XAS COMPARISON OF NITROGENASE MOFE PROTEIN MUTANTS

固氮酶 MOFE 蛋白突变体的 XAS 比较

• 批准号: 8362047
• 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: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362047
• 关键词: Data; Electronics; Funding; Genes; Grant; In Vitro; Incubated; Iron; Molybdoferredoxin; National Center for Research Resources; Nitrogenase; Principal Investigator; Radiation; Research; Research Infrastructure; Resources; Source; Structure; United States National Institutes of Health; absorption; base; cost; mutant; novel; research study; structural biology

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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 正在研究两个氮酶MOFE蛋白突变体，这是由于NiFH或NIFB基因的缺失而产生的。这些类型的突变体是铁溶血辅因子（FEMOCO）缺乏的，但对于[8FE-7] p群集含有足够的铁。晶体学数据支持存在“正常”的P簇的存在，类似于NIFB-MOFE蛋白中野生型MOFE蛋白观察到的。 EPR研究表明，NIFH-MOFE蛋白中的簇可能是P簇前体。 NIFB突变体可以通过添加分离的Femoco在体外直接激活，而NIFH-突变体不能直接激活。已经使用Fe K-Edge X射线吸收边缘和扩展的精细结构（EXAFS）分析研究了这两个突变体之间的电子和结构差异，该结构证实了NIFB-MOFE蛋白中的野生型P簇和新型[FE4S4]中的NIFH-MOFH-MOFE蛋白中的野生型P簇。在相关的一组实验中，表明与NIFH的孵育不会改变Nifh突变体中簇的结构，尽管它确实在Femoco插入后能够适度的硝化酶活性。这种预孵育的NIFH-MOFE蛋白是具有完整p簇的功能性MOFE蛋白的第一个例子。