title: NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU scaffold protein.
abstract: NifU is a homodimeric modular protein comprising N- and C-terminal domains and a central domain with a redox-active [2Fe-2S](2+,+) cluster. It plays a crucial role as a scaffold protein for the assembly of the Fe-S clusters required for the maturation of nif-specific Fe-S proteins. In this work, the time course and products of in vitro NifS-mediated iron-sulfur cluster assembly on full-length NifU and truncated forms involving only the N-terminal domain or the central and C-terminal domains have been investigated using UV-vis absorption and Mössbauer spectroscopies, coupled with analytical studies. The results demonstrate sequential assembly of labile [2Fe-2S](2+) and [4Fe-4S](2+) clusters in the U-type N-terminal scaffolding domain and the assembly of [4Fe-4S](2+) clusters in the Nfu-type C-terminal scaffolding domain. Both scaffolding domains of NifU are shown to be competent for in vitro maturation of nitrogenase component proteins, as evidenced by rapid transfer of [4Fe-4S](2+) clusters preassembled on either the N- or C-terminal domains to the apo nitrogenase Fe protein. Mutagenesis studies indicate that a conserved aspartate (Asp37) plays a critical role in mediating cluster transfer. The assembly and transfer of clusters on NifU are compared with results reported for U- and Nfu-type scaffold proteins, and the need for two functional Fe-S cluster scaffolding domains on NifU is discussed.
authors.affiliation: Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA.
authors.forename: Archer D
authors.initials: AD
authors.lastname: Smith
authors.forename: Guy N L
authors.initials: GN
authors.lastname: Jameson
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Dos Santos
authors.forename: Jeffrey N
authors.initials: JN
authors.lastname: Agar
authors.forename: Sunil
authors.initials: S
authors.lastname: Naik
authors.forename: Carsten
authors.initials: C
authors.lastname: Krebs
authors.forename: Jeverson
authors.initials: J
authors.lastname: Frazzon
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Dean
authors.forename: Boi Hanh
authors.initials: BH
authors.lastname: Huynh
authors.forename: Michael K
authors.initials: MK
authors.lastname: Johnson
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis / Q000737:chemistry*; D009586:Nitrogen Fixation; D009591:Nitrogenase / Q000096:biosynthesis*; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis; D000637:Transaminases / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; C088637:NifU protein, Bacteria; C080001:nifS protein, Bacteria; D009591:Nitrogenase; D000637:Transaminases