title: Iron-sulfur cluster biosynthesis: functional characterization of the N- and C-terminal domains of human NFU.
abstract: Human NFU (also known as HIRIP5) has been implicated in cellular iron-sulfur cluster biosynthesis. Bacterial and yeast forms are smaller than the human protein and are homologous to the C-terminal domain of the latter. This C-terminal domain contains a pair of redox active cysteines and demonstrates thioredoxin-like activity by mediating persulfide bond cleavage of sulfur-loaded NifS (an IscS-type protein), the sulfide donor for [2Fe-2S] cluster assembly on ISU-type scaffold proteins. Herein, the affinity of full-length human NFU and the individual N- and C-terminal domains for sulfide donor and cluster scaffold proteins is assessed. The influence of the N-terminal domain on C-terminal NFU binding to NifS and persulfide reductase activity is also examined. Only the C-terminal domain is required for persulfide reductase activity, while complex formation of NifS with full-length NFU is similar to that of the C-terminal domain alone (K(D) approximately 9.7 +/- 0.7 and 10.1 +/- 0.6 microM, respectively). There is negligible affinity between the isolated C- and N-terminal domains, while the N-terminal domain has negligible affinity for either sulfide donor or cluster scaffold proteins. The temperature dependence of the binding enthalpy for formation of the complex between NifS and the C-terminal domain of NFU yields a change in molar heat capacity (DeltaC(p) approximately 138 cal mol(-1) K(-1)) that suggests bonding at the protein-protein interface is dominated by electrostatic interactions. This is consistent with electrostatic potential maps for bacterial homologues of the N- and C-terminal domains of human NFU, which most likely reflect the structural characteristics expected for full-length human NFU.
authors.affiliation: Evans Laboratory of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
authors.forename: Yushi
authors.initials: Y
authors.lastname: Liu
authors.forename: Wenbin
authors.initials: W
authors.lastname: Qi
authors.forename: J A
authors.initials: JA
authors.lastname: Cowan
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D003461:Crystallography; D006801:Humans; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D007506:Iron-Sulfur Proteins; C426067:NFU1 protein, human; D010446:Peptide Fragments; C080001:nifS protein, Bacteria