Fig. 1: Cryo-EM structures elucidate various stages of sulfur transfer from NFS1 to ISCU2 during de novo [2Fe-2S] cluster formation.
a Model for the synthesis of a [2Fe-2S] cluster on the ISCU2 scaffold protein by the core ISC complex in various steps. Free ISCU2 (Fe-U) with Fe2+ (red circle) bound at its assembly site residues associates with the heterodimeric cysteine desulfurase complex NFS1-ISD11-ACP1 ((NIA)2; only one half of the dimer is shown) to form the (Fe-NIAU)2 complex. Addition of Cys enables rapid persulfidation of NFS1 forming the persulfide-containing (Fe-NSIAU)2 complex and Ala. FXN (X) addition stimulates sulfur transfer from NFS1 to a Cys residue at the ISCU2 assembly site creating the (Fe-NIAUSX)2 intermediate. FDX2-mediated persulfide reduction and interaction of two ISCU2 of different (Fe-NIAUSX)2 complexes leads to the formation of holo-ISCU2 with a bound [2Fe-2S] cluster. b 2.4 Å consensus cryo-EM map (C2 symmetry applied) of the (Fe-NIAUX)2 complex during persulfide transfer, segmented and colored by subunit. Unless indicated otherwise, subunit coloring is consistent throughout the manuscript. Symmetry expansion and subsequent focused 3D classification allowed further separation of the (Fe-NIAU)2 (c), (Fe-NIAUSX)2 (d), and (Fe-NSIAUSX)2 (e) reaction intermediates (left). Middle: the cartoon representation shows a detailed view of the NFS1-ISCU2 interface with the different locations of the Cys loop of NFS1, either in an outward position with Cys381NFS1 coordinating the Fe2+ at the ISCU2 assembly site (c, e), or in an inward orientation (d) with Cys381NFS1 close to the PLP cofactor of NFS1. Right: the close-up of the respective ISCU2 assembly site regions (with or without Cys381NFS1) depicts the different modes of Fe2+ coordination in these sulfur transfer intermediates. The cryo-EM maps reveal clear densities for a persulfide (CSS) on Cys381NFS1 and/or Cys138ISCU2 (d, e).
