X-RAY STUDIES OF NITROGENASE METALLOCLUSTER BIOSYNTHESIS

固氮酶金属簇生物合成的 X 射线研究

• 批准号: 8170209
• 负责人: KEITH O HODGSON
• 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/8170209
• 关键词: Anabolism; Biological; Biological Assay; Chemicals; Complex; Computer Retrieval of Information on Scientific Projects Database; Coupled; Electron Transport; Environment; Funding; Generations; Grant; Institution; Iron; Length; Metals; Molecular Conformation; Molybdoferredoxin; Nitrogen Fixation; Nitrogenase; Process; Proteins; Research; Research Personnel; Resources; Role; Scaffolding Protein; Series; Source; Structure; Study Subject; Sulfur; Time; United States National Institutes of Health; Work; absorption; metalloenzyme; microorganism; nitrogenase reductase; novel; oxidation; reconstitution

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. Atmospheric nitrogen fixation is a tremendous chemical feat accomplished by a few select microorganisms possessing nitrogenase, a unique metalloenzyme. Nitrogenase derives its competency from several iron-sulfur clusters that form a chain of electron transport. Two of these metal clusters, the P-cluster and FeMoco, possess an exceptional structure and composition that make them exciting subjects of study. Little is understood about the biosynthesis of these two complex metalloclusters. Progress made in unraveling the mechanism of their assembly would be invaluable in better understanding not only nitrogen fixation, but also biological metal cluster assembly as a whole. A series of x-ray absorption studies are proposed to ascertain the specific stepwise process through which these clusters are constructed. The first examination will focus on the biosynthesis of P-cluster. In a previous work, a non-native conformation of the Pcluster on a FeMoco-free nitrogenase MoFe protein (designated Av1) was identified. Pre-incubation of this form of MoFe protein at various time lengths with the nifH gene product, the nitrogenase Fe protein (designated Av2), will be performed prior to quench. Determination of the changes in the average iron coordination, oxidation state, and atomic environment by Fe K-edge XAS/EXAFS would render a time-lapse capture of P-cluster assembly and suggest a role of the Fe protein in P-cluster maturation. This will be coupled to concomitant EPR and activity assay analysis, providing a complete picture of P-cluster generation. The second examination will focus on the assembly of FeMoco. A unique assay has been developed that permits a previously unseen transformation of a significant fraction of the iron-only precursor into the mature FeMoco on the NifEN scaffold protein. Determination of the structure of this novel reconstituted precursor by Mo K-edge XAS/EXAFS may identify intermediates in FeMoco construction and establish structural and conditional requisites for FeMoco reactivity.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 大气氮固定是一种巨大的化学壮科，该氮由含氮酶（一种独特的金属酶）的一些精选的微生物实现。氮酶从形成电子传输链的几个铁硫簇中获得了能力。这些金属簇中的两个，P簇和Femoco具有特殊的结构和组成，使它们成为令人兴奋的研究主题。关于这两个复杂金属群的生物合成几乎没有理解。在阐明其组装机制方面取得的进展对于更好地理解氮的固定，而且是整个生物金属簇组装的更好理解。提出了一系列X射线吸收研究，以确定构建这些簇的特定逐步过程。首次检查将集中于p群集的生物合成。在先前的工作中，鉴定了PCLUSTER在无股骨氮酶MOFE蛋白（指定的AV1）上的非母构象。将这种形式的MOFE蛋白在各个时间长度上与NIFH基因产物，氮酶Fe蛋白（指定的AV2）进行预孵育。通过Fe K-Edge XAS/EXAFS来确定平均铁配位，氧化状态和原子环境的变化将使P簇组装的延时捕获，并提出Fe蛋白在P簇成熟中的作用。这将与伴随的EPR和活动分析分析相结合，从而提供了P群集生成的完整图片。第二次检查将集中于Feamoco的组装。已经开发了一种独特的测定法，该测定法可以将一小部分仅铁的前体的大部分转化为Nifen支架蛋白上成熟的Femoco。 Mo K-Edge XAS/EXAFS对这种新型重构前体的结构的确定可以鉴定Femoco构造中的中间体，并为Femoco反应性确定结构和条件要求。