Requirements for the biogenesis of [2Fe-2S] proteins in the human and yeast cytosol.

The biogenesis of iron-sulfur (Fe/S) proteins entails the synthesis and trafficking of Fe/S clusters, followed by their insertion into target apoproteins. In eukaryotes, the multiple steps of biogenesis are accomplished by complex protein machineries in both mitochondria and cytosol. The underlying biochemical pathways have been elucidated over the past decades, yet the mechanisms of cytosolic [2Fe-2S] protein assembly have remained ill-defined. Similarly, the precise site of glutathione (GSH) requirement in cytosolic and nuclear Fe/S protein biogenesis is unclear, as is the molecular role of the GSH-dependent cytosolic monothiol glutaredoxins (cGrxs). Here, we investigated these questions in human and yeast cells by various in vivo approaches. [2Fe-2S] cluster assembly of cytosolic target apoproteins required the mitochondrial ISC machinery, the mitochondrial transporter Atm1/ABCB7 and GSH, yet occurred independently of both the CIA system and cGrxs. This mechanism was strikingly different from the ISC-, Atm1/ABCB7-, GSH-, and CIA-dependent assembly of cytosolic-nuclear [4Fe-4S] proteins. One notable exception to this cytosolic [2Fe-2S] protein maturation pathway defined here was yeast Apd1 which used the CIA system via binding to the CIA targeting complex through its C-terminal tryptophan. cGrxs, although attributed as [2Fe-2S] cluster chaperones or trafficking proteins, were not essential in vivo for delivering [2Fe-2S] clusters to either CIA components or target apoproteins. Finally, the most critical GSH requirement was assigned to Atm1-dependent export, i.e. a step before GSH-dependent cGrxs function. Our findings extend the general model of eukaryotic Fe/S protein biogenesis by adding the molecular requirements for cytosolic [2Fe-2S] protein maturation.

A novel IBA57 variant is associated with mitochondrial iron-sulfur protein deficiency and necrotizing myelopathy in dogs.

Introduction: Hereditary necrotizing myelopathy (HNM) in young Kooiker dogs is characterized by progressive ataxia and paralysis with autosomal recessive inheritance. The basic genetic defect is unknown. We investigated the possible cause by a genome-wide analysis using six affected and 17 unrelated unaffected Kooiker dogs and by functional follow-up studies. Method: The HNM locus was mapped by a case-control study using a dense SNP array and confirmed by linkage analysis of two pedigrees. The gene exons in the critical region were analyzed by next-generation sequencing. The functional effect of the candidate canine IBA57 pathogenic variant was biochemically examined in an established HeLa cell culture model in which the endogenous IBA75 gene product was depleted by RNAi. Results: The basic defect was localized in the centromeric 5 Mb region of canine chromosome 14. The most associated SNP co-segregated fully with HNM and reached an LOD score of 6.1. A candidate pathogenic mutation was found in the iron-sulfur cluster assembly gene IBA57 and led to the amino acid substitution R147W. The expression of human IBA57 harboring the canine R147W exchange could only partially restore the biochemical defects of several mitochondrial [4Fe-4S] proteins upon IBA57 depletion, showing that the mutant protein is functionally impaired. Discussion: Pathogenic variants in human IBA57 cause multiple mitochondrial dysfunction syndrome 3 (MMDS3), a neurodegenerative disorder with distant similarities to HNM. The incomplete functional complementation of IBA57-depleted human cells by IBA57-R147W identifies the DNA mutation in affected Kooiker dogs as the genetic cause of HNM. Our findings further expand the phenotypic spectrum of pathogenic IBA57 variants.

Mitochondrial [2Fe-2S] ferredoxins: new functions for old dogs.

Ferredoxins (FDXs) comprise a large family of iron-sulfur proteins that shuttle electrons from NADPH and FDX reductases into diverse biological processes. This review focuses on the structure, function and specificity of mitochondrial [2Fe-2S] FDXs that are related to bacterial FDXs due to their endosymbiotic inheritance. Their classical function in cytochrome P450-dependent steroid transformations was identified around 1960, and is exemplified by mammalian FDX1 (aka adrenodoxin). Thirty years later the essential function in cellular Fe/S protein biogenesis was discovered for the yeast mitochondrial FDX Yah1 that is additionally crucial for the formation of haem a and ubiquinone CoQ6 . In mammals, Fe/S protein biogenesis is exclusively performed by the FDX1 paralog FDX2, despite the high structural similarity of both proteins. Recently, additional and specific roles of human FDX1 in haem a and lipoyl cofactor biosyntheses were described. For lipoyl synthesis, FDX1 transfers electrons to the radical S-adenosyl methionine-dependent lipoyl synthase to kickstart its radical chain reaction. The high target specificity of the two mammalian FDXs is contained within small conserved sequence motifs, that upon swapping change the target selection of these electron donors.

Functional spectrum and specificity of mitochondrial ferredoxins FDX1 and FDX2.

Ferredoxins comprise a large family of iron-sulfur (Fe-S) proteins that shuttle electrons in diverse biological processes. Human mitochondria contain two isoforms of [2Fe-2S] ferredoxins, FDX1 (aka adrenodoxin) and FDX2, with known functions in cytochrome P450-dependent steroid transformations and Fe-S protein biogenesis. Here, we show that only FDX2, but not FDX1, is involved in Fe-S protein maturation. Vice versa, FDX1 is specific not only for steroidogenesis, but also for heme a and lipoyl cofactor biosyntheses. In the latter pathway, FDX1 provides electrons to kickstart the radical chain reaction catalyzed by lipoyl synthase. We also identified lipoylation as a target of the toxic antitumor copper ionophore elesclomol. Finally, the striking target specificity of each ferredoxin was assigned to small conserved sequence motifs. Swapping these motifs changed the target specificity of these electron donors. Together, our findings identify new biochemical tasks of mitochondrial ferredoxins and provide structural insights into their functional specificity.

N-terminal tyrosine of ISCU2 triggers [2Fe-2S] cluster synthesis by ISCU2 dimerization.

Synthesis of iron-sulfur (Fe/S) clusters in living cells requires scaffold proteins for both facile synthesis and subsequent transfer of clusters to target apoproteins. The human mitochondrial ISCU2 scaffold protein is part of the core ISC (iron-sulfur cluster assembly) complex that synthesizes a bridging [2Fe-2S] cluster on dimeric ISCU2. Initial iron and sulfur loading onto monomeric ISCU2 have been elucidated biochemically, yet subsequent [2Fe-2S] cluster formation and dimerization of ISCU2 is mechanistically ill-defined. Our structural, biochemical and cell biological experiments now identify a crucial function of the universally conserved N-terminal Tyr35 of ISCU2 for these late reactions. Mixing two, per se non-functional ISCU2 mutant proteins with oppositely charged Asp35 and Lys35 residues, both bound to different cysteine desulfurase complexes NFS1-ISD11-ACP, restores wild-type ISCU2 maturation demonstrating that ionic forces can replace native Tyr-Tyr interactions during dimerization-induced [2Fe-2S] cluster formation. Our studies define the essential mechanistic role of Tyr35 in the reaction cycle of de novo mitochondrial [2Fe-2S] cluster synthesis.

Function and crystal structure of the dimeric P-loop ATPase CFD1 coordinating an exposed [4Fe-4S] cluster for transfer to apoproteins.

Maturation of iron-sulfur (Fe-S) proteins in eukaryotes requires complex machineries in mitochondria and cytosol. Initially, Fe-S clusters are assembled on dedicated scaffold proteins and then are trafficked to target apoproteins. Within the cytosolic Fe-S protein assembly (CIA) machinery, the conserved P-loop nucleoside triphosphatase Nbp35 performs a scaffold function. In yeast, Nbp35 cooperates with the related Cfd1, which is evolutionary less conserved and is absent in plants. Here, we investigated the potential scaffold function of human CFD1 (NUBP2) in CFD1-depleted HeLa cells by measuring Fe-S enzyme activities or 55Fe incorporation into Fe-S target proteins. We show that CFD1, in complex with NBP35 (NUBP1), performs a crucial role in the maturation of all tested cytosolic and nuclear Fe-S proteins, including essential ones involved in protein translation and DNA maintenance. CFD1 also matures iron regulatory protein 1 and thus is critical for cellular iron homeostasis. To better understand the scaffold function of CFD1-NBP35, we resolved the crystal structure of Chaetomium thermophilum holo-Cfd1 (ctCfd1) at 2.6-Å resolution as a model Cfd1 protein. Importantly, two ctCfd1 monomers coordinate a bridging [4Fe-4S] cluster via two conserved cysteine residues. The surface-exposed topology of the cluster is ideally suited for both de novo assembly and facile transfer to Fe-S apoproteins mediated by other CIA factors. ctCfd1 specifically interacted with ATP, which presumably associates with a pocket near the Cfd1 dimer interface formed by the conserved Walker motif. In contrast, ctNbp35 preferentially bound GTP, implying differential regulation of the two fungal scaffold components during Fe-S cluster assembly and/or release.

Biochemical Analyses of Human Iron-Sulfur Protein Biogenesis and of Related Diseases.

Maturation of Fe/S proteins in mammals is an intricate process mediated by two assembly systems located in the mitochondrial and cytosolic-nuclear compartments. Malfunction particularly of the mitochondrial system gives rise to severe neurological, metabolic, or hematological disorders, often with fatal outcome. In this chapter, we describe approaches for the differential biochemical investigation of cellular Fe/S protein maturation in mitochondria, cytosol, and nucleus. The analyses may also facilitate the identification of the affected Fe/S protein assembly step in diseased state. As Fe/S cluster insertion into target apoproteins is a frequent determinant of protein stability, examination of protein steady-state levels in biological samples frequently permits reliable first clues about the maturation process. In some specific cases, this approach allows the assessment of enzymatic or regulatory functions of Fe/S proteins, including the formation of lipoate cofactor by mitochondrial lipoic acid synthase or the posttranscriptional regulation of transferrin receptor and ferritin expression by the cytosolic iron regulatory proteins. More direct Fe/S protein maturation assays like enzymatic analyses may further validate the observed maturation defects. Here, we present a simple protocol for the determination of dihydropyrimidine dehydrogenase enzyme activity by thin-layer chromatography. In order to directly monitor Fe/S cluster insertion into target apoproteins, we have developed a 55Fe radiolabeling technique tracing the in vivo Fe/S cofactor formation in mammalian tissue culture. The combination of the presented techniques represents a comprehensive strategy to assess the multiple facets of Fe/S protein assembly for both mechanistic analyses and for the elucidation of specific defects in Fe/S diseases.

A novel de novo dominant mutation in ISCU associated with mitochondrial myopathy.

BACKGROUND: Hereditary myopathy with lactic acidosis and myopathy with deficiency of succinate dehydrogenase and aconitase are variants of a recessive disorder characterised by childhood-onset early fatigue, dyspnoea and palpitations on trivial exercise. The disease is non-progressive, but life-threatening episodes of widespread weakness, metabolic acidosis and rhabdomyolysis may occur. So far, this disease has been molecularly defined only in Swedish patients, all homozygous for a deep intronic splicing affecting mutation in ISCU encoding a scaffold protein for the assembly of iron-sulfur (Fe-S) clusters. A single Scandinavian family was identified with a different mutation, a missense change in compound heterozygosity with the common intronic mutation. The aim of the study was to identify the genetic defect in our proband. METHODS: A next-generation sequencing (NGS) approach was carried out on an Italian male who presented in childhood with ptosis, severe muscle weakness and exercise intolerance. His disease was slowly progressive, with partial recovery between episodes. Patient's specimens and yeast models were investigated. RESULTS: Histochemical and biochemical analyses on muscle biopsy showed multiple defects affecting mitochondrial respiratory chain complexes. We identified a single heterozygous mutation p.Gly96Val in ISCU, which was absent in DNA from his parents indicating a possible de novo dominant effect in the patient. Patient fibroblasts showed normal levels of ISCU protein and a few variably affected Fe-S cluster-dependent enzymes. Yeast studies confirmed both pathogenicity and dominance of the identified missense mutation. CONCLUSION: We describe the first heterozygous dominant mutation in ISCU which results in a phenotype reminiscent of the recessive disease previously reported.

Cellular requirements for iron-sulfur cluster insertion into the antiviral radical SAM protein viperin.

Viperin (RSAD2) is an interferon-stimulated antiviral protein that belongs to the radical S-adenosylmethionine (SAM) enzyme family. Viperin's iron-sulfur (Fe/S) cluster is critical for its antiviral activity against many different viruses. CIA1 (CIAO1), an essential component of the cytosolic iron-sulfur protein assembly (CIA) machinery, is crucial for Fe/S cluster insertion into viperin and hence for viperin's antiviral activity. In the CIA pathway, CIA1 cooperates with CIA2A, CIA2B, and MMS19 targeting factors to form various complexes that mediate the dedicated maturation of specific Fe/S recipient proteins. To date, however, the mechanisms of how viperin acquires its radical SAM Fe/S cluster to gain antiviral activity are poorly understood. Using co-immunoprecipitation and 55Fe-radiolabeling experiments, we therefore studied the roles of CIA2A, CIA2B, and MMS19 for Fe/S cluster insertion. CIA2B and MMS19 physically interacted with the C terminus of viperin and used CIA1 as the primary viperin-interacting protein. In contrast, CIA2A bound to viperin's N terminus in a CIA1-, CIA2B-, and MMS19-independent fashion. Of note, the observed interaction of both CIA2 isoforms with a single Fe/S target protein is unprecedented in the CIA pathway. 55Fe-radiolabeling experiments with human cells depleted of CIA1, CIA2A, CIA2B, or MMS19 revealed that CIA1, but none of the other CIA factors, is predominantly required for 55Fe/S cluster incorporation into viperin. Collectively, viperin maturation represents a novel CIA pathway with a minimal requirement of the CIA-targeting factors and represents a new paradigm for the insertion of the Fe/S cofactor into a radical SAM protein.

Compartmentalization of iron between mitochondria and the cytosol and its regulation.

Iron is essential for life. Its coordinated distribution between intracellular compartments and the adaptation of iron uptake to intracellular demands are central for a balanced iron homeostasis. Mitochondria take center stage in cellular iron metabolism as they harbor the two major iron-utilizing pathways, the synthesis of heme and the biogenesis of iron-sulfur (Fe/S) proteins. Consistent with this central role, mitochondria are also critical regulators of cellular iron homeostasis. They directly influence cellular iron uptake and the status of iron-utilizing metabolic processes through iron-dependent co-factors or by control of gene expression. For all these aspects of cellular iron metabolism, the uptake of iron into mitochondria is critical. During the last decade, considerable progress has been made with respect to the functional characterization of mitochondrial iron acquisition and the identification of transporters involved. The model organism Saccharomyces cerevisiae has been especially useful for the elucidation of this process. Here, we summarize the recent advances in the mechanism of mitochondrial iron transport and the impact of mitochondria on the regulation of cellular iron homeostasis.

The role of mitochondria and the CIA machinery in the maturation of cytosolic and nuclear iron-sulfur proteins.

Mitochondria have been derived from alpha-bacterial endosymbionts during the evolution of eukaryotes. Numerous bacterial functions have been maintained inside the organelles including fatty acid degradation, citric acid cycle, oxidative phosphorylation, and the synthesis of heme or lipoic acid cofactors. Additionally, mitochondria have inherited the bacterial iron-sulfur cluster assembly (ISC) machinery. Many of the ISC components are essential for cell viability because they generate a still unknown, sulfur-containing compound for the assembly of cytosolic and nuclear Fe/S proteins that perform important functions in, e.g., protein translation, DNA synthesis and repair, and chromosome segregation. The sulfur-containing compound is exported by the mitochondrial ABC transporter Atm1 (human ABCB7) and utilized by components of the cytosolic iron-sulfur protein assembly (CIA) machinery. An appealing minimal model for the striking compartmentation of eukaryotic Fe/S protein biogenesis is provided by organisms that contain mitosomes instead of mitochondria. Mitosomes have been derived from mitochondria by reductive evolution, during which they have lost virtually all classical mitochondrial tasks. Nevertheless, mitosomes harbor all core ISC components which presumably have been maintained for assisting the maturation of cytosolic-nuclear Fe/S proteins. The current review is centered around the Atm1 export process. We present an overview on the mitochondrial requirements for the export reaction, summarize recent insights into the 3D structure and potential mechanism of Atm1, and explain how the CIA machinery uses the mitochondrial export product for the assembly of cytosolic and nuclear Fe/S proteins.

Mitochondrial iron-sulfur protein biogenesis and human disease.

Work during the past 14 years has shown that mitochondria are the primary site for the biosynthesis of iron-sulfur (Fe/S) clusters. In fact, it is this process that renders mitochondria essential for viability of virtually all eukaryotes, because they participate in the synthesis of the Fe/S clusters of key nuclear and cytosolic proteins such as DNA polymerases, DNA helicases, and ABCE1 (Rli1), an ATPase involved in protein synthesis. As a consequence, mitochondrial function is crucial for nuclear DNA synthesis and repair, ribosomal protein synthesis, and numerous other extra-mitochondrial pathways including nucleotide metabolism and cellular iron regulation. Within mitochondria, the synthesis of Fe/S clusters and their insertion into apoproteins is assisted by 17 proteins forming the ISC (iron-sulfur cluster) assembly machinery. Biogenesis of mitochondrial Fe/S proteins can be dissected into three main steps: First, a Fe/S cluster is generated de novo on a scaffold protein. Second, the Fe/S cluster is dislocated from the scaffold and transiently bound to transfer proteins. Third, the latter components, together with specific ISC targeting factors insert the Fe/S cluster into client apoproteins. Disturbances of the first two steps impair the maturation of extra-mitochondrial Fe/S proteins and affect cellular and systemic iron homeostasis. In line with the essential function of mitochondria, genetic mutations in a number of ISC genes lead to severe neurological, hematological and metabolic diseases, often with a fatal outcome in early childhood. In this review we briefly summarize our current functional knowledge on the ISC assembly machinery, and we present a comprehensive overview of the various Fe/S protein assembly diseases.

Maturation of cytosolic and nuclear iron-sulfur proteins.

Eukaryotic cells contain numerous cytosolic and nuclear iron-sulfur (Fe/S) proteins that perform key functions in metabolic catalysis, iron regulation, protein translation, DNA synthesis, and DNA repair. Synthesis of Fe/S clusters and their insertion into apoproteins are essential for viability and are conserved in eukaryotes. The process is catalyzed in two major steps by the CIA (cytosolic iron-sulfur protein assembly) machinery encompassing nine known proteins. First, a [4Fe-4S] cluster is assembled on a scaffold complex. This step requires a sulfur-containing compound from mitochondria and reducing equivalents from an electron transfer chain. Second, the Fe/S cluster is transferred from the scaffold to specific apoproteins by the CIA targeting complex. This review summarizes our molecular knowledge on CIA protein function during the assembly process.

Viperin is an iron-sulfur protein that inhibits genome synthesis of tick-borne encephalitis virus via radical SAM domain activity.

Viperin is an interferon-induced protein with a broad antiviral activity. This evolutionary conserved protein contains a radical S-adenosyl-l-methionine (SAM) domain which has been shown in vitro to hold a [4Fe-4S] cluster. We identified tick-borne encephalitis virus (TBEV) as a novel target for which human viperin inhibits productionof the viral genome RNA. Wt viperin was found to require ER localization for full antiviral activity and to interact with the cytosolic Fe/S protein assembly factor CIAO1. Radiolabelling in vivo revealed incorporation of (55) Fe, indicative for the presence of an Fe-S cluster. Mutation of the cysteine residues ligating the Fe-S cluster in the central radical SAM domain entirely abolished both antiviral activity and incorporation of (55) Fe. Mutants lacking the extreme C-terminal W361 did not interact with CIAO1, were not matured, and were antivirally inactive. Moreover, intracellular removal of SAM by ectopic expression of the bacteriophage T3 SAMase abolished antiviral activity. Collectively, our data suggest that viperin requires CIAO1 for [4Fe-4S] cluster assembly, and acts through an enzymatic, Fe-S cluster- and SAM-dependent mechanism to inhibit viral RNA synthesis.

The role of mitochondria in cellular iron-sulfur protein biogenesis: mechanisms, connected processes, and diseases.

Iron-sulfur (Fe/S) clusters belong to the most ancient protein cofactors in life, and fulfill functions in electron transport, enzyme catalysis, homeostatic regulation, and sulfur activation. The synthesis of Fe/S clusters and their insertion into apoproteins requires almost 30 proteins in the mitochondria and cytosol of eukaryotic cells. This review summarizes our current biochemical knowledge of mitochondrial Fe/S protein maturation. Because this pathway is essential for various extramitochondrial processes, we then explain how mitochondria contribute to the mechanism of cytosolic and nuclear Fe/S protein biogenesis, and to other connected processes including nuclear DNA replication and repair, telomere maintenance, and transcription. We next describe how the efficiency of mitochondria to assemble Fe/S proteins is used to regulate cellular iron homeostasis. Finally, we briefly summarize a number of mitochondrial "Fe/S diseases" in which various biogenesis components are functionally impaired owing to genetic mutations. The thorough understanding of the diverse biochemical disease phenotypes helps with testing the current working model for the molecular mechanism of Fe/S protein biogenesis and its connected processes.

Human CIA2A-FAM96A and CIA2B-FAM96B integrate iron homeostasis and maturation of different subsets of cytosolic-nuclear iron-sulfur proteins.

Numerous cytosolic and nuclear proteins involved in metabolism, DNA maintenance, protein translation, or iron homeostasis depend on iron-sulfur (Fe/S) cofactors, yet their assembly is poorly defined. Here, we identify and characterize human CIA2A (FAM96A), CIA2B (FAM96B), and CIA1 (CIAO1) as components of the cytosolic Fe/S protein assembly (CIA) machinery. CIA1 associates with either CIA2A or CIA2B and the CIA-targeting factor MMS19. The CIA2B-CIA1-MMS19 complex binds to and facilitates assembly of most cytosolic-nuclear Fe/S proteins. In contrast, CIA2A specifically matures iron regulatory protein 1 (IRP1), which is critical for cellular iron homeostasis. Surprisingly, a second layer of iron regulation involves the stabilization of IRP2 by CIA2A binding or upon depletion of CIA2B or MMS19, even though IRP2 lacks an Fe/S cluster. In summary, CIA2B-CIA1-MMS19 and CIA2A-CIA1 assist different branches of Fe/S protein assembly and intimately link this process to cellular iron regulation via IRP1 Fe/S cluster maturation and IRP2 stabilization.

Crucial function of vertebrate glutaredoxin 3 (PICOT) in iron homeostasis and hemoglobin maturation.

The mechanisms by which eukaryotic cells handle and distribute the essential micronutrient iron within the cytosol and other cellular compartments are only beginning to emerge. The yeast monothiol multidomain glutaredoxins (Grx) 3 and 4 are essential for both transcriptional iron regulation and intracellular iron distribution. Despite the fact that the mechanisms of iron metabolism differ drastically in fungi and higher eukaryotes, the glutaredoxins are conserved, yet their precise function in vertebrates has remained elusive. Here we demonstrate a crucial role of the vertebrate-specific monothiol multidomain Grx3 (PICOT) in cellular iron homeostasis. During zebrafish embryonic development, depletion of Grx3 severely impairs the maturation of hemoglobin, the major iron-consuming process. Silencing of human Grx3 expression in HeLa cells decreases the activities of several cytosolic Fe/S proteins, for example, iron-regulatory protein 1, a major component of posttranscriptional iron regulation. As a consequence, Grx3-depleted cells show decreased levels of ferritin and increased levels of transferrin receptor, features characteristic of cellular iron starvation. Apparently, Grx3-deficient cells are unable to efficiently use iron, despite unimpaired cellular iron uptake. These data suggest an evolutionarily conserved role of cytosolic monothiol multidomain glutaredoxins in cellular iron metabolism pathways, including the biogenesis of Fe/S proteins and hemoglobin maturation.

MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity.

Instability of the nuclear genome is a hallmark of cancer and aging. MMS19 protein has been linked to maintenance of genomic integrity, but the molecular basis of this connection is unknown. Here, we identify MMS19 as a member of the cytosolic iron-sulfur protein assembly (CIA) machinery. MMS19 functions as part of the CIA targeting complex that specifically interacts with and facilitates iron-sulfur cluster insertion into apoproteins involved in methionine biosynthesis, DNA replication, DNA repair, and telomere maintenance. MMS19 thus serves as an adapter between early-acting CIA components and a subset of cellular iron-sulfur proteins. The function of MMS19 in the maturation of crucial components of DNA metabolism may explain the sensitivity of MMS19 mutants to DNA damage and the presence of extended telomeres.

The role of mitochondria in cellular iron-sulfur protein biogenesis and iron metabolism.

Mitochondria play a key role in iron metabolism in that they synthesize heme, assemble iron-sulfur (Fe/S) proteins, and participate in cellular iron regulation. Here, we review the latter two topics and their intimate connection. The mitochondrial Fe/S cluster (ISC) assembly machinery consists of 17 proteins that operate in three major steps of the maturation process. First, the cysteine desulfurase complex Nfs1-Isd11 as the sulfur donor cooperates with ferredoxin-ferredoxin reductase acting as an electron transfer chain, and frataxin to synthesize an [2Fe-2S] cluster on the scaffold protein Isu1. Second, the cluster is released from Isu1 and transferred toward apoproteins with the help of a dedicated Hsp70 chaperone system and the glutaredoxin Grx5. Finally, various specialized ISC components assist in the generation of [4Fe-4S] clusters and cluster insertion into specific target apoproteins. Functional defects of the core ISC assembly machinery are signaled to cytosolic or nuclear iron regulatory systems resulting in increased cellular iron acquisition and mitochondrial iron accumulation. In fungi, regulation is achieved by iron-responsive transcription factors controlling the expression of genes involved in iron uptake and intracellular distribution. They are assisted by cytosolic multidomain glutaredoxins which use a bound Fe/S cluster as iron sensor and additionally perform an essential role in intracellular iron delivery to target metalloproteins. In mammalian cells, the iron regulatory proteins IRP1, an Fe/S protein, and IRP2 act in a post-transcriptional fashion to adjust the cellular needs for iron. Thus, Fe/S protein biogenesis and cellular iron metabolism are tightly linked to coordinate iron supply and utilization. This article is part of a Special Issue entitled: Cell Biology of Metals.