Monomeric yeast frataxin is an iron-binding protein

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50,000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.     

Heat and cold denaturation of yeast frataxin: The effect of pressure

Yfh1 is a yeast protein with the peculiar characteristic to undergo, in the absence of salt, cold denaturation at temperatures above the water freezing point. This feature makes the protein particularly interesting for studies aiming at understanding the rules that determine protein fold stability. Here, we present the phase diagram of Yfh1 unfolding as a function of pressure (0.1–500 MPa) and temperature 278–313 K (5–40°C) both in the absence and in the presence of stabilizers using Trp fluorescence as a monitor. The protein showed a remarkable sensitivity to pressure: at 293 K, pressures around 10 MPa are sufficient to cause 50% of unfolding. Higher pressures were required for the unfolding of the protein in the presence of stabilizers. The phase diagram on the pressure-temperature plane together with a critical comparison between our results and those found in the literature allowed us to draw conclusions on the mechanism of the unfolding process under different environmental conditions.     

The serum ferroxidase activity and the iron mobilization by estrogens

The serum ferroxidase activity and the iron mobilization of estrogens were studied in chickens and turkeys. Serum iron and copper were determined in 20 male chickens, 5 adult females (not laying), 12 laying chickens, 13 male turkeys, 5 adult females (not laying), and 4 laying turkeys. Serum iron and copper increased simultaneously during the laying period as a consequence of a higher estrogen level during this period. Evidence of this was obtained by injecting estradiol benzoate (3 mg estradiol, 3 times/week for 2 weeks) which produced a 6-fold increase of serum iron and copper. The levels dropped to base line values after a third week without treatment. A maximum ferroxidase activity was noted during the laying period representing a 20-fold incre ase while iron showed a 7-fold increase. 2 mg diethylstilbestrol/kg adm inistered to 6 immature pullets demonstrated an increase in the ferroxid ase activity with a peak at 36 hours and a peak of iron at 60 hours. Serum mare gonadotrophin caused a significant increase in ferroxidase activity by Day 14 (p less than .0001). These results suggest that the ferroxidase induction by exogenous estrogens or by endogenous estrogens is an identical effect.     

The role of frataxin in fission yeast iron metabolism: Implications for Friedreich's ataxia

Background

The neurodegenerative disease Friedreich's ataxia is the result of frataxin deficiency. Frataxin is a mitochondrial protein involved in iron–sulfur cluster (Fe–S) cofactor biogenesis, but its functional role in this pathway is debated. This is due to the interconnectivity of iron metabolic and oxidative stress response pathways that make distinguishing primary effects of frataxin deficiency challenging. Since Fe–S cluster assembly is conserved, frataxin overexpression phenotypes in a simple eukaryotic organism will provide additional insight into frataxin function.

Methods

The Schizosaccharomyces pombe frataxin homologue (fxn1) was overexpressed from a plasmid under a thiamine repressible promoter. The S. pombe transformants were characterized at several expression strengths for cellular growth, mitochondrial organization, iron levels, oxidative stress, and activities of Fe–S cluster containing enzymes.

Results

Observed phenotypes were dependent on the amount of Fxn1 overexpression. High Fxn1 overexpression severely inhibited S. pombe growth, impaired mitochondrial membrane integrity and cellular respiration, and led to Fxn1 aggregation. Cellular iron accumulation was observed at moderate Fxn1 overexpression but was most pronounced at high levels of Fxn1. All levels of Fxn1 overexpression up-regulated oxidative stress defense and mitochondrial Fe–S cluster containing enzyme activities.

Conclusions

Despite the presence of oxidative stress and accumulated iron, activation of Fe–S cluster enzymes was common to all levels of Fxn1 overexpression; therefore, Fxn1 may regulate the efficiency of Fe–S cluster biogenesis in S. pombe.

General Significance

We provide evidence that suggests that dysregulated Fe–S cluster biogenesis is a primary effect of both frataxin overexpression and deficiency as in Friedreich's ataxia.     

Physical evidence that yeast frataxin is an iron storage protein

Frataxin is a conserved mitochondrial protein required for iron homeostasis. We showed previously that in the presence of ferrous iron recombinant yeast frataxin (mYfh1p) assembles into a regular multimer of approximately 1.1 MDa storing approximately 3000 iron atoms. Here, we further demonstrate that mYfh1p and iron form a stable hydrophilic complex that can be detected by either protein or iron staining on nondenaturing polyacrylamide gels, and by either interference or absorbance measurements at sedimentation equilibrium. The molecular mass of this complex has been refined to 840 kDa corresponding to 48 protein subunits and 2400 iron atoms. Solution density measurements have determined a partial specific volume of 0.58 cm(3)/g, consistent with the amino acid composition of mYfh1p and the presence of 50 Fe-O equivalents per subunit. By dynamic light scattering, we show that the complex has a radius of approximately 11 nm and assembles within 2 min at 30 degrees C when ferrous iron, not ferric iron or other divalent cations, is added to mYfh1p monomer at pH between 6 and 8. Iron-rich granules with diameter of 2-4 nm are detected in the complex by scanning transmission electron microscopy and energy-dispersive X-ray spectroscopy. These findings support the hypothesis that frataxin is an iron storage protein, which could explain the mitochondrial iron accumulation and oxidative damage associated with frataxin defects in yeast, mouse, and humans.     

Iron-dependent self-assembly of recombinant yeast frataxin: implications for Friedreich ataxia

Frataxin deficiency is the primary cause of Friedreich ataxia (FRDA), an autosomal recessive cardiodegenerative and neurodegenerative disease. Frataxin is a nuclear-encoded mitochondrial protein that is widely conserved among eukaryotes. Genetic inactivation of the yeast frataxin homologue (Yfh1p) results in mitochondrial iron accumulation and hypersensitivity to oxidative stress. Increased iron deposition and evidence of oxidative damage have also been observed in cardiac tissue and cultured fibroblasts from patients with FRDA. These findings indicate that frataxin is essential for mitochondrial iron homeostasis and protection from iron-induced formation of free radicals. The functional mechanism of frataxin, however, is still unknown. We have expressed the mature form of Yfh1p (mYfh1p) in Escherichia coli and have analyzed its function in vitro. Isolated mYfh1p is a soluble monomer (13,783 Da) that contains no iron and shows no significant tendency to self-associate. Aerobic addition of ferrous iron to mYfh1p results in assembly of regular spherical multimers with a molecular mass of approximately 1. 1 MDa (megadaltons) and a diameter of 13+/-2 nm. Each multimer consists of approximately 60 subunits and can sequester >3,000 atoms of iron. Titration of mYfh1p with increasing iron concentrations supports a stepwise mechanism of multimer assembly. Sequential addition of an iron chelator and a reducing agent results in quantitative iron release with concomitant disassembly of the multimer, indicating that mYfh1p sequesters iron in an available form. In yeast mitochondria, native mYfh1p exists as monomer and a higher-order species with a molecular weight >600,000. After addition of (55)Fe to the medium, immunoprecipitates of this species contain >16 atoms of (55)Fe per molecule of mYfh1p. We propose that iron-dependent self-assembly of recombinant mYfh1p reflects a physiological role for frataxin in mitochondrial iron sequestration and bioavailability.     

A study on ascorbate inhibition of ceruloplasmin ferroxidase activity

The ferroxidase activity of ceruloplasmin is often determined according to the method of Johnson et al. (1967), using apotransferrin for trapping ferric ions generated by the enzyme; spectrophotometrically monitoring the Fe–transferrin formation at pH6.0. Reports have shown that ascorbate inhibits this reaction, and it is hypothesized that the effect could be of physiological significance in individuals with a high ascorbate to ceruloplasmin ratio in plasma (e.g. premature babies).The present study shows that the inhibitory effect of ascorbate rapidly decreases with increasing pH. At pH7.4 no significant effect was observed, the result suggesting that ascorbate is not a physiological inhibitor of ceruloplasmin. Furthermore, experiments demonstrate that at acidic pH the inhibitory effect of ascorbate on the rate of Fe–transferrin formation is not primarily due to an interaction with ceruloplasmin, but to a reduction of enzymically generated ferric ions before they are bound to apotransferrin.     

Dynamics of protein damage in yeast frataxin mutant exposed to oxidative stress

Oxidative stress and protein carbonylation is implicated in aging and various diseases such as neurodegenerative disorders, diabetes, and cancer. Therefore, the accurate identification and quantification of protein carbonylation may lead to the discovery of new biomarkers. We have developed a new method that combines avidin affinity selection of carbonylated proteins with iTRAQ labeling and LC fractionation of intact proteins. This simple LC-based workflow is an effective technique to reduce sample complexity, minimize technical variation, and enable simultaneous quantification of four samples. This method was used to determine protein oxidation in an iron accumulating mutant of Saccharomyces cerevisiae exposed to oxidative stress. Overall, 31 proteins were identified with 99% peptide confidence, and of those, 27 proteins were quantified. Most of the identified proteins were associated with energy metabolism (32.3%), and cellular defense, transport, and folding (38.7%), suggesting a drop in energy production and reducing power of the cells due to the damage of glycolytic enzymes and decrease in activity of enzymes involved in protein protection and regeneration. In addition, the oxidation sites of seven proteins were identified and their estimated position also indicated a potential impact on the enzymatic activities. Predicted 3D structures of peroxiredoxin (TSA1) and thioredoxin II (TRX2) revealed close proximity of all oxidized amino acid residues to the protein active sites.     

Iron-induced oligomerization of yeast frataxin homologue Yfh1 is dispensable in vivo

The neurodegenerative disease Friedreich's ataxia is caused by reduced levels of frataxin, a mitochondrial matrix protein. The in vivo role of frataxin is under debate. Frataxin, as well as its yeast homologue Yfh1, binds multiple iron atoms as an oligomer and has been proposed to function as a crucial iron-storage protein. We identified a mutant Yfh1 defective in iron-induced oligomerization. This mutant protein was able to replace functionally wild-type Yfh1, even when expressed at low levels, when mitochondrial iron levels were high and in mutant strains having deletions of genes that had synthetic growth defects with a YFH1 deletion. The ability of an oligomerization-deficient Yfh1 to function in vivo suggests that oligomerization, and thus oligomerization-induced iron storage, is not a critical function of Yfh1. Rather, the capacity of this oligomerization-deficient mutant to interact with the Isu protein suggests a more direct role of Yfh1 in iron-sulphur cluster biogenesis.     

On the mechanism of citrate inhibition of ceruloplasmin ferroxidase activity

Ceruloplasmin is a plasma protein, which oxidizes ferrous ions in a catalytic manner. It is considered to function as a ferroxidasein vivo. Citrate was found to inhibit the reaction. The ceruloplasmin catalyzed oxidation ofp-phenylenediamines, however, was not affected by citrate. The inhibitory effect is proposed to be due to formation of Fe²⁺-citrate, which does not react with ceruloplasmin. The stability constant for the Fe²⁺-citrate complex estimated from the present inhibition study is in good agreement with previously published data.     

Acidic residues of yeast frataxin have an essential role in Fe-S cluster assembly

Friedreich ataxia is caused by decreased levels of frataxin, a mitochondrial acidic protein that is assumed to act as chaperone in the assembly of Fe-S clusters on the scaffold Isu protein. Frataxin has the in vitro capacity to form iron-loaded multimers, which also suggests an iron storage function. It has been reported that alanine substitution of residues in an acidic ridge of yeast frataxin (Yfh1) elicits loss of iron binding in vitro but has no effect on Fe-S cluster synthesis in vivo. Here, we show that a marked change in the electrostatic properties of a specific region of Yfh1 surface - by substituting two or four acidic residues by lysine or alanine, respectively - impairs Fe-S cluster assembly, weakens the interaction between Yfh1 and Isu1, and increases oxidative damage. Therefore, the acidic ridge is essential for the Yfh1 function and is likely to be involved in iron-mediated protein-protein interactions.     

Binding of yeast frataxin to the scaffold for Fe-S cluster biogenesis, Isu

Friedreich ataxia is caused by reduced activity of frataxin, a conserved iron-binding protein of the mitochondrial matrix, thought to supply iron for formation of Fe-S clusters on the scaffold protein Isu. Frataxin binds Isu in an iron-dependent manner in vitro. However, the biological relevance of this interaction and whether in vivo the interaction between frataxin and Isu is mediated by adaptor proteins is a matter of debate. Here, we report that alterations of conserved, surface-exposed residues of yeast frataxin, which have deleterious effects on cell growth, impair Fe-S cluster biogenesis and interaction with Isu while altering neither iron binding nor oligomerization. Our results support the idea that the surface of the beta-sheet, adjacent to the acidic, iron binding ridge, is important for interaction of Yfh1 with the Fe-S cluster scaffold and point to a critical role for frataxin in Fe-S cluster biogenesis.     

The structure and function of frataxin

Frataxin, a highly conserved protein found in prokaryotes and eukaryotes, is required for efficient regulation of cellular iron homeostasis. Humans with a frataxin deficiency have the cardio- and neurodegenerative disorder Friedreich's ataxia, commonly resulting from a GAA trinucleotide repeat expansion in the frataxin gene. While frataxin's specific function remains a point of controversy, the general consensus is that the protein assists in controlling cellular iron homeostasis by directly binding iron. This review focuses on the structural and biochemical aspects of iron binding by the frataxin orthologs and outlines molecular attributes that may help explain the protein's role in different cellular pathways.     

Evidence that yeast frataxin is not an iron storage protein in vivo

Yeast cells deficient in the yeast frataxin homolog (Yfh1p) accumulate iron in their mitochondria. Whether this iron is toxic, however, remains unclear. We showed that large excesses of iron in the growth medium did not inhibit growth and did not decrease cell viability. Increasing the ratio of mitochondrial iron-to-Yfh1p by decreasing the steady-state level of Yfh1p to less than 100 molecules per cell had very few deleterious effects on cell physiology, even though the mitochondrial iron concentration greatly exceeded the iron-binding capacity of Yfh1p in these conditions. Mössbauer spectroscopy and FPLC analyses of whole mitochondria or of isolated mitochondrial matrices showed that the chemical and biochemical forms of the accumulated iron in mitochondria of mutant yeast strains (Δyfh1, Δggc1 and Δssq1) displayed a nearly identical distribution. This was also the case for Δggc1 cells, in which Yfh1p was overproduced. In these mitochondria, most of the iron was insoluble, and the ratio of soluble-to-insoluble iron did not change when the amount of Yfh1p was increased up to 4500 molecules per cell. Our results do not privilege the hypothesis of Yfh1p being an iron storage protein in vivo.     

Dynamics, stability and iron-binding activity of frataxin clinical mutants

Friedreich's ataxia results from a deficiency in the mitochondrial protein frataxin, which carries single point mutations in some patients. In the present study, we analysed the consequences of different disease-related mutations in vitro on the stability and dynamics of human frataxin. Two of the mutations, G130V and D122Y, were investigated for the first time. Analysis by CD spectroscopy demonstrated a substantial decrease in the thermodynamic stability of the variants during chemical and thermal unfolding (wild-type > W155R > I154F > D122Y > G130V), which was reversible in all cases. Protein dynamics was studied in detail and revealed that the mutants have distinct propensities towards aggregation. It was observed that the mutants have increased correlation times and different relative ratios between soluble and insoluble/aggregated protein. NMR showed that the clinical mutants retained a compact and relatively rigid globular core despite their decreased stabilities. Limited proteolysis assays coupled with LC-MS allowed the identification of particularly flexible regions in the mutants; interestingly, these regions included those involved in iron-binding. In agreement, the iron metallochaperone activity of the Friedreich's ataxia mutants was affected: some mutants precipitate upon iron binding (I154F and W155R) and others have a lower binding stoichiometry (G130V and D122Y). Our results suggest that, in heterozygous patients, the development of Friedreich's ataxia may result from a combination of reduced efficiency of protein folding and accelerated degradation in vivo, leading to lower than normal concentrations of frataxin. This hypothesis also suggests that, although quite different from other neurodegenerative diseases involving toxic aggregation, Friedreich's ataxia could also be linked to a process of protein misfolding due to specific destabilization of frataxin.     

Iron toxicity protection by truncated Ras2 GTPase in yeast strain lacking frataxin

Yeast strain deleted for the YFH1 gene, which encodes the orthologue of human frataxin, accumulates iron in mitochondria, constitutively activates the high-affinity iron import system in the plasma membrane, and is sensitive to high iron media. We have performed a genetic screen for mutants of a yfh1 deleted strain with increased resistance to high levels of iron. One of the identified mutations caused the deletion of the hypervariable C-terminal region of Ras2p GTPase. The effect of ras2 mutation on the growth of yfh1 null strain was masked by the addition of caffeine. We found that the ras2 mutation does not alter the expression of the iron regulon nor prevent mitochondrial iron accumulation in a yfh1 mutant context. The double yfh1 ras2 mutant has increased mRNA levels of CIT2 gene and augmented catalase activity.     

Structural basis of the ferrous iron specificity of the yeast ferroxidase, Fet3p

Fet3p is a multicopper oxidase (MCO) that functions together with the iron permease, Ftr1p, to support high-affinity Fe uptake in yeast. Fet3p is a ferroxidase that, like ceruloplasmin and hephaestin, couples the oxidation of 4 equiv of Fe(II) to the reduction of O2 to 2 H2O. The ferrous iron specificity of this subclass of MCO proteins has not been delineated by rigorous structure-function analysis. Here the crystal structure of Fet3p has been used as a template to identify the amino acid residues that confer this substrate specificity and then to quantify the contributions they make to this specific reactivity by thermodynamic and kinetic analyses. In terms of the Marcus theory of outer-sphere electron transfer, we show here that D283, E185, and D409 in Fet3p provide a Fe(II) binding site that actually favors ferric iron; this site thus reduces the reduction potential of the bound Fe(II) in comparison to that of aqueous ferrous iron, providing a thermodynamically more robust driving force for electron transfer. In addition, E185 and D409 constitute parts of the electron-transfer pathway from the bound Fe(II) to the protein's type 1 Cu(II). This electronic matrix coupling relies on H-bonds from the carboxylate OD2 atom of each residue to the NE2 NH group of the two histidine ligands at the type 1 Cu site. These two acidic residues and this H-bond network appear to distinguish a fungal ferroxidase from a fungal laccase since the specificity that Fet3p has for Fe(II) is completely lost in a Fet3pE185A/D409A mutant. Indeed, this double mutant functions kinetically better as a laccase, albeit a relatively inefficient one.