NifS-Mediated Assembly of [4Fe−4S] Clusters in the N- and C-Terminal Domains of the NifU Scaffold Protein

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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.