X-RAY STUDIES OF NITROGENASE METALLOCLUSTER BIOSYNTHESIS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 大气固氮是一项巨大的化学壮举，由少数具有固氮酶的微生物完成，固氮酶是一种独特的金属酶。固氮酶的能力来源于形成电子传递链的几个铁-硫团簇。其中两个金属簇，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对象。关于这两个复杂金属簇的生物合成，人们知之甚少。在揭示它们的组装机制方面取得的进展不仅对于更好地理解固氮，而且对于更好地理解生物金属簇组装作为一个整体都将是非常宝贵的。提出了一系列的X射线吸收研究，以确定这些团簇形成的具体分步过程。第一个检查将集中在P-簇的生物合成上。在以前的工作中，在一个不含FeMoco的固氮酶MoFe蛋白(命名为Av1)上鉴定了P簇的非天然构象。这种形式的MoFe蛋白在不同的时间长度与nifH基因产物--固氮酶Fe蛋白(命名为AV2)预孵育，将在猝灭之前进行。用Fe K-Edge XAS/EXAFS测定铁的平均配位、氧化态和原子环境的变化，可以提供P-簇组装的时间推移捕获，并提示Fe蛋白在P-簇成熟中的作用。这将与伴随的EPR和活性分析相结合，提供P-簇生成的完整图景。第二项检查将集中在FeMoco的组装上。一种独特的检测方法已经被开发出来，它允许在Nifen支架蛋白上将相当一部分仅含铁的前体转化为成熟的FeMoco。用Mo K边XAS/EXAFS测定这一新型重组前驱体的结构，可以确定FeMoco结构中的中间体，并建立FeMoco反应的结构和条件条件。

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.