Smith Archer D, Jameson Guy N L, Dos Santos Patricia C, Agar Jeffrey N, Naik Sunil, Krebs Carsten, Frazzon Jeverson, Dean Dennis R, Huynh Boi Hanh, Johnson Michael K
Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA.
Biochemistry. 2005 Oct 4;44(39):12955-69. doi: 10.1021/bi051257i.
NifU is a homodimeric modular protein comprising N- and C-terminal domains and a central domain with a redox-active 2Fe-2S 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 and 4Fe-4S clusters in the U-type N-terminal scaffolding domain and the assembly of 4Fe-4S 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 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.
NifU是一种同二聚体模块化蛋白，由N端和C端结构域以及一个含有氧化还原活性2Fe-2S簇的中央结构域组成。作为一种支架蛋白，它在组装固氮特异性铁硫蛋白成熟所需的铁硫簇中起着关键作用。在这项工作中，利用紫外可见吸收光谱和穆斯堡尔光谱，并结合分析研究，研究了体外NifS介导的铁硫簇在全长NifU以及仅涉及N端结构域或中央和C端结构域的截短形式上的组装时间进程和产物。结果表明，不稳定的2Fe-2S和4Fe-4S簇在U型N端支架结构域中依次组装，而4Fe-4S簇在Nfu型C端支架结构域中组装。NifU的两个支架结构域都被证明能够在体外使固氮酶组分蛋白成熟，这一点通过预先组装在N端或C端结构域上的4Fe-4S簇快速转移到脱辅基固氮酶铁蛋白上得到证明。诱变研究表明，一个保守的天冬氨酸（Asp37）在介导簇转移中起关键作用。将NifU上簇的组装和转移与已报道的U型和Nfu型支架蛋白的结果进行了比较，并讨论了NifU上两个功能性铁硫簇支架结构域的必要性。
