XAS COMPARISON OF NITROGENASE MOFE PROTEIN MUTANTS

固氮酶 MOFE 蛋白突变体的 XAS 比较

• 批准号: 8169920
• 负责人: BRITT HEDMAN
• 金额: $ 0.03万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169920
• 关键词: Computer Retrieval of Information on Scientific Projects Database; Data; Electronics; Funding; Genes; Grant; In Vitro; Incubated; Institution; Iron; Molybdoferredoxin; Nitrogenase; Research; Research Personnel; Resources; Source; Structure; United States National Institutes of Health; absorption; base; mutant; novel; research study

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 目前正在研究由 nifH 或 nifB 基因缺失产生的两种固氮酶 MoFe 蛋白突变体。这些类型的突变体缺乏铁钼辅助因子 (FeMoco)，但含有足够的铁来形成 [8Fe-7S] P 簇。晶体学数据支持 nifB-MoFe 蛋白中存在“正常”P 簇，类似于在野生型 MoFe 蛋白中观察到的情况。 EPR 研究表明 nifH-MoFe 蛋白中的簇可能是 P 簇前体。 nifB-突变体可以通过添加分离的FeMoco在体外直接激活，而nifH-突变体则不能。使用 Fe K 边 X 射线吸收边和扩展精细结构 (EXAFS) 分析研究了这两种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，并且nifH-MoFe 蛋白中一种新型的基于 [Fe4S4] 的簇。在一组相关的实验中，结果表明，与 NifH 一起孵育不会改变 nifH 突变体中簇的结构，尽管在 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 蛋白是第一个没有完整 P 簇的功能性 MoFe 蛋白的例子。