Structural/Functional Properties of Human NFU1, an Intermediate [4Fe-4S] Carrier in Human Mitochondrial Iron-Sulfur Cluster Biogenesis

Human mitochondrial NFU1 functions in the maturation of iron-sulfur proteins, and NFU1 deficiency is associated with a fatal mitochondrial disease. We determined three-dimensional structures of the N- and C-terminal domains of human NFU1 by nuclear magnetic resonance spectroscopy and used these structures along with small-angle X-ray scattering (SAXS) data to derive structural models for full-length monomeric apo-NFU1, dimeric apo-NFU1 (an artifact of intermolecular disulfide bond formation), and holo-NFUI (the [4Fe-4S] cluster-containing form of the protein). Apo-NFU1 contains two cysteine residues in its C-terminal domain, and two apo-NFU1 subunits coordinate one [4Fe-4S] cluster to form a cluster-linked dimer. Holo-NFU1 consists of a complex of three of these dimers as shown by molecular weight estimates from SAXS and size-exclusion chromatography. The SAXS-derived structural model indicates that one N-terminal region from each of the three dimers forms a tripartite interface. The activity of the holo-NFU1 preparation was verified by demonstrating its ability to activate apo-aconitase. [Display omitted] •We determined NMR structures of the isolated N- and C-terminal domains of human NFU1•The domain structures combined with SAXS data yielded a model for full-length apo-NFU1•A holo-NFU1 preparation was shown to be functional in activating apo-aconitase•A structural model for holo-NFU1 was derived from combined NMR and SAXS data Cai et al. used NMR spectroscopy and small-angle X-ray scattering data to determine the 3D structure of human mitochondrial NFU1 in its apo- and iron-sulfur cluster-containing holo-form. Apo-NFU1 is monomeric, whereas holo-NFU1 consists of a trimer of three [4Fe-4S] cluster-linked dimers.