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: 8362249
• 负责人: KEITH O HODGSON
• 金额: $ 0.66万
• 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/8362249
• 关键词: Anabolism; Biological; Biological Assay; Chemicals; Complex; Coupled; Electron Transport; Environment; Funding; Generations; Grant; Iron; Length; Metals; Molecular Conformation; Molybdoferredoxin; National Center for Research Resources; Nitrogen Fixation; Nitrogenase; Principal Investigator; Process; Proteins; Radiation; Research; Research Infrastructure; Resources; Roentgen Rays; Role; Scaffolding Protein; Series; Source; Structure; Study Subject; Sulfur; Time; United States National Institutes of Health; Work; absorption; cost; metalloenzyme; microorganism; nitrogenase reductase; novel; oxidation; reconstitution; 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. 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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 大气固氮是一项巨大的化学壮举，由少数拥有固氮酶（一种独特的金属酶）的精选微生物完成。固氮酶的能力来自于形成电子传输链的几个铁硫簇。其中两种金属团簇，即 P 团簇和 FeMoco，具有特殊的结构和成分，使其成为令人兴奋的研究对象。关于这两种复杂金属簇的生物合成知之甚少。在阐明其组装机制方面取得的进展对于更好地理解固氮以及整个生物金属簇组装具有不可估量的价值。提出了一系列 X 射线吸收研究，以确定构建这些簇的具体逐步过程。第一次检查将集中于 P 簇的生物合成。在之前的工作中，鉴定了不含 FeMoco 的固氮酶 MoFe 蛋白（称为 Av1）上的 Pcluster 的非天然构象。这种形式的 MoFe 蛋白与 nifH 基因产物固氮酶 Fe 蛋白（指定为 Av2）在不同的时间长度下预孵育，将在淬灭之前进行。通过 Fe K 边缘 XAS/EXAFS 确定平均铁配位、氧化态和原子环境的变化，可以实现 P 簇组装的延时捕获，并表明 Fe 蛋白在 P 簇成熟中的作用。这将与伴随的 EPR 和活性测定分析相结合，提供 P 簇生成的完整图片。第二次检查将重点关注 FeMoco 的组装。已经开发出一种独特的检测方法，可以将大部分纯铁前体以前所未见的转化为 NifEN 支架蛋白上的成熟 FeMoco。通过 Mo K-edge XAS/EXAFS 确定这种新型重构前体的结构可以识别 FeMoco 构建中的中间体，并建立 FeMoco 反应性的结构和条件要求。