NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU Scaffold protein

Archer D. Smith, Guy N.L. Jameson, Patricia C. Dos Santos, Jeffrey N. Agar, Sunil Naik, Carsten Krebs, Jeverson Frazzon, Dennis R. Dean, Boi Hanh Huynh, Michael K. Johnson

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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.

Original languageEnglish (US)
Pages (from-to)12955-12969
Number of pages15
JournalBiochemistry
Volume44
Issue number39
DOIs
StatePublished - Oct 4 2005

All Science Journal Classification (ASJC) codes

  • Biochemistry

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    Smith, A. D., Jameson, G. N. L., Dos Santos, P. C., Agar, J. N., Naik, S., Krebs, C., Frazzon, J., Dean, D. R., Huynh, B. H., & Johnson, M. K. (2005). NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU Scaffold protein. Biochemistry, 44(39), 12955-12969. https://doi.org/10.1021/bi051257i