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Maturation of Mitochondrial [4Fe-4S]-Containing Proteins: History
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Contributor: Francesca Camponeschi , Simone Ciofi-Baffoni , Vito Calderone , Lucia Banci

The importance of mitochondria in mammalian cells is widely known. Several biochemical reactions and pathways take place within mitochondria: among them, there are those involving the biogenesis of the iron–sulfur (Fe-S) clusters. The latter are evolutionarily conserved, ubiquitous inorganic cofactors, performing a variety of functions, such as electron transport, enzymatic catalysis, DNA maintenance, and gene expression regulation. The synthesis and distribution of Fe-S clusters are strictly controlled cellular processes that involve several mitochondrial proteins that specifically interact each other to form a complex machinery (Iron Sulfur Cluster assembly machinery, ISC machinery hereafter). This machinery ensures the correct assembly of both [2Fe-2S] and [4Fe-4S] clusters and their insertion in the mitochondrial target proteins. 

  • iron–sulfur cluster
  • mitochondrial proteins
  • multiple mitochondrial dysfunction syndromes
  • rare diseases

1. Introduction

Mitochondria play pivotal roles in mammalian cells. A number of processes occur within mitochondria, including ATP production, metabolic pathways, such as citric acid and urea cycles, biosynthesis of amino acids, lipids and of essential protein cofactors such as heme, biotin, lipoic acid, molybdenum cofactor, and iron–sulfur (Fe-S) clusters [1][2]. The latter are evolutionarily conserved, ubiquitous inorganic cofactors, performing a multiplicity of functions, such as electron transport, enzymatic catalysis, DNA maintenance, and gene expression regulation [3][4][5]. In mammalian cells, the synthesis and distribution of Fe-S clusters is a tightly controlled process, performed in mitochondria by several proteins that specifically interact each other to form a complex machinery, the so-called Iron Sulfur Cluster assembly machinery (ISC machinery hereafter) [6][7][8][9]. The latter ensures the correct assembly and insertion of Fe-S clusters in mitochondrial target proteins.
The ISC machinery can be dissected into three main steps (Figure 1), the first of which is the de novo synthesis of a [2Fe-2S]2+ cluster on a multimeric protein complex, formed by the scaffold protein ISCU2, the cysteine desulfurase complex NFS1/ISD11/ACP1, and frataxin (FXN) [10][11][12][13]. The cluster is assembled from ferrous ions and inorganic sulfur. While the latter is provided by the cysteine desulfurase NFS1/ISD11/ACP1 complex that converts Cys into Ala [14][15], the entry mechanism of Fe2+ into the multimeric complex is still not clearly defined. Electrons are also required for the formation of the [2Fe-2S]2+ cluster and are provided by the mitochondrial ferredoxin (FDX2)/ferredoxin reductase (FDXR) system (Figure 1) [16][17][18]. The newly synthesized cluster is then released and transferred downstream of the ISCU2/NFS1/ISD11/ACP1 complex with the help of adaptors, such as HSPA9 and HSC20 [19], to the monothiol glutaredoxin GLRX5, for the following insertion into target [2Fe-2S]-binding proteins (Figure 1). An in vitro study recently showed that [2Fe-2S] ISCU2 can also transfer the cluster to apo GLRX5-BOLA3 complex [20] to form a heterodimeric [2Fe-2S] GLRX5-BOLA3 complex, which might have a secondary role in the following step of the machinery to assemble [4Fe-4S] clusters. Indeed, the main route of the ISC machinery involves the GLRX5-bound [2Fe-2S]2+ cluster that is used as a starting point to form a [4Fe-4S]2+ cluster. Specifically, the [2Fe-2S]2+ cluster bound to homodimeric GLRX5 is transferred to an accessory protein system able to assemble a [4Fe-4S]2+ cluster [21][22] and to insert it into recipient apoproteins (Figure 1). This accessory protein system forms the last step of the ISC machinery, involving several proteins (ISCA1, ISCA2, IBA57, FDX2, IND1, BOLA3, and NFU1) that work in parallel pathways, each maturing specific final target(s) (Figure 1) [6].
Figure 1. The three steps of the mitochondrial iron–sulfur cluster assembly machinery required to mature mitochondrial [4Fe-4S] target proteins. In the first step, a [2Fe-2S] cluster is assembled de novo on the scaffold protein ISCU2. The biosynthesis involves six additional ISC proteins including the cysteine desulfurase complex NFS1-ISD11-ACP1 as a sulfur donor, frataxin (FXN), and the electron (e−) transfer chain from NADPH via ferredoxin reductase (FDXR) to ferredoxin FDX2. Only one half of the symmetric core ISC complex is depicted. In the second step, a dedicated chaperone system (HSPA9, HSC20, and GRPLE1/2) facilitates the transfer of the [2Fe-2S] cluster from the ISCU2 scaffold to the monothiol glutaredoxin GLRX5, which binds the cluster in glutathione (GS)-dependent fashion. A secondary route (indicated by dashed arrows) involves BOLA3, which forms an apo complex with GLRX5 able to receive a [2Fe-2S] cluster from ISCU2 to form the homodimeric [2Fe-2S] GLRX5 complex; or, alternatively, the latter complex can be formed via the interaction of BOLA3 with the homodimeric [2Fe-2S] GLRX5 complex. The third step involves [4Fe-4S] cluster synthesis and apoprotein insertion. GLRX5 delivers its [2Fe-2S] cluster to three late-acting ISC proteins (ISCA1, ISCA2, and IBA57) for [4Fe-4S] cluster biosynthesis, which additionally requires the ferredoxin FDX2 electron transfer chain. Subsequently, the newly formed [4Fe-4S] cluster is delivered to recipient apoproteins by dedicated Fe-S-targeting proteins (e.g., NFU1, IND1 directly binding the [4Fe-4S] cluster) via parallel pathways. The major role of BOLA3 protein is in lipoyl synthase (LIAS) maturation. BOLA3 might be involved in such function via an alternative pathway, which consists on the [2Fe-2S] cluster donation from [2Fe-2S] GLRX5-BOLA3 complex to apo NFU1 to assemble a [4Fe-4S] cluster on NFU1 thanks to the delivery of two electrons, whose physiological source needs, however, to be identified.
The correct functioning of the ISC machinery is of vital importance for the correct activity of mitochondria. Indeed, mutations in the genes encoding for the components of the ISC machinery cause rare but severe diseases in humans [23][24]. These include Friedreich’s ataxia (FRDA), a neurodegenerative disease caused by mutations in frataxin (FXN), myopathies caused by mutations in ISCU2 and FDX2, a rare form of sideroblastic anemia caused by mutation in the GLRX5 gene, a form of encephalomyopathy caused by dysfunction of the respiratory chain complex I as a consequence of mutation in the gene encoding for IND1, and multiple mitochondrial dysfunction syndromes (MMDSs) caused by mutations in the genes encoding for the proteins acting in the last step of the ISC machinery (see later).

2. [4Fe-4S] Cluster Assembly in Mitochondria

The mechanism responsible for the maturation of mitochondrial [4Fe-4S] proteins is not yet fully defined. Indeed, both transient protein–protein interactions and stable protein complexes involved in such processes are still only partially characterized at the molecular level, as well as the picture of the operative protein–protein interaction network is still argument of debate in the literature, where two main models have been proposed. One model proposed that NFU1 operates with ISCU2 and ISCA1 proteins to assemble a [4Fe-4S]2+ cluster [25]. Specifically, the latter proteins provide a [2Fe-2S]2+ cluster each to NFU1 forming a ISCU2-ISCA1-NFU1 complex; this ternary complex receives two electrons from FDX2 to assemble a [4Fe-4S]2+ cluster on dimeric NFU1 [25]. The [4Fe-4S]2+ cluster assembled on NFU1 was proposed to be then transferred to apo recipient proteins [25]. However, at the moment, the most accredited model, based on experimental data collected by different research groups, supports a different protein–protein interaction network, described hereafter. This model has been here considered in the description of the effects that pathogenic mutations have on the maturation of mitochondrial [4Fe-4S] proteins.
ISCA1 and ISCA2 preferentially form a stable hetero-dimeric complex responsible for assembling a [4Fe-4S] cluster [21][26]. The latter is assembled through a reductive coupling of two [2Fe-2S] clusters that are donated to the apo ISCA1-ISCA2 hetero-complex by homo-dimeric [2Fe-2S]2+ GLRX5 (Figure 1) [21][22][27]. The latter bridges a [2Fe-2S]2+ cluster coordinated by two glutathione molecules and a conserved cysteine per each subunit of the homodimer, and transiently interacts with ISCA1 and ISCA2 to transfer the two [2Fe-2S]2+ clusters cargo to the apo ISCA1-ISCA2 hetero-complex via sequential molecular events [21][22]. Electrons required to couple the two received [2Fe-2S]2+ clusters are donated by FDX2 (Figure 1) [27], but no information is yet available on which is the specific electron acceptor. IBA57 has been shown to be required to assemble the [4Fe-4S]2+ cluster on ISCA1-ISCA2 complex, but its specific molecular function is still not clarified. IBA57 was shown to form a hetero-dimeric complex with ISCA2, but not with ISCA1, via bridging a [2Fe-2S] cluster [28][29]. Considering that the ISCA2-IBA57 complex can stabilize both reduced [2Fe-2S]+ and oxidized [2Fe-2S]2+ bound clusters [28], a possibility is that the [2Fe-2S] IBA57-ISCA2 heterodimeric complex is the entry point of the electrons donated by FDX2, but this still needs experimental evidence. The assembled [4Fe-4S]2+ cluster can then be inserted into apo recipient proteins, such as aconitase, without the requirement of further accessory proteins [6][30][31] or can be transferred to NFU1 or IND1 that then mediate the [4Fe-4S]2+ cluster insertion specifically into recipient proteins such as complex I, complex II and lipoyl synthase (LIAS) (Figure 1) [6][30][31]. While the molecular mechanism involving IND1 in the insertion of the [4Fe-4S]2+ cluster into complex I is not yet characterized, the insertion of the [4Fe-4S]2+ cluster into LIAS has been deeply investigated. Sequential protein–protein interactions involving ISCA1 and NFU1 have been shown to be responsible for the insertion of the [4Fe-4S] cluster into LIAS [32][33]. LIAS is a member of the radical S-adenosylmethionine (SAM) superfamily [34][35]. It catalyzes the final step of the biosynthesis of lipoyl cofactor [36][37][38] and binds two [4Fe-4S] clusters [39][40]: a [4Fe-4S] cluster (FeSRS), typical of all radical SAM enzymes, and a [4Fe-4S] cluster (FeSaux) that provides two sulfur atoms to the lipoyl cofactor [41][42]. It has been shown that the C-domain of NFU1 drives first [4Fe-4S]2+ cluster delivery from the ISCA1-ISCA2 complex, where the [4Fe-4S]2+ cluster is assembled, to a [4Fe-4S]2+ ISCA1-NFU1 intermediate complex, which then specifically directs the cluster into the FeSRS site of LIAS [32][33]. According to this molecular function, NFU1 has been shown to form two hetero-complexes with ISCA1 and LIAS [32][33]. These two complexes have been recently characterized at a molecular level, and it has been shown that, in both cases, only the C-domain of NFU1 is involved in the protein–protein interaction [32][33]. This domain therefore drives the assembled [4Fe-4S]2+ cluster from the ISCA1-ISCA2 complex to the final destination. BOLA3 has been implicated in the latter transfer step to LIAS (Figure 1) on the basis of clinical phenotypes of patients with pathogenic variants in the BOLA3 gene similar to those of patients with pathogenic variants in the NFU1 gene [43]. However, how BOLA3 contributes to this process is yet not defined. Indeed, in vitro studies showed that NFU1 and BOLA3 do not interact with each other in various experimental conditions, i.e., BOLA3 with either apo or [4Fe-4S]2+ NFU1 [20][44]. The only well-defined partner of BOLA3 is GLRX5, which operates upstream in the [4Fe-4S] cluster maturation pathway as a [2Fe-2S] cluster donor. GLRX5 forms an apo hetero-dimeric complex with BOLA3 [45][46]. This apo complex is able to bridge a [2Fe-2S]2+ cluster between the two proteins being coordinated, on the GLRX5 side, by the conserved Cys67 and by the cysteine of a GLRX5-bound glutathione (GSH) molecule, and on the BOLA3 side by the conserved Cys59 and His96. This holo-complex has been shown in vitro to function in [2Fe-2S] cluster trafficking in the mitochondrial iron–sulfur protein biogenesis. The [2Fe-2S]2+ BOLA3-GLRX5 complex was shown indeed to transfer the cluster to both apo human ferredoxins FDX1 and FDX2 with rate constants comparable to other cluster donors to FDX proteins [20], as well as to transfer its cluster to apo NFU1 to form a [4Fe-4S]2+ NFU1 dimer [44]. However, considering that the yeast homologue of human BOLA3 was shown not to be required for the maturation of mitochondrial [2Fe-2S] proteins [46], the cluster transfer to FDXs is very likely not physiologically relevant. On the other hand, cluster transfer and assembly from [2Fe-2S]2+ GLRX5-BOLA3 to NFU1 was proposed to be alternative to the pathway involving the [4Fe-4S] cluster transfer from the ISCA1-ISCA2 complex to NFU1, being exclusively activated under oxidative cellular conditions [44] (Figure 1).

3. Mutations on Components Maturing ISC Proteins Cause Severe Congenital Diseases

Mutations in GLRX5, which connects the first step of the ISC machinery to its last step, as well as mutations in the accessory proteins, which are involved in the late-acting step of the ISC machinery devoted to mitochondrial [4Fe-4S]-binding protein biogenesis, cause different Fe-S cluster-related diseases, such as sideroblastic anemia, muscle myopathy, multiple mitochondrial disfunction syndromes 1 to 5, and complex I deficiency [23][30][47][48][49]. All of the aforementioned diseases are associated with severe, often lethal outcomes due to defects in mitochondrial [4Fe-4S] proteins, documenting the importance of these late-acting accessory proteins for cell viability. Here, below, researchers report a description of all pathogenic mutations identified up to now in GLRX5 and in the late-acting accessory proteins, and researchers describe, from a structural point of view, the effects that these mutations can have on the mutated proteins and on impairing their protein–protein interaction networks.

3.1. Structural Aspects of Pathogenic Missense Mutations in GLRX5, a Protein Involved in a Rare Form of Congenital Sideroblastic Anemia

Mutations in GLXR5 gene have been associated so far with two different phenotypes, i.e., the variant nonketotic hyperglycinemia (NKH, MIM# 605899) [50] and the congenital sideroblastic anemia (SIDBA3, MIM#616860) [51]. NKH was associated with the non-sense p.K51del variant of GLRX5 [50]. On the contrary, missense mutations were found in patients affected by SIDBA3. Congenital sideroblastic anemia comprises a heterogeneous group of genetic disorders, characterized by reduced heme synthesis, mitochondrial iron overload and the presence of ringed sideroblasts [24]. In total, four pathogenic missense mutations (two pairs of heterologous pathogenic missense mutations) were identified in GLRX5 gene (Table 1). Two heterozygous missense mutations were found in the GLRX5 gene of a Chinese patient showing SIDBA3: Lys101Gln and Leu148Ser substitution in the GLRX5 protein [51]. Lys101 in GLRX5 protein is highly conserved from yeast to humans [51], whereas Leu148 is less conserved. Decreased holo IRP1 protein levels were observed in peripheral blood mononuclear cells (PBMCs), and, consequently, decreased activity of cytosolic aconitase was evident in PBMCs, together with increased expression of Transferrin receptor 1 (TfR1) protein and reduced expression of H-ferritin protein. Decreased ferrochelatase (FECH) level suggested mitochondrial Fe-S biogenesis impairment in PBMCs of the patient [51]. Daher et al. identified in a 14-year-old girl with SIDBA3, featuring heterozygous mutations in the GLRX5 gene (Cys69Tyr and Met128Lys, Table 1). Functional studies of these variants were not performed, but studies in patient lymphoblastoid cells showed decreased activity in several Fe-S containing enzymes, including mitochondrial respiratory chain complexes I and mitochondrial aconitase (mACO), and in heme-containing enzymes, such as respiratory chain Complex IV [52].
Table 1. Missense mutations of the late-acting ISC components with the associated mitochondrial disorders.
Gene/
Protein		 Missense Mutation Predicted Protein Mutations Associated Disease References
GLRX5 c.301A > C; c.443T > C p.Lys101Gln; p.Leu148Ser Nonsindromic sideroblastic anemia 3 [51]
c.200G > A; c.383T > A p.Cys67Tyr; p.Met128Lys Nonsindromic sideroblastic anemia 3 [52]
NFU1 c.545G > A; c.545G > A p.Arg182Gln; p.Arg182Gln MMDS1 [43]
c.622G > T; c.622G > T p.Gly208Cys; p.Gly208Cys MMDS1 [53][54]
c.565G > A; c.565G > A p.Gly189Arg; p.Gly189Arg MMDS1 [55]
c.179G > T; c.179G > T p.Phe60Cys; p.Phe60Cys MMDS1 [56]
c.545G > A; c.622G > T p.Arg182Gln; p.Gly208Cys MMDS1 [57]
c.544C > T; c.622G > T p.Arg182Trp; p.Gly208Cys MMDS1 [58]
c.629G > T; c.622G > T p.Cys210Phe; p.Gly208Cys MMDS1 [56]
c.565G > A; c.622G> T p.Gly189Arg; p.Gly208Cys MMDS1 [56][59]
c.565G > A; c.629G > T p.Gly189Arg; p.Cys210Phe MMDS1 [60]
c.565G > A; c.568G > A p.Gly189Arg; p.Gly190Arg MMDS1 [61]
c.62G > C; c.622G > T p.Arg21Pro; p.Gly208Cys MMDS1 [61]
c.299C > G; c.398T > C p.Ala100Gly; p.Leu133Pro MMDS1 [62]
c.721G > T; c.303_369del p.Val241Phe; ? MMDS1 [63]
BOLA3 c.200T > A; c.200T > A p.Ile67Asn; p.Ile67Asn MMDS2 [64][65]
c.287A > G; c.287A > G p.His96Arg; p.His96Arg MMDS2 [66][67][68]
c.295C > T; c.295C > T p.Arg99Trp; p.Arg99Trp MMDS2 [69]
c.176G > A; c.136C > T p.Cys59Tyr; p.Arg46 *a MMDS2 [70]
IBA57 c.706C > T; c.706C > T p.Pro236Ser; p.Pro236Ser MMDS3 [71]
c.941A > C; c.941A > C p.Gln314Pro; p.Gln314Pro MMDS3 [72]
c.286T > C; c.188G > A p.Tyr96His; p.Gly63Asp MMDS3 [73][74]
c.316A > G; c.286T > C p.Thr106Ala; p.Tyr96His MMDS3 [73][74]
c.738C > G; c.316A > G p.Asn246Lys; p.Thr106Ala MMDS3 [56]
c.757G > C; c.316A > G p.Val253Leu; p.Thr106Ala MMDS3 [56]
c.335T > G; p.437G > C p.Leu112Trp; p.Arg146Pro MMDS3 [56]
c.335T > C; c.588dup p.Leu112Ser; p.Arg197Alafs MMDS3 [75]
c.386A > T; c.731A > C p.Asp129Val; p.Glu244Ala MMDS3 [76]
c.436C > T; c.436C > T p.Arg146Trp; p.Arg146Trp MMDS3 [77]
c.586T > G;c.686C > T p.Trp196Gly; p.Pro229Leu MMDS3 [71]
c.656 > G; c.706C > T p.Tyr219Cys; p.Pro236Ser MMDS3 [69]
c.701A > G; c.782T > C p.Asp234Gly; p.Ile261Thr MMDS3 [73][74]
c.738C > G; c.802C > T p.Asn246Lys; p.Arg268Cys MMDS3 [69]
c.286T > C; c.754G > T p.Tyr96His; p.Gly252Cys MMDS3 [73][74]
c.323A > C; c.150C > A p.Tyr108Ser; pCys50 *a MMDS3 [76]
c.87insGCCCAAGGTGC; c.313C > T p.Arg30Alafs; p.Arg105Trp MMDS3 [71]
c.236C > T; c.307C > T p.Pro79Leu; p.Gln103 *a MMDS3 [74]
c.580A > G; c.286T > C p.Met194Val; p.Tyr96His MMDS3 [78]
ISCA2 c.154C > T; c.154C > T p.Leu52Phe; p.Leu52Phe MMDS4 [56]
c.313A > G; c.313A > G p.Arg105Gly; p.Arg105Gly MMDS4 [56]
c.G229 > A; c.G229 > A p.Gly77Ser; p.Gly77Ser MMDS4 [79][80][81]
c.355G > A; c.355G > A p.Ala119Thr; p.Ala119Thr MMDS4 [82]
c.5C > A; c.413C > G p.Ala2Asp; p.Pro138Arg MMDS4 [83]
c.295delT; c.334A > G p.Phe99Leufs*18; b p.Ser112Gly   [84]
ISCA1 c.259G > A; c.259G > A p.Glu87Lys; p.Glu87Lys MMDS5 [85][86]
c.29T > G; c.29T > G p.Val10Gly; p.Val10Gly MMDS5 [87]
c.302A > G; c.302A > G p.Tyr101Cys; p.Tyr101Cys MMDS5 [88]
IND1 c.815-27T > C; c.G166 > A p.Asp273Glnfs*31; b p.Gly56Arg Complex I deficiency [89][90][91]
c.313G > T; c.166G > A; c.815-27T > C p.Asp105Tyr; p.Gly56Arg; p.Asp273Glnfs*31 b Complex I deficiency [89][90][91]
c.579A > C; c.G166 > A p.Leu193Phe; p.Gly56Arg Complex I deficiency [89][90][91]
c.311T > C; c.815-27T > C p.Leu104Pro; p.Asp273Glnfs*31 b Complex I deficiency [92]
c.815-27T > C; c.545T > C p.Val182Ala; p.Val182Ala Complex I deficiency [92]
FDX2 c.1A > T; c.1A > T p.Met1Leu; p.Met1Leu MEOAL [93]
c.431C > T; c.431C > T p.Pro144Leu; p.Pro144Leu MEOAL [94]
a The “*” symbol indicates a nonsense mutation; b frameshift mutation
In order to analyze the effects of the pathogenic missense mutations from a structural point of view, two GLRX5 PDB entries (2WUL and 2MMZ) have been considered: the first is the crystallographic structure of the holo form, i.e., the protein in a homo-dimeric state in complex with a [2Fe-2S]2+ cluster and two glutathione molecules [95], while the second is a solution NMR structure of the monomeric apo protein [22]. The four identified missense pathogenic mutations were here mapped on the dimeric [2Fe-2S]2+ GLRX5 structure (Figure 2A). The two pathogenic mutations Lys101Gln and Cys67Tyr appears to have a major structural effect: the first one involves the disruption of the electrostatic interaction between the carboxylate group of glutathione and the ε-amino group of Lys101 sidechain in both [2Fe-2S] homo-dimeric GLRX5 and hetero-dimeric GLRX5-BOLA3 complexes (Figure 2 and Figure 4B). The second one is even more critical since it affects the coordination of the [2Fe-2S] cluster to GLRX5, as Cys67 is involved in iron coordination in both [2Fe-2S] homo-dimeric GLRX5 and hetero-dimeric GLRX5-BOLA3 complexes (Figure 2A,B and Figure 4B). The Met128Lys mutation involves a methionine that points to the interior in both apo and holo structures establishing hydrophobic contacts with nearby hydrophobic residues (Figure 2C). Thus, it is likely that the introduction of a charged Lys residue significantly impacts on these hydrophobic interaction patterns, thus destabilizing the structure of GLRX5. Mutation Leu148Ser does not provide clear structural evidence for an impairment of the protein function: it is located at the C-terminus in a mobile region with very poor electron density in 2WUL and distant from GSH and the cluster binding site (Figure 2A); likewise, the mutation is situated in a very mobile region also in the apo GLRX5 structure.
Figure 2. Pathogenic missense mutations mapped on the crystallographic structure of human GLRX5. (A) The backbone of the residues involved in pathogenic missense mutations of GLRX5 is shown in red on the ribbon structure of homodimeric [2Fe-2S] GLRX5 (PDB ID 2WUL). The sidechain of the conserved cluster coordinating Cys67 residue is shown in yellow. The iron and sulfur atoms of the [2Fe-2S] cluster are in red and yellow spheres, respectively; and glutathione (GS) ligand is shown in blue. (B) The electrostatic interaction established between the sidechain of Lys101 (in magenta) and the carboxylate group of the glutathione cluster ligand, shown in ball and stick mode, is shown as a dotted line. (C) The sidechains of the hydrophobic residues (in blue) interacting with the sidechain of Met128 (in magenta) are shown.
The pathogenicity of both pairs of mutations might therefore arise from a reduced [2Fe-2S] cluster chaperone activity of GLRX5 in mitochondria, having severe consequences on the efficiency of the ISC machinery and on the maturation of mitochondrial and extra-mitochondrial Fe-S proteins, the latter requiring both the mitochondrial and the cytosolic Fe-S cluster assembly machineries [7]. GLRX5 deficiency has been also linked to the impairment of heme biosynthesis and to the depletion of cytosolic iron [96], this possibly explaining the effects observed on Complex IV in the presence of pathogenic GLRX5 mutations causing SA [51][52].

3.2. Pathogenic Missense Mutations in the Late Acting Accessory Proteins of the ISC Machinery

Multiple Mitochondrial Dysfunction Syndromes (MMDS) types 1 to 5 are a group of rare but severe autosomal recessive diseases, caused by variants in the genes encoding for NFU1, BOLA3, IBA57, ISCA2 and ISCA1 proteins, respectively. The hallmark of these diseases is a decreased energy metabolism, which results in defects in neurologic development, muscle weakness, lactic acidosis, respiratory failure. Generally, the pathology manifests in early infancy and results in early death [23]. All of the MMDSs 1–5 have the main impact on enzymes whose functions rely on the presence of bound [4Fe-4S] clusters, such as LIAS and respiratory complexes I and II.
In addition to MMDS 1 to 5, two other genetic diseases are linked to genes of the late-acting step of the ISC machinery. The first is called Complex I deficiency, and is associated with mutations in the mitochondrial P-loop NTPase IND1, that is directly responsible for the maturation of the [4Fe-4S] cluster-containing subunits of respiratory chain complex I. A second is called Episodic mitochondrial myopathy with or without optic atrophy and reversible leukoencephalopathy (MEOAL), which has been linked to deficiency of FDX2. FDX2 is a protein that binds a [2Fe-2S] cluster that provides electrons to assemble the [4Fe-4S] cluster and is thus responsible for the maturation of all mitochondrial [4Fe-4S] target proteins.

3.2.1. Structural Aspects of Pathogenic Missense Mutations of NFU1 Involved in MMDS1

Bi-allelic pathogenic variants in the NFU1 gene cause MMDS type 1 (MMDS1, MIM#605711), clinically characterized by severe encephalopathy that manifests in the first year of life, with evidences of decreased energetic metabolism. Eleven missense (Table 1) and six non-sense disease-causing variants in NFU1 have been identified to date in patients affected by MMDS1. The metabolic profiles of these patients are characterized by a decreased activity of several mitochondrial proteins, such as pyruvate dehydrogenase (PDH), Glycine Cleavage System (GCS) and respiratory chain complexes. PDH and GCS decreased activity was due to lipoic acid synthesis impairment.
The structural characterization of apo NFU1 showed that the apo form is monomeric in solution and adopts a dumbbell-shaped structure with well-structured N- and C-domains connected by a linker [96]. It has been also shown that NFU1 binds a [4Fe-4S]2+ cluster, which induces the formation of a homo-dimer through the bridging [4Fe-4S]2+ cluster, coordinated by two cysteines of the conserved CXXC motif of each of the two C-domains [44]. Moreover, the presence of an equilibrium between the [4Fe-4S]2+ NFU1 dimer with the cluster coordinated by the Cys residues of the two CXXC motifs, and a [4Fe-4S]2+ NFU1 dimer where a Cys ligand of the CXXC motif is replaced by a S-donor small molecule ligand, such as GSH or DTT, was observed [44]. In order to analyze the effects of the pathogenic missense mutations on the protein structure, researchers have considered the solution NMR structures of the single N- and C-domains of NFU1 (2LTM and 2M5O, respectively), since no structure of the full-length protein is yet available. The missense pathogenic mutations are mainly located on the C-domain of NFU1 in the surrounding of the cluster binding CXXC motif (7 out of 11 mutations, Figure 3A).
Figure 3. Pathogenic missense mutations mapped on the solution structures of the isolated N- and C-terminal domains of human NFU1 and on a structural docking model describing their interaction. (A–C) The backbone of the residues involved in pathogenic missense mutations in the C-terminal (A) and N-terminal (C) domains of NFU1 is shown in red on the ribbon structures of these domains (PDB ID 2LTM and 2M5O, respectively). The sidechains of the two conserved cluster coordinating Cys residues on the C-terminal domain of NFU1 are shown in yellow. (B) The electrostatic interactions established by sidechain of Arg182 (magenta) and the sidechains of the surrounding Asp residues (in orange) are shown as dotted black lines. (D) Docking structural model of the interaction between the N-terminal and C-terminal domains of NFU1 is shown. Ala100 of the N-terminal domain (in red) is close to Arg101 and Arg 105 (both in blue), which are in turn involved in electrostatic interactions with Glu and Asp residues of the C-terminal domain (all in orange). Interacting hydrophobic patches between the two domains (Van der Waals surfaces are in blue and red for the C- and N-domains, respectively), which involve both aromatic and aliphatic residues (sidechains are in green) and are close in space to Ala100 (sidechain is in red), are also shown.
The c.545G > A (p.Arg182Gln) pathogenic missense mutation was found in two cases. One is in homozygosity, where no mature protein could be detected in fibroblast mitochondria as a consequence of defective mRNA splicing caused by this mutation [43]. It was suggested that the mutant protein might undergo degradation [43]. Another patient carried both the c.622G > T (p.Gly208Cys) and the c.545G > A (p.Arg182Gln) heterozygous mutations in NFU1 gene. Metabolic findings were high blood lactate and urine glutaric acid levels, but no information about the impairing of any mitochondrial Fe-S protein is available [57]. Arg182 is also involved in another pathogenic missense mutation, i.e., c.544C > T (p.Arg182Trp). This mutation is found in heterozygosity with the Gly208Cys missense mutation [61]. It was shown that in the processed mRNA, only the c.544C > T gene mutation was detectable in the patient’s fibroblasts, suggesting that the protein can also be detected in fibroblast mitochondria at variance with the c.545G > A missense mutation [61]. Both Arg182 mutations appear to be critical from the chemical point of view since they involve the disappearance of a charged residue. Two Asp residues are located nearby to Arg182, and this charge loss in the pathogenic mutants can thus perturb these electrostatic interactions, destabilizing the overall structure as well as protein–protein recognition (Figure 3B). Arg182 is solvent exposed and is located on the helix of the C-domain of NFU1 involved in the complex formation with both ISCA1 and LIAS [32][33]. Its mutation into Gln or Trp might negatively affect the protein–protein recognition between the C-domain of NFU1 and both ISCA1 and LIAS and the [4Fe-4S] cluster transfer to LIAS. Thus, the two pathogenic mutations, destabilizing the protein complex, might determine a significant decrease in the cellular content of mature LIAS, and consequently a defective lipoylation of the acid-dependent 2-oxoacid dehydrogenases and of the glycine cleavage system. This model is confirmed by the high deficiency of those enzymes requiring lipoate in patients carrying mutations on Arg182 [43][57][61].
Gly208Cys was found both in homozygous and heterozygous states. This variant was specifically linked to pulmonary arterial hypertension [53]. The mutation modifies the native Fe-S binding CXXC pattern of NFU1 into CXCXXC. The mutation does not significantly impact on the protein stability and leads to a slight decrease in alpha helix and beta sheet content with a corresponding increase in the random coil part [97]. Differential scanning calorimetry and analytical ultracentrifugation experiments also suggested an increased tendency of the protein to oligomerize, specifically to the dimeric form. From in vitro cluster transfer experiments, it was argued that Gly208Cys NFU1 is not able to receive a cluster in vivo and would therefore be unable to transfer a cluster to downstream partners [97].
Another missense pathogenic mutation found in the C-domain is Gly189Arg in homozygosis, which is the second most frequent variant (5 patients). It has been suggested that this variant is associated with a milder progressive disease [61], as, out of the five patients, one survived till the age of 41 months [55], and another one is still alive in its adulthood [98]. The latter is carrying a second heterozygous variant (Cys210Phe) and showed an atypical clinical phenotype, with a later onset of the first neurological manifestation of the disease and partial recovery. The patient showed decreased activity of PDH in the fibroblasts. The Gly189Arg mutation is located close to the cluster binding site and thus the introduction of a charged and bulky residue might drastically compromise [4Fe-4S] cluster binding. This mutation has been characterized in a quite recent paper [99]; it has been shown that the mutation induces structural changes that increase flexibility, decrease stability, and alter the monomer–dimer equilibrium toward the monomeric form, thus impairing the ability of the Gly189Arg moieties to receive the cluster from physiologically relevant partners.
Next to the Gly189Arg mutation, there is one of the same nature, i.e., Gly190Arg, that is in heterozygosity with Gly189Arg [61]. Both amino acids are highly conserved, and their mutation was predicted to have a major impact on protein function by several programs (PolyPhen2, SIFT, MutationTaster) [61]. As in the case of Gly189Arg, this mutation implies the non-native presence of a charged and bulky residue, potentially negatively affecting [4Fe-4S] cluster binding ability.
Cys210Phe was found in heterozygosity with i) Gly208Cys mutation [56], with the patient showing the same clinical features as those usually found in patients with the Gly208Cys mutation only, and moderately reduced activity of respiratory chain complex IV, and ii) Gly189Arg [60]. In the latter case, Western blot analysis performed on patient’s fibroblasts showed no reduction in the NFU1 level, but a partial reduction of that of the subunits of complex II, SDHA (70 kDa), and SDHB (30 kDa), whereas the levels of the subunits of the other respiratory chain complexes are not affected [60]. Cys210 is one coordinating ligand of the [4Fe-4S]2+ cluster in the NFU1 homodimer [44][96]. Therefore, this pathogenic mutation abolishes the ability of NFU1 to bind the [4Fe-4S]2+ cluster.
The missense pathogenic mutation Val241Phe was found in heterozygosis with exon 4 deletion of the NFU1 gene. Val241 is located close to the C-terminus of the C-domain of NFU1 at the end of the last β-strand and might therefore play a role in the C-domain structural conformation and stability [63] as a consequence of the introduction of a bulkier aromatic residue with respect to the original one.
There are only four pathogenic missense mutations in the N-domain of NFU1. At variance with the C-domain, the functional role of the N-domain of NFU1 in the maturation of mitochondrial [4Fe-4S] proteins is still debated, as it does not directly contribute to the interactions with LIAS and ISCA1. The structural effect of these mutations are mapped and analyzed on the solution NMR structure of the single N-domain of NFU1 (Figure 3C).
Arg21Pro is in heterozygosity with Gly208Cys. Biochemical analysis showed clear complex II deficiency, and decreased activity of complex I in liver tissues [61]. Phe60Cys was found in homozygosis in a patient showing clinical features typical of MMSD1, but normal α-ketoglutarate dehydrogenase (α-KGDHc) activity and protein lipoylation [56]. Arg21 and Phe60 are located at the N-terminal unstructured segment of NFU1, which constitutes the mitochondrial targeting sequence composed by the first 58 N-terminal residues and, once processed by mitochondrial processing peptidases, produces the mature form of NFU1 with a molecular mass of ~22 kDa [100]. Researchers can predict that these pathogenic mutations do not affect the structural feature of NFU1 as well as its functional interaction network, but rather they might be crucial in the import in the mitochondrial matrix.
Ala100Gly was found in heterozygosity with the Leu133Pro missense mutation. No biochemical analysis has been reported. Both residues are located in the core of the domain and form hydrophobic interactions with nearby non-polar residues. In this respect, the Ala-to-Gly mutation should have a minor impact on the local folding properties. However, this mutation might be critical in the interaction between the N- and C-domains of NFU1. Indeed, Ala100 is located in a region involved in the interaction between the two domains [44]. A docking structural model of the two domains [44] shows that they interact through a hydrophobic patch and a charged patch, and therefore, the Ala100Gly mutation might negatively affect this inter-domain interaction in such a way to impair the functional role of the N-domain (Figure 3D). On the contrary, the Leu133Pro missense mutation could be critical for the structural stability of the N-domain since the presence of a Pro can break the α-helix where Leu 133 is located.

This entry is adapted from the peer-reviewed paper 10.3390/biom12071009

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