Biochemical and structural basis of iron-sulfur cluster biogenesis to elucidate the molecular function of frataxin – FRATAXUR

Iron-sulfur (Fe-S) clusters are essential prosthetic groups of proteins and defects in their synthesis lead to severe human diseases such as Friedreich’s ataxia, a fatal neurodegenerative and cardiac disease due to impaired expression of frataxin (FXN). Several drugs aiming to restore the level of FXN or mimic its function are currently under clinical trials. Although promising results are now reported for gene therapy to increase FXN expression, the requirement to deliver these drugs locally at the site of affection (cerebellum and heart) is still challenging for the development of these therapies. Several drugs attempting to mimic the function of FXN are also tested, but have not yet produce conclusive results. The main reason is that the molecular function of FXN is still elusive. FXN was proposed to function as an iron chaperone for the mitochondrial Fe-S cluster assembly machinery, but this model is controversial. Our previous studies rather suggest that FXN is involved in the mobilization of sulfur for the scaffold protein ISCU, on which Fe-S clusters are assembled. However, a clear picture of the mechanism of Fe-S cluster assembly by the mitochondrial machinery is still missing which precludes the elucidation of the molecular function of FXN and consequently the development of novel therapies based on the replacement of FXN.

To start addressing these issues, the mitochondrial Fe-S cluster assembly machinery was reconstituted in vitro. We managed for the first time, to characterize the iron containing form of ISCU that is competent for Fe-S cluster assembly under physiologically relevant conditions. Using this system, we performed a stepwise study. Our data revealed that FXN is not required for iron insertion but operates at a later step of Fe-S cluster assembly. The process is initiated by an unprecedented iron-assisted persulfide transfer reaction between the cysteine desulfurase NFS1 and ISCU. This reaction most probably enables synchronization of iron and sulfur supplies to ISCU. In a second step, the persulfide transferred to ISCU is reduced into sulfide by the ferredoxin FDX2. This reaction also seems to be assisted by iron, most probably to trap the nascent sulfide ion and thus prevent its diffusion. Importantly, our data indicate that FXN stimulates Fe-S cluster assembly by enhancing the rate of the iron-assisted persulfide transfer reaction, possibly by modulating the structure of the iron center. These data thus point to a change of paradigm for the molecular function of FXN and provide a rationale description of the Fe-S cluster assembly process. The aim of our proposal is to examine in details the mechanism of Fe-S cluster assembly and especially the effect of FXN on this reaction using biochemical, spectroscopic and structural methods along with cellular approaches to i) validate the physiological relevance of our reconstituted machinery, ii) elucidate the role of FXN in this process and iii) provide the structural basis of this reaction at atomic resolution.

The main outcomes of our proposal are 1) to demonstrate how FXN stimulates Fe-S cluster assembly to better define the physiopathology of FA, 2) to establish a physiologically relevant assay for large scale screening of FXN-mimetic drugs, 3) to provide the structural basis of the effect of FXN to open new perspectives for the development of FXN-mimetic compounds by drug-design and 4) to provide a clear description of the mitochondrial Fe-S cluster assembly process as a basis to elucidate the whole cellular process of Fe-S cluster biogenesis in mitochondria and the cytoplasm.