XAS COMPARISON OF NITROGENASE MOFE PROTEIN MUTANTS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 两个固氮酶MoFe蛋白突变体，从缺失的nifH或nifB基因，正在研究中。这些类型的突变体是铁钼辅因子（FeMoco）缺乏，但含有足够的铁[8 Fe-7S] P-簇。晶体学数据支持在nifB-MoFe蛋白中存在“正常”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-簇前体。nifB-突变体可以通过加入分离的FeMoco在体外直接活化，而nifH-突变体不能。这两个突变体之间的电子和结构的差异进行了研究，使用Fe K-边X射线吸收边和扩展精细结构（EXAFS）分析，这证实了存在的野生型P-簇的nifB-MoFe蛋白和一个新的[Fe 4S 4]为基础的簇的nifH-MoFe蛋白。在一组相关的实验中，显示与NifH孵育不改变nifH-突变体中簇的结构，尽管其在FeMoco插入后确实能够实现适度的固氮酶活性。这种预孵育的nifH-MoFe蛋白是没有完整P簇的功能性MoFe蛋白的第一个例子。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.