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The insertion of iron initiating the biosynthesis of Fe-S centers is a conserved process

​In a collaborative study, a team from SBIGeM (I2BC) shows, by several spectroscopic approaches, that the assembly of a Fe-S cluster, the catalytic center of many essential proteins, is a highly conserved process, initiated by the insertion of ferrous iron at the cysteine-rich assembly site of the scaffold protein ISCU (Iron-sulfur cluster assembly enzyme). A step towards the elucidation of this particularly complex vital mechanism.

Published on 9 December 2022


Iron-sulfur (Fe-S) clusters are some of the oldest inorganic protein co-factors. The cellular machineries allowing to make them would be prior to the oxygenation of the Earth's atmosphere! Fe-S clusters, which have the ability to transfer electrons, are the catalytic center of many redox enzymes and signaling proteins with essential biological functions (energy production, protein synthesis, maintenance of genome integrity...). However, the particularly complex assembly mechanism of Fe-S clusters remains poorly understood and, for several years, Benoit D'Autréaux's team has been trying to elucidate this mechanism by reconstituting in vitro the assembly machinery of Fe-S centers with all its components (for more information, see The mechanism of Iron-Sulfur Cluster assembly, on the team page of B D'Autréaux).


In mitochondria, the multiprotein Fe-S center assembly machinery, ISC (Iron-Sulfur Cluster), comprises a central complex composed of i) the scaffold protein ISCU on which Fe-S clusters are assembled, ii) an NFS1-ISD11-ACP complex containing the cysteine desulfurase NFS1, a pyridoxal-phosphate (PLP) cofactor enzyme providing sulfur in the form of a persulfide, iii) ferredoxin 2 (FDX2) with its reductase (FDXR) and iv) Frataxin (FXN), a gas pedal of Fe-S center assembly (see our actuality on the FXN role). These proteins operate sequentially. It is known that the biosynthesis of Fe-S centers in the ISCU scaffold protein is initiated by the insertion of ferrous iron (Fe2+), followed by the acquisition of sulfur in two steps (FXN-accelerated persulfide transfer from NFS1, followed by FDX2-mediated reduction to sulfide ions), but the mechanisms underlying each step are largely unknown. In particular, it remains unclear whether iron initially binds to the cysteine-rich assembly site of ISCU or to a cysteine-free helper site via unknown ligands.


In this study, researchers examined the binding properties of iron to ISCU proteins from the bacteria E. coli, the fungus Chaetomium thermophilum, and the mammals Mus musculus and H. sapiens. Using CD (circular dichroism) and Mössbauer* spectroscopies, they found that iron binds exclusively to the cysteine-rich assembly site** of the monomeric form of prokaryotic and eukaryotic ISCU proteins. Iron outside this cysteine-rich assembly site is either free or bound to aggregated ISCUs, calling into question the existence of an auxiliary site in these proteins. Analysis of the properties of the assembly site revealed that iron binding is sensitive to pH, the nature of the buffer and the oligomeric state of the protein. Its structural characterization by directed mutagenesis and CD, NMR, XAS (X-ray absorption spectroscopy), EPR (Electron Paramagnetic Resonance) and Mössbauer spectroscopies shows that iron is bound by four strictly conserved amino acids of the assembly site: one aspartate, one histidine and two cysteine residues, a third conserved cysteine, Cys104, persulfide receptor, is not binding but is very close and able to bind directly to iron when histidine is missing.


Taken together, these results provide evidence that ferrous iron insertion into the ISCU assembly site is a highly conserved process. By establishing the structural basis through extensive spectroscopic analyses, these data pave the way to elucidate the assembly process of Fe-S clusters.

Contact : Benoît D'Autréaux ( )

* Mössbauer spectroscopy is a method based on the absorption of gamma rays by atomic nuclei in a solid. By measuring the transitions between the energy levels of these nuclei, it can provide information about the local environment of the atom.

** The ISCU assembly site contains five strictly conserved amino acids that are essential for the biogenesis of Fe-S centers in vivo: Cys35, Asp37, Cys61, His103 and Cys104 (murine ISCU numbering).

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