EXAFS SPECTROSCOPIC STUDIES OF SUBSTRATE BOUND MOFE NITROGENASE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。氮素的生物还原为氨是在MoFe固氮酶中以Fe7S9MoX簇实现的，称为FeMocofactor。尽管已经有了高分辨率的晶体结构，但底物结合的位置和随后还原的机制仍然不清楚。Fe和Mo位都被认为是底物化学的中心，并认为辅因子可能采用更开放的构象作为周转的一部分。EXAFS显然是帮助解决这些问题的理想技术，底物结合应该是直接可见的，辅助因子的构象变化应该通过钼-铁和铁-铁相互作用的长程变化来明显。然而，以前的EXAFS结构研究一直受到酶固有的不能在纯还原底物结合状态下分离的限制。与我们的合作者Lance Seefeldt一起，我们建议使用EXAFS来结合适当的SGM修饰的MoFe固氮酶。Seefeldt实验室的工作已经制备了SGM变体，如70Ala，它允许高产率地制备结合中间态。目前，这些物质包括结合的丙炔醇和联氨以及其他中间体。此外，与我们的合作者Paul Ludden一起，我们计划开发FeMoco生物合成蛋白Nafy与FeMoco配位的能力，提供一个MoFe活性中心的小蛋白质模型。这些研究应该使我们能够确定是铁还是钼(或两者都有！)协调底物和中间体，以及FEMO辅因子是否作为周转的一部分进行了结构修饰。这些信息应该有助于最终阐明这种对环境重要的酶的机制。此外，所发现的新的生物无机化学很可能导致新型催化剂的开发。

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. The biological reduction of dinitrogen to ammonia is achieved in MoFe nitrogenase at a remarkable Fe7S9MoX cluster termed FeMocofactor. Despite the availability of high resolution crystal structures, the site of substrate binding and mechanism of subsequent reduction remain unclear. Both Fe and Mo sites have been proposed as the locus of substrate chemistry, and it been suggested that the cofactor may adopt a more open conformation as part of turnover. EXAFS is clearly an ideal technique to help resolve these issues ¿¿ substrate binding should be directly observable and cofactor conformational change should be apparent through changes in ¿¿longrange¿¿ Mo-Fe and Fe-Fe interactions. However, previous EXAFS structural studies have been limited by the inherent inability of the enzyme to be isolated in pure reduced, substrate bound states. With our collaborator Lance Seefeldt, we propose to use EXAFS to substrates bound to appropriately SGM modified MoFe nitrogenase. Work in the Seefeldt laboratory has prepared SGM variants, such as 70Ala, which, allow preparation of bound intermediate states in high yield. These currently include bound propargyl alcohol and hydrazine as well as other intermediates. In addition, with our collaborator Paul Ludden, we plan to exploit the ability of the FeMoco biosynthesis protein NafY to coordinate FeMoco, provide a small protein model of the MoFe active center. These studies should enable us to determine the whether Fe or Mo sites (or both!) coordinate substrates and intermediates and whether the FeMo-cofactor is structurally modified as part of turnover. This information should assist with the eventual elucidation of the mechanism of the environmentally important enzyme. In addition, the novel bioinorganic chemistry uncovered could well lead to the development of novel catalysts.