Tangled web of interactions among proteins involved in iron–sulfur cluster assembly as unraveled by NMR, SAXS, chemical crosslinking, and functional studies

Abstract

Proteins containing iron–sulfur (Fe–S) clusters arose early in evolution and are essential to life. Organisms have evolved machinery consisting of specialized proteins that operate together to assemble Fe–S clusters efficiently so as to minimize cellular exposure to their toxic constituents: iron and sulfide ions. To date, the best studied system is the iron sulfur cluster (isc) operon of Escherichia coli, and the eight ISC proteins it encodes. Our investigations over the past five years have identified two functional conformational states for the scaffold protein (IscU) and have shown that the other ISC proteins that interact with IscU prefer to bind one conformational state or the other. From analyses of the NMR spectroscopy-derived network of interactions of ISC proteins and small-angle X-ray scattering (SAXS), chemical crosslinking experiments, and functional assays, we have constructed working models for Fe–S cluster assembly and delivery. Future work is needed to validate and refine what has been learned about the E. coli system and to extend these findings to the homologous Fe–S cluster biosynthetic machinery of yeast and human mitochondria. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery

“…The mass spectrometer was set to perform a Fourier transform full scan from 340 to 1600 m/z at a resolution of 120,000 (400 m/z resolving power), followed by higher energy collisional dissociation orbitrap MS/MS scans on the top 15 ions at a resolution of 15,000. The MS scan 1 automatic gain control target was set to 1e 6 , and the MS scan 2 target was set to 5e 5 with maximum ion injection times of 50 and 100 ms, respectively. Dynamic exclusion placed selected ions on an exclusion list for 30 s (50,51).…”

“…Interestingly, the structure of an asymmetric IscU trimer from A. aeolicus revealed one [2Fe-2S] cluster bound on the surface of one subunit but buried inside the interface formed by all three subunits (34). These species may not be fully representative of the active forms of IscU-type scaffolds, however, as cluster assembly activity requires binding of the scaffold to the iron and the sulfur donor as well as other specialized proteins (5,6), which may result in conformational changes that are only partially defined (39).…”

“…Even though Fe-S clusters can assemble spontaneously in vitro (4), protected and safe Fe-S cluster synthesis in mitochondria requires several specialized proteins, most of which were inherited from the main bacterial Fe-S cluster assembly pathway (ISC pathway) (5,6). Thus, in both bacteria and mitochondria, Fe-S cluster assembly is centered around IscU-type protein scaffolds (bacterial IscU/yeast Isu1) upon which new Fe-S clusters are assembled using the following proteins: (i) a pyridoxal phosphate-dependent cysteine desulfurase (bacterial IscS/yeast Nfs1) with a stabilizing binding partner in eukaryotes (yeast Isd11) that serve as the sulfur donor (1); (ii) the ironbinding protein frataxin (bacterial CyaY/yeast Yfh1) that serves as the iron donor (7)(8)(9)(10) as well as a regulator of the cysteine desulfurase activity (11,12); and (iii) the electron donor chain formed by ferredoxin reductase and ferredoxin (13).…”

Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery

“…The mechanism of sulfur transfer to each of these sites involves a flexible loop of NFS1 containing the invariant cysteine residue, Cys-381, which is persulfurated through the cysteine desulfurase reaction (10,12,20,21 2 accelerates persulfide formation on NFS1 and is also thought to induce a conformational change in ISCU that enhances the transfer of sulfur from NFS1 to Cys-138, the primary or main persulfide acceptor of ISCU (23)(24)(25) (note that Cys-138 corresponds to Cys-104 if the ISCU amino acid sequence is numbered starting from the mature protein N terminus instead of the precursor protein N terminus). Studies of the yeast system further suggest that sulfur-donation to ISCU may require conformational changes in NFS1 to (i) expose the NFS1 substrate-binding site, (ii) bring the flexible loop of NFS1 close to the substrate-binding site to enable formation of persulfurated Cys-381, and (iii) bring the NFS1 flexible loop close to the Fe-S cluster assembly site to enable sulfur transfer from persulfurated Cys-381 to ISCU (10,26).…”

“…In yeast and animal cells, mitochondria are a main site of Fe-S cluster assembly (1), which involves highly conserved protein systems derived from the main bacterial Fe-S cluster assembly system (2,3). All of these systems utilize IscUtype proteins as scaffolds upon which new Fe-S clusters are assembled and additional components, including the following: (i) pyridoxal phosphate-dependent cysteine desulfurases (bacterial IscS/yeast Nfs1/human NFS1), which in eukaryotes require a stabilizing binding partner (yeast Isd11/human ISD11) that provides elemental sulfur (4); (ii) members of the frataxin family (bacterial CyaY/yeast Yfh1/human FXN) that provide elemental iron (5)(6)(7)(8) and also regulate the cysteine desulfurase activity (9,10); and (iii) the electron donor chain formed by ferredoxin reductase and ferredoxin (11).…”