EXAFS SPECTROSCOPIC STUDIES OF SUBSTRATE BOUND MOFE NITROGENASE

底物结合 MOFE 固氮酶的 EXAFS 光谱研究

• 批准号: 7598053
• 负责人: SIMON J GEORGE
• 金额: $ 0.22万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598053
• 关键词: Adopted; Ammonia; Binding; Bioinorganic Chemistry; Biological; Chemistry; Computer Retrieval of Information on Scientific Projects Database; Development; Enzymes; Funding; Grant; Hydrazine; Hydrazines; Institution; Laboratories; Lead; Modeling; Molecular Conformation; Molybdoferredoxin; Nitrogenase; Preparation; Protein Biosynthesis; Proteins; Research; Research Personnel; Resolution; Resources; Site; Source; Structure; Techniques; United States National Institutes of Health; Variant; Work; catalyst; cofactor; novel; propargyl alcohol

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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 生物还原的二氮氨是在一个显着的Fe 7S 9 MoX集群被称为FeMocofactor在MoFe固氮酶。尽管有高分辨率的晶体结构，底物结合的位点和随后的还原机制仍然不清楚。铁和钼网站已被提议为基板化学的轨迹，有人建议，辅因子可能会采取更开放的构象作为营业额的一部分。EXAFS显然是一种理想的技术，以帮助解决这些问题-底物结合应直接观察和辅因子构象变化应通过变化在<$远程<$Mo-Fe和Fe-Fe相互作用明显。然而，以前的EXAFS结构研究受到限制的酶的固有的无法被分离在纯还原，底物结合状态。与我们的合作者Lance Seefeldt一起，我们建议使用EXAFS来检测与适当SGM修饰的MoFe固氮酶结合的底物。Seefeldt实验室的工作已经制备了SGM变体，例如70 Ala，其允许以高产率制备结合的中间态。这些目前包括结合的炔丙醇和肼以及其他中间体。此外，我们计划与我们的合作者Paul Ludden一起利用FeMoco生物合成蛋白NafY与FeMoco的协调能力，提供一个MoFe活性中心的小蛋白模型。这些研究应该使我们能够确定是否铁或钼网站（或两者兼而有之！）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结构修饰。这些信息将有助于最终阐明这种对环境具有重要意义的酶的作用机制。此外，新的生物无机化学发现可能会导致新的催化剂的发展。