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

Frataxin deficiency is the primary cause of Friedreich ataxia (FRDA), an au tosomal recessive cardiodegenerative and neurodegenerative disease. Frataxi n is a nuclear-encoded mitochondrial protein that is widely conserved among eukaryotes. Genetic inactivation of the yeast frataxin homologue (Yfh1p) r esults in mitochondrial iron accumulation and hypersensitivity to oxidative stress. Increased iron deposition and evidence of oxidative damage have al so been observed in cardiac tissue and cultured fibroblasts from patients w ith FRDA. These findings indicate that frataxin is essential for mitochondr ial iron homeostasis and protection from iron-induced formation of free rad icals. The functional mechanism of frataxin, however, is still unknown. We have expressed the mature form of Yfh1p (mYfh1p) in Escherichia coli and ha ve 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-as sociate. Aerobic addition of ferrous iron to mYfh1p results in assembly of regular spherical multimers with a molecular mass of similar to 1.1 MDa (me gadaltons) and a diameter of 13 +/- 2 nm. Each multimer consists of similar to 60 subunits and can sequester >3,000 atoms of iron. Titration of mYfh1p with increasing iron concentrations supports a stepwise mechanism of multi mer assembly. Sequential addition of an iron chelator and a reducing agent results in quantitative iron release with concomitant disassembly of the mu ltimer, indicating that mYfh1p sequesters iron in an available form. In yea st mitochondria, native mYfh1p exists as monomer and a higher-order species with a molecular weight >600,000. After addition of Fe-55 to the medium, i mmunoprecipitates of this species contain >16 atoms of Fe-55 per molecule o f mYfh1p. We propose that iron-dependent self-assembly of recombinant mYfh1 p reflects a physiological role for frataxin in mitochondrial iron sequestr ation and bioavailability.