Mechanistic Analysis of the Role of Frataxin in Bacterial and Eukaryotic Fe-S Cluster Biosynthetic Pathways

Iron-sulfur (Fe-S) clusters are essential cofactors and are found in all branches of life. In eukaryotes, Fe-S assembly complex is composed of NFS1, ISD11, ACP, ISCU2, and FXN subunits. NFS1 is a cysteine desulfurase that utilizes a PLP cofactor to extract sulfur from its cysteine substrate and produce a persulfide species on a cysteine residue of a mobile S-transfer loop. Sulfur is then transferred from the mobile S-transfer loop of NFS1 to ISCU2, where it is combined with ferrous iron and electrons to generate [2Fe-2S] clusters. ISD11 and ACP are critical for the stability of the Fe-S assembly complex and may modulate NFS1 function. Eukaryotic ISC pathway is characterized by low activity of SDA and significant rate enhancement by FXN in presence of ISCU2. Prokaryotic cysteine desulfurase IscS (59% identity) stable without accessory proteins and highly active. Interestingly, both FXN and homolog CyaY activates/inhibits the eukaryotic/prokaryotic Fe-S assembly respectively. Here, we investigated the role of FXN/CyaY in each step of Fe-S biosynthesis. We found that S-transfer loop cysteine participates in cys-aldimine formation, cys-quinonoid decay, persulfide formation on itself, and transfer of this sulfane sulfur to scaffold protein. Interestingly, in eukaryotes, FXN enhances all these steps but not as fast as IscS. All these data support a model in which monomeric cysteine desulfurase architecture (such as in SDAvec) promotes non-productive conformations of S-transfer loop of cysteine desulfurase resulting in low activity. FXN binding (in eukaryotic system) excludes non-productive conformations and directs the trajectory of the NFS1 mobile S-transfer loop to position the cysteine to function as a general acid, nucleophile, and sulfur delivery agent in different steps and accelerates Fe-S cluster biosynthesis. In prokaryotes, because of the closed architecture, the other subunit of cysteine desulfurase regulates S-transfer loop trajectory (better than the FXN in eukaryotic system) and therefore have high activity even in absence of FXN. Consistent with this, IscS^S10Q variant exhibits significantly weaker dimer interface, and concentration dependent enhancement of dimer concentration and activity. Also, data indicate that reduced FDX2 is probably not the electron source but sensitizer of persulfide on scaffold protein towards reduction by reduced glutathione