X-RAY STUDIES OF NITROGENASE METALLOCLUSTER BIOSYNTHESIS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 大气固氮是由少数具有固氮酶（一种独特的金属酶）的微生物完成的一项巨大的化学壮举。固氮酶的能力来源于几个形成电子传递链的铁硫簇。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课题。关于这两种复杂的生物合成酶的生物合成知之甚少。在解开它们的组装机制方面取得的进展将是非常宝贵的，不仅可以更好地理解固氮，而且可以更好地理解整个生物金属簇组装。提出了一系列的X射线吸收研究，以确定具体的逐步过程，通过这些集群的构建。第一个检查将集中在P-簇的生物合成。在以前的工作中，一个非天然构象的Pcluster上的FeMoco无固氮酶MoFe蛋白（指定Av 1）被确定。在淬灭之前，将这种形式的MoFe蛋白与nifH基因产物、固氮酶Fe蛋白（指定为Av 2）以不同的时间长度进行预孵育。通过Fe K-边XAS/EXAFS测定平均铁配位、氧化态和原子环境的变化将呈现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。第二次检查将集中在FeMoco的组装上。已经开发了一种独特的测定方法，该方法允许以前未见过的仅含铁前体的显著部分转化为NifEN支架蛋白上的成熟FeMoco。通过Mo K-边XAS/EXAFS确定这种新型重构前体的结构可以识别FeMoco结构中的中间体，并建立FeMoco反应性的结构和条件分析。

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.