Monomeric Yeast Frataxin Is an Iron-Binding Protein

Abstract

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 nuclear encoded, frataxin 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 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 monomeric frataxin might function as the common iron donor.In order 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 2 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 6 coordinate iron-(oxygen and 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. Based on our results, we have developed a model for how we believe yeast frataxin interacts with iron. Keywords Briefs:We present a characterization of monomeric yeast frataxin's iron binding ability. Various spectroscopic techniques were applied to help characterize the iron binding affinity of yeast frataxin, the oligomeric state of the protein, specific amino acids affected by the presence of iron and finally the metal-ligand coordination geometry. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 August 19. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript patients, is a direct result of a trinucleotide repeat expansion in the first intron of the gene for the protein frataxin; this expansion disrupts gene transcription leading to a frataxin deficiency in humans (4). The inability to produce frataxin is associated with mitochondrial accumulation of iron that is biologically unavailable. In humans, the breakdown in normal iron regulation pathways resulting from frataxin deficiency is exemplified physiologically by the degeneration of sensory neurons in the dorsal root ganglia, hypertrophic cardiomyopathy and diabetes mellitus (5). In yeast, reintroduction of frataxin into knockout cells results in restored bioavailability of the accumulated metal (6). These data indicate frataxin is required for retaining bi...

Frataxin Depletion in Yeast Triggers Up-regulation of Iron Transport Systems before Affecting Iron-Sulfur Enzyme Activities

“…The second one suggests that, rather than acting as the iron donor for ironsulfur biogenesis, frataxin senses mitochondrial iron content and regulates the rate of iron-sulfur biogenesis (53). This second hypothesis would explain the interactions observed between frataxin and members of the iron-sulfur biosynthetic machinery (11)(12)(13), as well as its iron binding properties (54). Of course, one of the early consequences of losing either an iron storage protein or an iron-sensing protein could be the deregulation of iron metabolism, as observed in the present study.…”

Metal Ion availability in mitochondria

“…Frataxin adopts a well defined αβ-sandwich structural motif and is capable of binding Fe(II) ions in a N-terminal conserved acidic patch (He et al 2004). Frataxin binds 2 Fe(II) ions as a monomeric protein with micromolar affinities (Cook et al 2006). Thus, frataxin satisfies numerous criteria for being a non-essential iron metallochaperone in its ability to bind target molecules and iron in a transient manner.…”

Lateral-flow immunoassay for the frataxin protein in Friedreich’s ataxia patients and carriers

“…The decrease in frataxin protein has broad, far-reaching effects because the protein is an essential iron chaperone required for the biogenesis of iron-sulfur clusters, aconitase activation, and heme biosynthesis [18][19][20][21], and further performs a critical role in iron detoxification and anti-oxidant protection [22][23][24]. Thus a loss of frataxin leads to widespread impairment of energy metabolism, increased oxidative stress, and a generally dysregulated iron metabolism, including accumulation of iron in the heart and nervous system [25].…”