Iron Insertion at the Assembly Site of the ISCU Scaffold Protein Is a Conserved Process Initiating Fe-S Cluster Biosynthesis.

Iron-sulfur (Fe-S) clusters are prosthetic groups of proteins biosynthesized on scaffold proteins by highly conserved multi-protein machineries. Biosynthesis of Fe-S clusters into the ISCU scaffold protein is initiated by ferrous iron insertion, followed by sulfur acquisition, via a still elusive mechanism. Notably, whether iron initially binds to the ISCU cysteine-rich assembly site or to a cysteine-less auxiliary site via N/O ligands remains unclear. We show here by SEC, circular dichroism (CD), and Mössbauer spectroscopies that iron binds to the assembly site of the monomeric form of prokaryotic and eukaryotic ISCU proteins via either one or two cysteines, referred to the 1-Cys and 2-Cys forms, respectively. The latter predominated at pH 8.0 and correlated with the Fe-S cluster assembly activity, whereas the former increased at a more acidic pH, together with free iron, suggesting that it constitutes an intermediate of the iron insertion process. Iron not binding to the assembly site was non-specifically bound to the aggregated ISCU, ruling out the existence of a structurally defined auxiliary site in ISCU. Characterization of the 2-Cys form by site-directed mutagenesis, CD, NMR, X-ray absorption, Mössbauer, and electron paramagnetic resonance spectroscopies showed that the iron center is coordinated by four strictly conserved amino acids of the assembly site, Cys35, Asp37, Cys61, and His103, in a tetrahedral geometry. The sulfur receptor Cys104 was at a very close distance and apparently bound to the iron center when His103 was missing, which may enable iron-dependent sulfur acquisition. Altogether, these data provide the structural basis to elucidate the Fe-S cluster assembly process and establish that the initiation of Fe-S cluster biosynthesis by insertion of a ferrous iron in the assembly site of ISCU is a conserved mechanism.

Recent Advances in the Elucidation of Frataxin Biochemical Function Open Novel Perspectives for the Treatment of Friedreich's Ataxia.

Friedreich's ataxia (FRDA) is the most prevalent autosomic recessive ataxia and is associated with a severe cardiac hypertrophy and less frequently diabetes. It is caused by mutations in the gene encoding frataxin (FXN), a small mitochondrial protein. The primary consequence is a defective expression of FXN, with basal protein levels decreased by 70-98%, which foremost affects the cerebellum, dorsal root ganglia, heart and liver. FXN is a mitochondrial protein involved in iron metabolism but its exact function has remained elusive and highly debated since its discovery. At the cellular level, FRDA is characterized by a general deficit in the biosynthesis of iron-sulfur (Fe-S) clusters and heme, iron accumulation and deposition in mitochondria, and sensitivity to oxidative stress. Based on these phenotypes and the proposed ability of FXN to bind iron, a role as an iron storage protein providing iron for Fe-S cluster and heme biosynthesis was initially proposed. However, this model was challenged by several other studies and it is now widely accepted that FXN functions primarily in Fe-S cluster biosynthesis, with iron accumulation, heme deficiency and oxidative stress sensitivity appearing later on as secondary defects. Nonetheless, the biochemical function of FXN in Fe-S cluster biosynthesis is still debated. Several roles have been proposed for FXN: iron chaperone, gate-keeper of detrimental Fe-S cluster biosynthesis, sulfide production stimulator and sulfur transfer accelerator. A picture is now emerging which points toward a unique function of FXN as an accelerator of a key step of sulfur transfer between two components of the Fe-S cluster biosynthetic complex. These findings should foster the development of new strategies for the treatment of FRDA. We will review here the latest discoveries on the biochemical function of frataxin and the implication for a potential therapeutic treatment of FRDA.