solution structures of human ISC targeting factors. C, C-terminal domain of NFU1 (PDB code2M5O); D, BOLA1 (PDB code 5LCI); and E, BOLA3 (PDB code 2NCL). Potential Fe/S cluster coordinating residues are shown in orange as sticks (yellow, sulfur; blue,nitrogen).

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oxygen pressure. As an alternative to Grx5-mediated traffick-ing, Jac1 and Ssq1 have been suggested to support the release ofclusters from Isu1 directly to target proteins (Fig. 2) (68, 75).However, higher eukaryotes require efficient Grx5-dependent[2Fe-2S] trafficking reactions, because mutations in humanGLRX5 are associated with human disease (7, 76). In mechanis-tic and molecular terms, much remains to be explored in thepathways of [2Fe-2S] cluster trafficking and insertion in themitochondrion.

Synthesis and trafficking of the [4Fe-4S] cluster inmitochondria

In vivo studies in S. cerevisiae and human cell culture haveshown that a dedicated ISC complex is needed for cellular [4Fe-4S] cluster synthesis. This late-acting complex is composed ofIsa1-Isa2-Iba57 and does not interact with the early ISCmachinery, but it is dependent on the delivery of a [2Fe-2S]cluster species (Fig. 2) (77–79). Specifically, deficiency of anyconstituent of the Isa1-Isa2-Iba57 complex results in mutantyeast or human cells that lack a functional respiratory chain andlipoic acid (see below). Yeast cells additionally are lysine andglutamine auxotrophic because mitochondrial homoaconitase(Lys4) and glutamate synthase (Glt1) are not matured. The ISCproteins Isa1 and Isa2 are proposed to accept a [2Fe-2S] clusterfrom Grx5, connecting the [2Fe-2S] trafficking step to the lateISC machinery (Figs. 1 and 2) (78, 80 – 82). The two Isa proteinsbelong to the A-type family of ISC proteins that have beenshown to be either homo- or heterodimers, which can coordi-nate Fe/S clusters and/or mononuclear iron via three conservedcysteine residues (78, 82– 84). In vitro reconstitution of [2Fe-2S] cluster release from Grx5 to a Isa1-Isa2 heterodimer andsubsequent DTT-dependent reductive coupling of two [2Fe-2S] clusters to form a [4Fe-4S] cluster have been reported basedon NMR and UV-visible studies (Fig. 2) (82). This reconstitu-tion assay, however, occurred at slow rates and did not includethe third required component, Iba57, leaving its function unac-counted for. All three proteins are required for the synthesis ofthe [4Fe-4S] cluster in the cell, yet their stoichiometry, struc-ture, interaction modes, and cluster-binding sites remainmolecularly undefined. Depending on the oxidation states ofthe cluster, [2Fe-2S]1� or [2Fe-2S]2�, a biological electronsource may not or may be required for [4Fe-4S]2� cluster syn-thesis, respectively.

Additional ISC targeting factors facilitate the trafficking ofthe [4Fe-4S] clusters from the Isa1-Isa2-Iba57 complex to ded-icated apoproteins. At present, two mitochondrial [4Fe-4S]cluster-binding proteins are known, Nfu1 and Ind1 (Fig. 2) (12,13, 85– 88). Recent in vivo evidence indicates that the mito-chondrial protein Nfu1 can interact with the [4Fe-4S] clustermachinery and with potential [4Fe-4S] target proteins, thussupporting its targeting function (89). These in vivo observa-tions agree with the in vitro characterization of the C-terminaldomain of Nfu1 coordinating a bridging [4Fe-4S] cluster, via aCXXC motif, between two monomers, as observed in the plantand human homologues (Fig. 4) (90 –92). Furthermore, [4Fe-4S] cluster-bound Nfu1 was capable of inserting the cluster intoplant target proteins in vitro (90). Yeast and human cells lackingNfu1 contain partially defective Fe/S target proteins aconitase,

succinate dehydrogenase, and lipoic acid synthase, indicating anon-essential role of Nfu1 as a [4Fe-4S]-targeting protein (Figs.2 and 4) (12, 89, 93).

A further set of mitochondrial ISC targeting factors termedBol1 and Bol3 are proposed to provide specificity for Fe/S clus-ter insertion into apoproteins. These factors are members ofthe Bol (BOLA) protein family, which also includes Bol2 in thecytosol (94). Two recent in vivo studies in yeast and a BOLA3patient analysis suggested that Bol3 and Bol1 are involved in thematuration of a sub-class of mitochondrial [4Fe-4S] proteins,especially succinate dehydrogenase and lipoic acid synthasewith its two [4Fe-4S] clusters, whereas several [2Fe-2S] proteinswere assembled independently of the Bol proteins (Fig. 2) (13,89, 93). Like Nfu1, the mitochondrial Bol proteins are notessential for viability of S. cerevisiae, and residual maturation oftheir target proteins was observed even in BOL double nullmutants. Bol3 was co-immunoprecipitated with both late ISCmachinery and [4Fe-4S] target proteins, a protein set overlap-ping with the Nfu1 interactome (89). Furthermore, deletingboth BOL genes in S. cerevisiae did not show the tell-tale sign ofearly ISC gene disruption, i.e. a general cellular Fe/S proteindefect and the activation of the iron regulon, as described forthe GRX5 mutant cells, for example (31). Together, these prop-erties place the Bol protein function in the late part of the ISCpathway after Isa1-Isa2-Iba57 function (Fig. 2).

A combination of in vitro and in vivo studies have shown thatboth Bol1 and Bol3 interact with Grx5, raising the question ofwhether Grx5 performs another function in the late ISCmachinery or, alternatively, whether the Bol-Grx5 heterocom-plexes could also have undisclosed functions in [2Fe-2S] clustertrafficking and/or insertion reactions (89, 93). A physiologicalrelevance of a Grx-Bol heterodimer has been established onlyfor the yeast cytosolic monothiol glutaredoxin Grx3 and Bol2(formerly known as Fra2), which are involved in iron homeo-stasis in the yeast cytosol (94 –96). Similar Fe/S cluster-contain-ing Bol-Grx heterocomplexes have been observed in bacteria(BolA with GrxD or Grx4), plants (BolA1 and BolA2 withGrxS14 and GrxS17), and recently in humans (BOLA1 andBOLA3 with GLRX5) (93, 97, 98). Mitochondrial Bol1 proteinscontain three conserved histidine residues, and Bol3 contains ahistidine and a cysteine residue as candidates for coordinationof the [2Fe-2S] cluster in the heterodimers with Grx5 (Fig. 4)(94, 99). In vitro characterization and NMR studies have shownthat human BOLA1 and BOLA3 can specifically interact withboth the apo- and holo-forms of GLRX5, yet the affinities of therespective hetero-clusters differ characteristically (89, 93). Thedifferent biochemical characteristics of the human GLRX5-BOLA complexes suggest that the two complexes may havedistinct functions, e.g. different reactivities/affinities/specifici-ties for Fe/S cluster target apoproteins.

The physical interaction between Nfu1 and Bol3 and the dra-matic increase of Bol3 levels upon overexpression of Nfu1 fur-ther suggest the roles of these proteins late in the ISC pathway(89, 93). In further support of their function in the late ISCsteps, deletion of all three ISC genes (bol1�bol3�nfu1� yeastmutant cells) resulted in a synergistic growth and Fe/S proteinbiogenesis defect approaching the phenotype observed for cellslacking the Isa1-Isa2-Iba57 proteins. However, the Bol3 and

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Nfu1 protein functions do not overlap, because overexpressionof one factor did not ameliorate the defects arising from thedeletion of the other ISC gene (93). In the triple deletion cells,severe effects were observed for �-ketoglutarate dehydroge-nase and pyruvate dehydrogenase, two proteins dependent onthe Fe/S protein lipoic acid synthase (Fig. 2). Intriguingly, thesynthesis of lipoic acid is dependent on an Acp1-derived octa-noyl fatty acyl chain, an aspect that links the early ISC machin-ery and the Fe/S target protein lipoic acid synthase (45). Thecombined defects in protein lipoylation and the respiratorychain complexes are a hallmark of Fe/S diseases, especially inthe so-called multiple mitochondrial dysfunction syndromes,and are now used for diagnostic purposes (7, 9).

Conclusions and perspectives

Fe/S protein biogenesis in mitochondria continues to be animportant area of active research with many key questions to beaddressed. The early stage of the ISC assembly pathway is thebest understood to date, yet much is to be elucidated concern-ing the structure of the biosynthetic ISC complex and the step-wise biochemical mechanisms of Fe/S cluster synthesis. Itseems clear that the various multimeric ISC complexes play keyroles in orchestrating and protecting Fe/S cluster synthesis andtrafficking. Future challenges will involve understanding theassociated dynamics of the individual proteins in these largercomplexes. Following this, the thermodynamics and kinetics oftrafficking species that connect the four ISC stages must also beclearly described, in addition to determining how the early ISCsystem supplies the enigmatic X-S compound for mitochon-drial export.

Despite the high number of known ISC proteins, new fac-tors may still be discovered. The non-essential function ofthe transfer protein Grx5 in S. cerevisiae would support thisidea in addition to the newest biogenesis factor, Acp1, beingadded in 2016. This latest discovery also adds a new avenueof research in the mitochondrion, specifically understandinghow Fe/S cluster biogenesis is regulated, e.g. by connectionto mitochondrial fatty acid synthesis. Furthermore, onepressing question to address is the source and trafficker ofiron to the ISC biosynthetic complex. Ferrous iron may sim-ply diffuse to the active site of Fe/S cluster synthesis; how-ever, the amount of energy the cell puts forth to constructingFe/S clusters and the required metal specificity would argueagainst such a nonspecific mechanism. In vitro reconstitu-tion assays will continue to be instrumental in progressingour understanding further to a complete Fe/S protein bio-genesis model. However, it seems crucial to verify these find-ings in vivo to confirm their validity in the mitochondrialsetting across various model organisms. Fruitful times arestill ahead in the field of mitochondrial Fe/S protein biogen-esis with many fundamental questions still to be addressed.

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Joseph J. Braymer and Roland Lillsulfur cluster biogenesis and trafficking in mitochondria−Iron

doi: 10.1074/jbc.R117.787101 originally published online June 14, 20172017, 292:12754-12763.J. Biol. Chem. 

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