| dc.description.abstract | Iron-sulfur (Fe-S) clusters are inorganic cofactors found throughout all domains of life. These clusters are often ligated to proteins allowing Fe-S clusters to participate in a variety of essential processes such as oxidative respiration and photosynthesis. In eukaryotes, Fe-S clusters are assembled by a multi-subunit protein complex, and the synthesized clusters are subsequently distributed to target apoproteins. Specifically, in human mitochondria, the cysteine desulfurase, NFS1, catalyzes the conversion of cysteine to alanine and sulfane sulfur, which is transferred as a persulfide intermediate to acceptor proteins. Unexpectedly, NFS1 forms a tight complex with the LYRM adaptor protein (ISD11) and with the mitochondrial acyl-carrier protein (ACP). This complex is responsible for the transfer of persulfide sulfur to ISCU2 where a [2Fe-2S] cluster can be synthesized. Prior to these studies, little was known about the structure of the biosynthetic subcomplex consisting of NFS1, ISD11, and ACP and how structural differences between the eukaryotic and prokaryotic cysteine desulfurases might be linked to the frataxin (FXN) activation requirement in the human system. A variety of biochemical and biophysical approaches were applied to determine structure-function properties for the human Fe-S cluster biosynthetic subcomplex (NFS1- ISD11-ACP). We found that the human Fe-S cluster biosynthetic subcomplex displays a unique, open cysteine desulfurase architecture that provides a structural explanation for the complex’s low activity and demonstrates clear roles for ISD11 and ACP within eukaryotic Fe-S cluster biosynthesis. Additional crystallographic, functional, and mass spectrometry studies suggest the NFS1-ISD11-ACP subcomplex exists as a mixture of forms in solution with distinct quaternary structures. We show that multiple species can be separated by cation exchange chromatography and that all species, including those generated by treatment with the substrate L-cysteine, are in equilibrium with one another.
Cation-exchange coupled small-angle X-ray scattering was used to investigate the conformational landscape of the NFS1-ISD11-ACP subcomplex. These studies demonstrated a unique species that has an extended conformation in solution upon treatment with the substrate. Taken together, our studies provide evidence for a morpheein-like regulatory model in which FXN stimulates the cysteine desulfurase and Fe-S cluster assembly activities by driving a subunit rearrangement for the NFS1-ISD11- ACP-ISCU2 complex. | en |
