Human Frataxin Activates Fe–S Cluster Biosynthesis by Facilitating Sulfur Transfer Chemistry

Abstract

Iron–sulfur clusters are ubiquitous protein cofactors with critical cellular functions. The mitochondrial Fe–S assembly complex, which consists of the cysteine desulfurase NFS1 and its accessory protein (ISD11), the Fe–S assembly protein (ISCU2), and frataxin (FXN), converts substrates l-cysteine, ferrous iron, and electrons into Fe–S clusters. The physiological function of FXN has received a tremendous amount of attention since the discovery that its loss is directly linked to the neurodegenerative disease Friedreich’s ataxia. Previous in vitro results revealed a role for human FXN in activating the cysteine desulfurase and Fe–S cluster biosynthesis activities of the Fe–S assembly complex. Here we present radiolabeling experiments that indicate FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting persulfide species is viable in the subsequent synthesis of Fe–S clusters. Additional mutagenesis, enzyme kinetic, UV–visible, and circular dichroism spectroscopic studies suggest conserved ISCU2 residue C104 is critical for FXN activation, whereas C35, C61, and C104 are all essential for Fe–S cluster formation on the assembly complex. These results cannot be fully explained by the hypothesis that FXN functions as an iron donor for Fe–S cluster biosynthesis, and further support an allosteric regulator role for FXN. Together, these results lead to an activation model in which FXN accelerates persulfide formation on NFS1 and favors a helix-to-coil interconversion on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2 as an initial step in Fe–S cluster biosynthesis.

Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols

“…We thus conclude that FXN is not required for persulfide formation of NFS1 as confirmed by mass spectrometry analysis. These results challenge two recent studies reporting a stimulatory effect of FXN and its yeast homologue, Yfh1, on NFS1 persulfide formation 12,31 . In these studies, the formation of persulfide was monitored using 35 S labelling of L-cysteine.…”

“…These results rule out the hypothesis that FXN stimulates sulfide release by enhancing persulfide formation on NFS1, as proposed by others 11,12,14,31 . FXN has also been proposed to stimulate iron entry within the NIU complex in Fe-S cluster reconstitution assays with DTT 11 .…”

“…In the bacterial system, the sulfane sulfur of the persulfide carried by IscS, the homologue of NFS1, is transferred to IscU by a trans-persulfuration process leading to poly-persulfurated IscU [28][29][30] . A similar trans-persulfuration process has been recently decribed with the mammalian system 31 . Unfortunately, mechanistic details of the subsequent steps leading to Fe-S cluster assembly are still lacking.…”

Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin

“…The first step crucially requires the cysteine desulphurase complex Nfs1-Isd11 (bacterial IscS; no Isd11 homologue known) that releases sulphur from cysteine by generating a Nfs1-bound persulphide (-SSH) intermediate 19 . Frataxin (yeast Yfh1) is crucial for mitochondrial Fe/S protein biogenesis, and has been proposed to serve as an iron donor and/or positive regulator of the desulphurase enzyme [20][21][22] . In Salmonella, a frataxin homologue also facilitates Fe/S protein assembly 23 , yet the Escherichia coli frataxin homologue CyaY inhibits Fe/S cluster synthesis in vitro and was suggested to be a negative regulator 24 .…”

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery

“…Additional studies demonstrated similar bacterial and yeast heterotetramers in which a dimer of IscS/ Nfs1 binds to one IscU/Isu1 monomer at each end, and it was further proposed that one CyaY/Yfh1 monomer could bind in a pocket between the cysteine desulfurase and the scaffold (10, 16 -18). Models based on these structures have also been pro-posed for the human proteins (19,20 (10,17). The mechanism of sulfur transfer to each of these sites is thought to involve a flexible loop of Nfs1 containing the invariant cysteine residue Cys-421, which becomes persulfurated at the end of the cysteine desulfurase reaction (12).…”