NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU scaffold protein.
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.
Smith
Archer D
AD
Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA.
Jameson
Guy N L
GN
Dos Santos
Patricia C
PC
Agar
Jeffrey N
JN
Naik
Sunil
S
Krebs
Carsten
C
Frazzon
Jeverson
J
Dean
Dennis R
DR
Huynh
Boi Hanh
BH
Johnson
Michael K
MK
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*
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; C088637:NifU protein, Bacteria; C080001:nifS protein, Bacteria; D009591:Nitrogenase; D000637:Transaminases