Partial conservation of functions between eukaryotic frataxin and the Escherichia coli frataxin homolog CyaY

Frataxin is a mitochondrial protein structurally conserved from bacteria to humans. Eukaryotic frataxins are known to be involved in the maintenance of mitochondrial iron balance via roles in iron delivery and iron detoxification. The prokaryotic frataxin homolog, CyaY, has been shown to bind and donate iron for the assembly of [2Fe-2S] clusters in vitro. However, in contrast to the severe phenotypes associated with the partial or complete loss of frataxin in humans and other eukaryotes, deletion of the cyaY gene does not cause any obvious alteration of iron balance in bacterial cells, an effect that probably reflects functional redundancy between CyaY and other bacterial proteins. To study CyaY function in a nonredundant setting, we have expressed a mitochondria-targeted form of CyaY in a Saccharomyces cerevisiae strain depleted of the endogenous yeast frataxin protein (yfh1Delta). We show that in this strain CyaY complements to a large extent the loss of iron-sulfur cluster enzyme activities and heme synthesis, and thereby maintains a nearly normal respiratory growth. In addition, CyaY effectively protects yfh1Delta from oxidative damage during treatment with hydrogen peroxide but is less efficient in detoxifying excess labile iron during aerobic growth.     

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.     

Solution structure of YggX: a prokaryotic protein involved in Fe(II) trafficking

We report the three-dimensional structure of YggX from Salmonella enterica, determined by solution nuclear magnetic resonance (NMR) spectroscopy from protein labeled with carbon-13 and nitrogen-15 produced by Escherichia coli cells. The protein has a beta1beta2alpha1alpha2alpha3 fold that is unique to YggX and one of its homologs, a protein from Pseudomonas aeruginosa with 45% sequence identity whose X-ray structure [Protein Data Bank (PDB) 1T07] was determined by a structural genomics center. The NMR structure, which revealed that the C-terminal region of YggX is dynamically disordered, explains why electron density from the corresponding region was missing in the X-ray structure of the Pseudomonas protein. Because it has been hypothesized that YggX has a role in iron trafficking, we investigated the influence of Fe(II) on the (1)H-(15)N NMR fingerprint region of nitrogen-15-labeled YggX. Several signals shifted or broadened upon the addition of excess Fe(II) under anoxic conditions, with His81 showing the largest effect. These results indicate that Fe(II) binds weakly to this protein at a region of the sequence conserved only in the subset of the YggX proteins from organisms similar to Salmonella. The finding that iron binds only weakly to YggX, and not to a highly conserved region of the structure, suggests that the role of this protein in iron homeostasis is more complex than previously thought.