• Friedreich's ataxia;
  • iron metabolism;
  • mitochondria;
  • oxidative stress


  1. Top of page
  2. Abstract
  3. Structural and biophysical studies on frataxin
  4. Frataxin function
  5. Acknowledgements
  6. Conflicts of interest
  7. References

Reduced levels of the protein frataxin cause the neurodegenerative disease Friedreich's ataxia. Pathology is associated with disruption of iron–sulfur cluster biosynthesis, mitochondrial iron overload, and oxidative stress. Frataxin is a highly conserved iron-binding protein present in most organisms. Despite the intense interest generated since the determination of its pathology, identification of the cellular function of frataxin has so far remained elusive. In this review, we revisit the most significant milestones that have led us to our current understanding of frataxin and its functions. The picture that emerges is that frataxin is a crucial element of one of the most essential cellular machines specialized in iron-sulfur cluster biogenesis. Future developments, therefore, can be expected from further advancements in our comprehension of this machine.

Abbreviations used

Friedreich's ataxia


mitochondrial processing peptidase


root mean square deviations


X-ray absorption spectroscopy

Frataxin is a small protein which owes its ‘notoriety’ to its link to the neurodegenerative disease Friedreich's ataxia (FRDA) (Campuzano et al. 1996). This pathology is caused by an abnormal expansion of a non-coding GAA triplet repeat in the first intron of the FRDA gene, leading to lower expression levels of frataxin through heterochromatization of the locus (Campuzano et al. 1997). In healthy individuals, the number of repeats range from 6–36, whereas in FRDA patients, the number of repeats is in the 70–1700 range, most commonly 600–900 (Pandolfo 2009). The severity of the disease and the age at onset inversely correlate with the number of repeats. Nothing besides the primary sequence was known about frataxin when the FRDA gene was first linked to FRDA (Campuzano et al. 1996). The pace of advancement in the field of FRDA has been rapid. Sixteen years later, we have accumulated a fair amount of knowledge about the protein localization, its cellular forms and its interactome, even though we seem to be still far from a complete understanding of the frataxin cellular function. In this review, we shall highlight the most important steps that have led us to our current knowledge and discuss possible future developments. Here, we have underlined some of the main contributions to the investigation of frataxin's function. We apologize in advance to the colleagues who we have not cited. Other recent reviews may complement the information provided here (Ye and Rouault 2010; Martelli et al. 2012; Vaubel and Isaya 2012).

Structural and biophysical studies on frataxin

  1. Top of page
  2. Abstract
  3. Structural and biophysical studies on frataxin
  4. Frataxin function
  5. Acknowledgements
  6. Conflicts of interest
  7. References

The frataxin sequence and its cellular localization

Frataxin is a small acidic protein (with isoelectric point on average around 4.9) highly conserved in most organisms from bacteria to mammalians (Gibson et al. 1996; Adinolfi et al. 2002) (Fig. 1a). A frataxin homolog was also identified in the human parasite Trichomonas vaginalis (Dolezal et al. 2007). This finding is interesting since these unicellular eukaryote organisms do not have mitochondria but hydrogenosomes which share common ancestry with mitochondria. Sequence alignment of the frataxin family shows two distinct regions. An N-terminal block of 70–90 residues is completely absent in prokaryotes and poorly conserved also among eukaryotes (Huynen et al. 2001). Its sequence has the features typical of intrinsically unfolded proteins. The C-terminus of the protein comprises a highly conserved block of ca. 100–120 amino acids that is conserved in most organisms. The sequence identity of this region is as high as ~ 25%, whereas the similarity is 40–70%, indicating that this is the functionally important part of the molecule. Among the most conserved residues are three tryptophans that are residues with a relatively low occurrence in proteins. Their conservation suggests that they could have an important structural and/or functional role. Semi-conserved is a stretch of negatively charged residues.


Figure 1. Frataxin sequence and structure. (a) Sequence alignment of bacterial, yeast, and human frataxin. (b) Four representative structures taken from the 19 entries present in PDB. Red: X-ray structure of E. coli CyaY (1ew4), blue: X-ray structure of human frataxin (1ekg), yellow: NMR structure of yeast Yfh1 (2ga5), green: X-ray structure of Yfh1 (3oeq). The differences between the NMR and X-ray structures of Yfh1 are almost certainly because of the appreciable lower accuracy of the NMR structure.

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Clues on the cellular localization of frataxin were provided already from the sequence alignment of the family (Gibson et al. 1996). The distribution of the FRDA gene in different genomes, and some clinical similarities with another ataxia linked to vitamin E deficiency and with neuropathies with mitochondrial DNA instability caused by mutations in nuclear genes, suggested that frataxin could be a protein imported to mitochondria. It was argued that the absence of the non-conserved N-terminus in most prokaryotes and the presence of homologs in purple bacterial genomes, but not in other bacteria, suggest a mitochondrial localization. This hypothesis was confirmed by tagging experiments which showed that human frataxin co-localizes with a mitochondrial protein (Koutnikova et al. 1997). It is now well accepted that frataxin is nuclearly encoded, expressed in the cytoplasm and imported in the mitochondrion through an import signal contained in the N-terminus. This explains the poorer conservation of this region.

Frataxin maturation in mitochondria

The mature form of the yeast frataxin ortholog, Yfh1, was established early on to start at residue 52 of the transcript (Branda et al. 1999). On the other hand, the exact nature of the human mature frataxin has been the subject of a long-standing controversy. Human frataxin is synthesized as a precursor of 210 amino acids imported to the mitochondrion, and undergoes maturation by the mitochondrial processing peptidase (MMP) through a two-step process that leads to the successive generation of an intermediate form of 19 kDa cleaved between G41 and L42 (residues 42–210) and a mature form of 14 kDa (residues 81–210) (Koutnikova et al. 1998; Condò et al. 2007; Schmucker et al. 2008). Another form starting at S56 was also reported (Cavadini et al. 2000), but it is now widely accepted that the 81–210 form is the most abundant species both in normal individuals and in FRDA patients (Condò et al. 2007; Schmucker et al. 2008; Gakh et al. 2010). The frataxin 81–210 mature form is fully functional for cell survival and is thus the most suitable species to investigate in further functional studies.

The 3D structure of frataxin

Recombinant frataxins from E. coli, S. cerevisiae and H. sapiens have been intensively studied structurally: there are 19 entries responding to the keyword frataxin in the PDB Database (Fig. 1b). The structure of the conserved C-terminal domain of the human protein was the first to be characterized both by NMR and crystallography, together with the crystal structure of the E. coli ortholog CyaY (Cho et al. 2000; Dhe-Paganon et al. 2000; Musco et al. 2000). The solution structures of bacterial frataxin and of Yfh1 in crystal and solution states were solved later (He et al. 2004; Nair et al. 2004; Karlberg et al. 2006). These structures superpose with root mean square deviations (RMSD) of 1.7–2.4 Å in agreement with the high degree of sequence conservation. The structure consists of two N- and C-terminal regions. The N-terminal eukaryotic tail of the human protein (residues 81–92) was shown to be intrinsically unfolded and highly flexible (Musco et al. 2000; Prischi et al. 2009). Interestingly, the equivalent region in Yfh1 seems more rigid. In the X-ray structure, the region is visible and involved in intermolecular interactions with other copies of the molecule (Karlberg et al. 2006). In the solution structure, it bends back in a conformation that is shared by all copies of the NMR bundle, whereas it should adopt different conformations in different models if it were flexible (He et al. 2004). The conserved C-terminal domain folds in a mixed αβ structure that consists of two helices packing against a contiguous anti-parallel beta-sheet assembled in the sequence alpha-(beta)5–7-alpha. When first solved, this fold was recognized as novel. Despite its simplicity, it remains almost unique. Only much later, the fold was also observed in the crystal structure of complex I of the Thermus thermophilus mitochondrial respiratory chain: the N3 domain of the protein Nqo15 has a fold similar to that observed in frataxin. The two proteins lack any detectable sequence homology (Sazanov and Hinchliffe 2006). Lack of conservation of residues known to be functionally important in frataxins suggests that, in this case, the fold similarity does not imply a conserved function.

Same fold but different stability

Despite having the same fold, bacterial, yeast, and human frataxins have very different thermodynamic stabilities. Yeast frataxin (Yfh1) is unusually unstable when studied at low ionic strength. The midpoint of the thermal unfolding of the protein (Tm) is ca. 35°C (Adinolfi et al. 2004). This protein also undergoes a low temperature transition giving rise to the phenomenon of cold denaturation at temperatures well above the water freezing point (5°C), making it a useful model system to study protein stability (Pastore et al. 2007; Adrover et al. 2010, 2012). For all three proteins, the Tms increase appreciably when salts are added. As protein stability is the net balance of different stabilizing and destabilizing contributions, it is not possible to pinpoint a single cause accounting for these very different stabilities. One of the factors that, nevertheless, influences protein stability is undoubtedly the length of the C-terminus that is variable in the three proteins. In human frataxin, this region folds back and inserts between the two helices, thus protecting the hydrophobic core. In E. coli CyaY, the C-terminus is shorter but has a similar structural role, whereas this additional region is absent in yeast Yfh1. This hypothesis was proven by artificially elongating the yeast protein with a tail spanning the human sequence and showing that this is sufficient to stabilize the fold (Adinolfi et al. 2004). Conversely, truncation of human frataxin resulted in destabilization.

Frataxin mutants

Knowledge of the frataxin structure allowed us to rationalize the role of residues conserved among species. These residues can suggest the regions of the protein that are important for structural stability and/or function. Conserved buried residues are usually important parts of the hydrophobic core: any variation might destabilize the fold. On the contrary, the conserved residues exposed on the protein surface are usually involved in protein function. Examples of this concept are the proximal histidine in globins or residues in the active sites of enzymes. In the frataxin family, the semi-conserved negatively charged residues and the other exposed conserved residues cluster on the first helix and on the β-sheet (Musco et al. 2000). Among these residues are a well-exposed and conserved aspartate (D31 in E. coli) and the exposed and completely conserved tryptophan (e.g., position 61 in E. coli and 155 in human frataxin). FRDA is usually caused by the presence of an expansion of GAA repeats in the non-coding regions of both alleles of the frataxinc gene which results in reduced levels of protein expression. However, a small number of FRDA patients (4%) have been found to be heterozygous for triplet expansion on one allele and a point mutation on frataxin on the other. At least 15 missense point mutations have been reported. However, there are a few prevalent mutations that result either in the classical FRDA phenotype (I154F and W155R) or in atypical clinical presentation (G130V and D122Y). Interestingly, all clinically important missense mutations observed in heterozygous FRDA patients affect conserved residues (Cossée et al. 1999; Labuda et al. 1999).

In vitro studies demonstrated that these mutants retain a native fold under physiological conditions but have reduced thermodynamic stabilities that follow, in this order, the trend: wild type > W155R > I154F > D122Y > G130V (Correia et al. 2006, 2008). Unfolding is reversible, which rules out the possibility of the formation of insoluble aggregates. The mutants also differ by protein dynamics, propensities toward aggregation and tendency toward proteolytic digestion. These results suggest that the development of FRDA in heterozygous patients may result from the combined effect of reduced protein folding efficiency and accelerated degradation in vivo, that in turn lead to lower frataxin concentrations. In addition, some of the mutations, including W155R and N146K, have been suggested to affect the binding capacity of frataxin for its protein partners (Musco et al. 2000; Leidgens et al. 2010; Schmucker et al. 2011).

Monomeric versus oligomeric structures

Another difference among frataxin orthologs concerns their tendency to form large assemblies. All three orthologs studied so far are highly soluble monomeric proteins in the absence of iron. When iron excesses are added (e.g., 1 : 20) under aerobic conditions, oligomers are observed both for CyaY and Yfh1 (Cook et al. 2006, 2010; Adinolfi et al. 2009). The structures of the Yfh1 oligomers have been thoroughly characterized by Isaya and co-workers (Adamec et al. 2000; Gakh et al. 2002). Oligomerization proceeds through formation of trimers, hexamers, 12-mers, 24-mers up to the final 48-mers with the trimer representing the basic unit. The final spheroidal structures can store approximately 50–75 iron atoms per subunit in 1–2 nm cores, which are structurally similar to ferrihydrite. This is the main iron form found in vertebrate ferritins (Nichol et al. 2003; Park et al. 2003). Yfh1 oligomers disassemble into monomers upon reduction in the ferric iron cores (Park et al. 2003) indicating the importance of iron in holding these structures together. Further characterization of the oligomer structure was made possible by the identification of mutations in the mature form of Yfh1 that would enable the yeast frataxin monomer to oligomerize in an iron-independent manner (O'Neill et al. 2005; Gakh et al. 2006). Mutations in two regions (the N-terminus and strands β2–β3) resulted in oligomers when the yeast protein was expressed in E. coli, yielding trimers and lower quantities of larger oligomers. The trimer and a 24 subunit oligomer formed by one of these proteins (Y73A) were extensively characterized by X-ray crystallography and small angle X-ray scattering (SAXS) studies (Söderberg et al. 2011). E. coli CyaY forms iron-promoted aggregates but only under aerobic conditions, non-physiological ionic strengths and large excesses of iron (Adinolfi et al. 2002; Layer et al. 2006). Iron-induced oligomerization of Yfh1 is dispensable in S. cerevisiae since an oligomerization-deficient form of Yfh1, which is unable to store iron in vitro, can fully rescue the ∆Yfh1 yeast under normal conditions (Aloria et al. 2004). However, mutation enhances the sensitivity of yeast cells to oxidative stress and causes lethality when combined with lack of copper-zinc superoxide dismutase (Gakh et al. 2006). A trimeric form of Yfh1 seems to be the predominant species present in wild-type cells, but larger oligomers form when Yfh1 is overproduced (Seguin et al. 2009). Yfh1 oligomerization in vivo seems to be associated with stress conditions such as over-expression of wild-type Yfh1 monomer, mitochondrial iron uptake, mutations that stabilize the Yfh1 trimer, or heat stress (Gakh et al. 2008). Yfh1 is otherwise monomeric, suggesting that this is the form that is functionally important. Unlike CyaY and Yfh1, human frataxin assembles only under extreme conditions and in an iron-independent manner (O'Neill et al. 2005). Essential for oligomerization of this ortholog are residues in the non-conserved N-terminus of the protein. Furthermore, the mature form of human frataxin (81–210), which has been shown not to oligomerize, rescues cellular viability of murine fibroblasts deleted for frataxin (Schmucker et al. 2008), indicating that the formation of oligomers is not a requisite for frataxin to be functional.

Frataxins are iron-binding proteins

Isaya and co-workers were the first to suggest that Yfh1 is able to trap iron in spheroidal oligomeric structures similar to that observed in ferritins (Adamec et al. 2000). However, it was initially unclear whether trapping was of colloidal nature or a specific and stoichiometric interaction. It was thanks to studies of protein stability that it became clear that frataxins bind iron directly. Comparison of the stabilities of CyaY, Yfh1, and human frataxin and screening of conditions that might stabilize or destabilize these proteins showed that any increase in ionic strength has a profound effect on protein stabilization (Adinolfi et al. 2004). The effect is comprehensibly more evident for the inherently less stable yeast frataxin where it can be as large as an increase of 10–15°C of Tm. Both Fe2+ and Fe3+ bind as well as other divalent cations. Phosphate too has a strong effect. CyaY and Yhf1 were shown to bind two Fe2+ ions with comparable dissociation constants (Kd) (Bou-Abdallah et al. 2004; Cook et al. 2006). Monomeric CyaY binds six Fe3+ ions. Additional weaker binding sites allow further loading of up to 25–26 cations per monomer (Bou-Abdallah et al. 2004). Different constructs of human frataxin were reported to bind 6–7 Fe2+ or Fe3+per monomer, but with largely discrepant dissociation constants (Yoon and Cowan 2003; Yoon et al. 2007; Huang et al. 2008).

The binding surface was identified by NMR which indicated that iron binding does not require aggregation. The primary binding site involves the semi-conserved negatively charged ridge previously identified and is centered on residues 18, 19, and 22 of CyaY and the corresponding residues in human and yeast frataxins. X-ray absorption spectroscopy (XAS) together with extended X-ray absorption fine structure (EXAFS ) data also suggested that the monomeric protein binds high-spin state Fe2+ coordinated in a highly centrosymmetric metal-ligand with a six-coordination geometry (Cook et al. 2006). Binding is thus prevalently electrostatic in nature and does not involve cysteines and/or histidines which are found in most iron-binding proteins. This makes ion binding weak and relatively unspecific. CyaY, for instance, binds other divalent and trivalent cations such as lanthanides, copper, and cobalt. Interestingly, crystals of CyaY soaked in the presence of divalent cations do adsorb cobalt and europium but the coordination involves non-conserved residues. Zn2+ and Al3+ promote protein aggregation already at low molar ratios. Mutation of D86, E89, D101, and E103 of Yfh1 into lysines leads to severe effects on yeast metabolism that range from impairment of Fe-S cluster assembly to weakening of the interaction with Fe-S cluster proteins and increase in oxidative damage (Foury et al. 2007). Milder mutation of the same residues to alanine does not lead to a phenotype (Aloria et al. 2004; Gakh et al. 2006).

Frataxin function

  1. Top of page
  2. Abstract
  3. Structural and biophysical studies on frataxin
  4. Frataxin function
  5. Acknowledgements
  6. Conflicts of interest
  7. References

The iron chaperone hypothesis

The first functional hypothesis on frataxin was suggested by Grazia Isaya and co-workers alongside her discovery of iron binding and aggregate formation. Frataxin was put forward as a ferritin-like scavenger that keeps iron in a bio-available form (Adamec et al. 2000; Cavadini et al. 2002). This hypothesis was supported by the evidence that ferritin can partially complement absence of frataxin (Zanella et al. 2008). However, independent evidence suggests that this is not the major function of frataxin. First, the effect of other ions on oligomerization is such that physiological concentrations of magnesium or calcium salts within the mitochondria would stabilize CyaY and Yfh1 in an iron bound monomeric state and destabilize oligomerization (Adinolfi et al. 2002). Second, it was independently shown that oligomerization is unessential when the protein functions as an iron chaperone during heme and Fe-S cluster assembly (Aloria et al. 2004). In higher organisms, mitochondria contain a specialized ferritin (Levi et al. 2001), that, when expressed in yeast, can prevent iron accumulation caused by Yfh1 deletion (Campanella et al. 2004), suggesting that iron storage is a redundant function, at least in mammals.

In search for functional interactions

Clues on the function of proteins often come from identification of interacting partners. This approach was attempted also for frataxin. The first indication of functional interactions with other proteins came from a bioinformatics study which investigated whether the frataxin gene co-occurred with other genes (Huynen et al. 2001). It was found that, over the 56 genomes available at the time, two genes, hscA and hscB/JAC1, had identical phylogenetic distribution with the frataxin/CyaY gene. The hit was particularly clear as, in the authors' words, ‘these genes have not only emerged in the same evolutionary lineage as the frataxin gene, they have also been lost at least twice with it, and they have been horizontally transferred with it in the evolution of the mitochondria'. The corresponding gene products encode hscA and hscB, two proteins involved in the synthesis of Fe-S clusters in proteobacteria suggesting a role of frataxin in this machinery (Fig. 2a).


Figure 2. A link between frataxin and the proteins from the Isc machinery. (a) Schematic representation of the E. coli Isc operon. It is worth notice that frataxin does not belong to the operon. (b) Human frataxin interactome as provided by the STRING database ( Frataxin is shown as a red node (FXN). It has been linked to the following proteins: LYRM4, LYR motif (or Isd11); IscU, scaffold protein; INS, insulin; GAA, alpha-glucosidase; IscS, Nfs1 desulfurase; LEP, leptin; ACO2, mitochondrial aconitase; TTPA, alpha-tocopherol transfer protein; EPAS1, endothelian PAS domain protein 1; PIP5K1B, phosphatidylinositol-4-phosphate-5-kinase. The links in the network are color coded as follows: linked in databases (green connectors), from text mining (black) and from experimental evidence (pink). Notice that there is no node for ferrochelatase that is found instead when starting from yeast Yfh1 (not shown). (c) Model of the triple complex between CyaY (in yellow), IscS (blue) and IscU (red) as described in Prischi et al. (2010). Top: CPK representation to show the surface complementarity; bottom: ribbon representation to show some of the details of the molecular recognition.

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This elegant study initiated a long search for experimental links between frataxin with this metabolic pathway. Several different hits now define a rather complex interactome (Fig. 2b). Independent experimental evidence was published proving interactions between frataxin and other components of the same pathway. Early pathophysiological studies in a conditional mouse model reproducing the heart cardiomyopathy of FRDA also pointed to a primary involvement of frataxin in Fe-S cluster biogenesis (Puccio et al. 2001).

It was shown that, in the absence of Yfh1, activity of Fe-S-containing enzymes (aconitase, succinate dehydrogenase) and cluster incorporation into apo-ferredoxin decrease specifically (Chen et al. 2002; Duby et al. 2002; Mühlenhoff et al. 2002). Finally, it was found that yeast and human frataxin bind to the central Fe-S cluster assembly complex, which is composed of the Nfs1 enzyme and the scaffold protein Isu (Gerber et al. 2003; Ramazzotti et al. 2004). These two proteins are, respectively, the cysteine desulfurase that converts cysteine to alanine and a highly reactive persulfide used in the synthesis of sulfur bio-organic derivatives and the transient scaffold protein on which the cluster assembles (Bandyopadhyay et al. 2008; Py et al. 2011) (Fig. 2a). They are highly conserved in most organisms. The constitutive Nfs1 homodimer and two copies of monomeric Isu (or their bacterial orthologs IscS and IscU) form a binary complex.

The direct frataxin partner however remained controversial together with the state under which the proteins interact. Early genetic and biochemical studies were careful to generically indicate interaction between frataxin and the Nfs1/Isu complex (Gerber et al. 2003; Ramazzotti et al. 2004). Later on, it was reported that both Yfh1 and human frataxin interact with Isu (Cook et al. 2006; Bencze et al. 2007). It was suggested that human frataxin binds Isu with high affinity and in an iron-dependent way. It is however rather difficult to reconcile these reports with the interaction surface that was mapped on the frataxin β-sheet, since this region is not involved in iron binding. This interaction could also not be reproduced in the bacterial system where CyaY primarily pulls down endogenous IscS while IscU is pulled-down indirectly through its interaction with IscS (Layer et al. 2006; Adinolfi et al. 2009). The surface of interaction between IscS and CyaY was suggested by mutagenesis in an elegant study also presenting the X-ray structure of the IscS/IscU complex (Shi et al. 2010). A more accurate, although low-resolution, description of the interaction was obtained by a combination of SAXS, NMR, and modeling techniques (Prischi et al. 2010) (Fig. 2c). This study showed that two CyaY monomers bind IscS in a pocket that forms between the active site and the IscS dimer interface. The interaction between CyaY and IscS is iron independent as it was mapped in regions of the two proteins with complementary charges. CyaY packs mainly against IscS, which acts as a mold. A direct interaction with IscU is possible but only in the context of the ternary complex. CyaY binding increases the affinity of the IscS/IscU complex. Independent work using mammalian recombinant proteins confirmed these results by showing that human frataxin too interacts with a preformed complex composed of Nfs1, Isu and Isd11 in an iron-independent fashion (Tsai and Barondeau 2010; and Schmucker et al. 2011). Furthermore, mutagenesis experiments in the mammalian system are consistent with the surface of interaction suggested in the yeast and bacterial systems (Leidgens et al. 2010).

Another functional link was suggested by an interaction between frataxin and ferrochelatase, the enzyme that catalyzes the final step of heme biosynthesis by inserting the ferrous ion into porphyrin (Foury and Cazzalini 1997; Lesuisse et al. 2003; Taketani 2005). A direct high-affinity interaction between frataxin and ferrochelatase was reported both for Yfh1 and for human frataxin (Yoon and Cowan 2004; Bencze et al. 2007). These observations could explain why frataxin deficiency leads to kinetic inhibition of the heme pathway (Schoenfeld et al. 2005).

Finally, independent reports have linked frataxin to mitochondrial aconitase (Bulteau et al. 2004), subunits of complex II of the mitochondrial respiratory chain (Gonzalez-Cabo et al. 2005) and several chaperones (GRP75, Ssc1) (Shan et al. 2007). The relevance of these interactions has, however, recently been questioned, as different approaches were unable to identify or reproduce some of the previously described interactions (Schmucker et al. 2011; AP unpublished results). The potential involvement of frataxin in other mitochondrial pathways thus needs to be further validated through complementary in vivo and in vitro approaches. The only pathway that seems to be consolidated by increasing experimental evidence is that of Fe-S cluster biogenesis.

The role of frataxin in iron-sulfur cluster formation

Identification of interactions between frataxin and iron–sulfur cluster components led to different hypotheses on the level of involvement of the frataxin protein in this metabolic pathway. It was first suggested that frataxin is the iron donor which helps to solubilize and transport iron to the cluster biogenesis machine (Yoon and Cowan 2003). While cysteine is the accepted preferential donor of sulfur, nothing is known about how the largely insoluble iron gets to the complex. It was shown that cluster formation on the scaffold Isu is appreciably more efficient in the presence of frataxin leading to the conclusion that frataxin is an iron chaperone. This study was however limited by the absence of IscS/Nfs1, a component which may be crucial for understanding the biological functioning of the complex.

Later on, it was shown that the role of frataxin goes well beyond that of an iron transporter (Adinolfi et al. 2009). These studies greatly benefitted from the establishment of an enzymatic assay followed by different spectroscopies which, under strict anaerobic conditions, follow the kinetics of iron-sulfur cluster formation on the IscU/Isu. When the experiment was repeated comparing the rates of enzymatic cluster formation on IscU, it became clear that frataxin has a role in regulating the kinetics. Extensive work at this level was carried out using a prokaryotic model (Prischi et al. 2010; Iannuzzi et al. 2011). This work showed unequivocally that CyaY acts as an enzyme inhibitor. The structural information suggests at least two non-mutually exclusive mechanisms to explain these data. The presence of frataxin increases the affinity of the interaction between the scaffold IscU and the desulfurase IscS. This implies that detachment of IscU, which is needed to continue the reaction, is less favored. The effect could also be the consequence of a slower movement of the loop that brings the persulfide group from the active site to IscU, because of the steric impediment caused by the presence of CyaY.

Further studies however showed that also this interpretation was probably too simple: the effects of eukaryotic frataxin on the same enzymatic process seem to be opposite of those observed in bacteria: frataxin increases the rates of the enzymatic cluster formation thus acting as an activator (Gakh et al. 2010; Tsai and Barondeau 2010). Although confirming a role of frataxin as a regulator, it proposes the question of whether this is an unusual example of an enzyme that inverts or reverts its mode of control through evolution or if there is a different factor that is responsible for the observed behavior. To resolve the discrepancy, the enzyme kinetics were measured in parallel for the human and E. coli systems individually interchanging all the components (Bridwell-Rabb et al. 2012). This study unambiguously revealed that the effect of frataxin as an activator or an inhibitor is solely determined by which cysteine desulfurase is present, and not by the identity of the frataxin ortholog. A possible explanation is that the two systems are not entirely equivalent since the recombinant Nfs1 used in these studies was produced in E. coli where it is soluble only when co-expressed with Isd11, a protein absent in prokaryotes. Isd11 is poorly characterized: little remains known about the stoichiometry and the binding site on Nfs1. Native mass spectrometry studies on the mammalian ternary and quaternary complex suggest a stoichiometry of two ISD11 per NFS1 (Schmucker et al. 2011). This stoichiometry is however not easily compatible with the symmetry of the NFS1 dimer. Isd11 was proposed to stabilize Nfs1 through a direct interaction and to activate the enzyme through a conformational change (Adam et al. 2006; Wiedemann et al. 2006; Pandey et al. 2011). It is possible that Isd11 could interfere with frataxin or alter the Nfs1 conformation. New data are necessary to clarify this crucial issue. More recently, it was shown that the presence of human frataxin helps the efficiency of formation of [Fe4S4] clusters that can be transferred to mammalian aconitase (ACO2) to generate an active enzyme (Colin et al. 2013).

Conclusions and future perspectives

The identification of the FRDA gene in 1996 has finally opened new avenues for the comprehension of the molecular causes of FRDA. Yet, we still seem to be a long way away from the moment when we can be sure of the precise role of frataxin. This small and yet highly conserved essential protein has so far eluded most efforts along this direction thanks to its novel fold and its unusual properties. Two important milestones, however, have been the determination of its iron-binding properties and the link to a precise, although still understudied metabolic pathway. At this point, the most promising perspective seems to be that of dissecting the machine responsible for Fe-S cluster biogenesis and understanding the precise role of frataxin in it. This task will require, among other things, the development of new tools to study not just an isolated molecule or a protein complex but a complex network of competing multiple interactions. While making the project much more demanding, this constitutes one of the main challenges for the next several decades. In the words of Bruce Alberts (2012), we can conclude that ‘we now know that the most interesting properties of cells are “emergent” properties, resulting from elaborate networks of interactions between many different molecules that include the protein machines. Scientists currently lack the ability to decipher this complexity, and gaining this ability will require great ingenuity and many new developments whose exact nature is unpredictable. Much of the work will need to be done through small-science research in relatively simple systems, such as the E. coli bacterium, with the hope that what is learned will lead to new approaches and principles that can be transferred to the more complicated cells of mammals.'


  1. Top of page
  2. Abstract
  3. Structural and biophysical studies on frataxin
  4. Frataxin function
  5. Acknowledgements
  6. Conflicts of interest
  7. References

This review has received funding from the European Community's Seventh Framework Program FP7/2007-2013 under the grant agreement no 242193 EFACTS and from MRC (Grant ref. U117584256).


  1. Top of page
  2. Abstract
  3. Structural and biophysical studies on frataxin
  4. Frataxin function
  5. Acknowledgements
  6. Conflicts of interest
  7. References
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