XAS COMPARISON OF NITROGENASE MOFE PROTEIN MUTANTS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 两个固氮酶MoFe蛋白突变体正在研究中，它们是由nifH或nifB基因缺失引起的。这些类型的突变体缺乏铁钼辅因子(FeMoco)，但含有足够的铁来形成[8Fe-7S]P-簇。结晶学数据支持nifB-MoFe蛋白中存在一个“正常”的P-簇，类似于在野生型MoFe蛋白中观察到的。EPR研究表明，nifH-MoFe蛋白中的簇可能是P簇的前体。NifB-突变体可以通过加入分离的FeMoco在体外直接激活，而nifH-突变体不能。用Fe K边X射线吸收边和扩展精细结构(EXAFS)分析了这两个突变体之间的电子和结构差异，证实了nifB-MoFe蛋白中存在野生型P-簇，nifH-MoFe蛋白中存在新的基于[Fe4S4]的簇。在一组相关的实验中，研究表明，与nifH孵育并不改变nifH-突变体中的簇结构，尽管它确实在FeMoco插入后能够产生适度的固氮酶活性。这种预先孵育的nifH-MoFe蛋白是第一个没有完整P-簇的功能MoFe蛋白的例子。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Two nitrogenase MoFe protein mutants, resulting from the deletion of either the nifH or nifB gene, are being studied. Mutants of these types are iron-molybdenum cofactor (FeMoco) deficient, yet contain sufficient iron for an [8Fe-7S] P-cluster. Crystallographic data support the presence of a "normal" P-cluster, similar to that observed in wild-type MoFe protein, in the nifB- MoFe protein. EPR studies suggest that the cluster in the nifH- MoFe protein could be a P-cluster precursor. nifB- mutants can be directly activated in vitro by the addition of isolated FeMoco whereas nifH- mutants cannot. The electronic and structural difference between these two mutants have been studied using Fe K-edge x-ray absorption edge and extended fine structure (EXAFS) analyses, which confirmed the presence of a wild-type P-cluster in the nifB- MoFe protein and a novel [Fe4S4]-based cluster in the nifH- MoFe protein. In a related set of experiments, it was shown that incubation with NifH does not change the structure of the cluster in the nifH- mutant, although it does enable modest nitrogenase activity after FeMoco insertion. This pre-incubated nifH- MoFe protein is the first example of a functioning MoFe protein without an intact P-cluster.