Biophysical Studies of Metalloenzymes

金属酶的生物物理研究

• 批准号: 0723330
• 负责人: Brian Hoffman
• 金额: $ 63.85万
• 依托单位: Northwestern University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0723330&HistoricalAwards=false
• 关键词: Biophysical; Studies; Metalloenzymes

Biological nitrogen fixation - the reduction of N2 to yield ammonia - is catalyzed by a complex metalloenzyme called nitrogenase. The agronomic, economic, and social significance of this enzyme can be appreciated by recognizing that the lives of about two-thirds of today''s human population depend on plant growth that involves biologically fixed nitrogen. Nitrogenase is, however, the most complex metalloenzyme, and despite nearly 40 years of intense investigation, it has repulsed all efforts to characterize the mechanism of biological nitrogen reduction, and nothing was known about the intermediate stages in the process. Recently, however, this project has participated in the first successes at trapping catalytic intermediates, and it has obtained the first details of their structure. This information is acquired through the use of electron-nuclear double resonance (ENDOR) and electron spinecho envelope modulation (ESEEM) spectroscopies. These techniques permit the NMR determination of the complete coordination geometry and bonding of the metal ions of the N2-binding/reduction active site, the FeMo-cofactor ([7Fe-9S-Mo-X-homocitrate], and can supply detailed structures of metal-bound substrates, intermediates, and products.This project has three components:(i) ENDOR/ESEEM Characterization of Trapped N2 Reduction Intermediates: This research aims to determine the structures of substrate-derived species bound to the active-site FeMo-cofactor in multiple intermediates throughout the catalytic cycle of nitrogenase.(ii) Electronic Structure of the Active-Site Molybdenum-Iron cofactor (FeMo-co) Active Site. The parallel goal is to determine the metal-ion valencies of FeMo-co throughout the catalytic cycle.(iii)The Identity of the Interstitial Atom of FeMo-co. Long after the basic structure of FeMo-co had been determined, a higher-resolution X-ray structure revealed the presence of a single N, O, or C atom at its center. This component of the project is aimed at determining the identity of this atom.Broader Impact:This project will involve pioneering development of the tools, ENDOR and ESEEM, and of the analysis procedures and algorithms that together give these techniques their power. The development of these capabilities is diseminated through publications and presentations, through formal and informal collaborations, as well as distribution of analysis programs via the web. Most importantly, it is involved in training the next generation of practitioners as graduate students and postdoctoral fellows. Through these contributions it is fundamentally altering the disciplines of metallobiochemistry/bioinorganic chemistry.

生物固氮-N2还原产生氨-由一种称为固氮酶的复合金属酶催化。这种酶的农学、经济和社会意义可以通过认识到今天大约三分之二的人类人口的生命取决于涉及生物固定氮的植物生长来理解。然而，固氮酶是最复杂的金属酶，尽管近40年的深入研究，它已经击退了所有的努力，以表征生物氮还原的机制，并没有什么知道的中间阶段的过程。然而，最近，该项目参与了捕获催化中间体的第一次成功，并获得了它们结构的第一个细节。这些信息是通过使用电子核双共振（ENDOR）和电子自旋回波包络调制（ESEEM）光谱学获得的。这些技术允许NMR测定N2-结合/还原活性位点的金属离子的完整配位几何形状和键合，FeMo-辅因子（[7 Fe-9 S-Mo-X-homocitrate]），并可提供金属结合底物、中间体和产物的详细结构。该项目有三个组成部分：（i）被捕获的N2还原中间体的ENDOR/ESEEM表征：本研究旨在确定固氮酶催化循环过程中多个中间体中与活性中心FeMo-辅因子结合的底物衍生物种的结构。(ii)活性位点钼-铁辅因子（FeMo-co）活性位点的电子结构。平行的目标是确定整个催化循环中FeMo-Co的金属离子价态。（iii）FeMo-co的间隙原子的身份。在FeMo-co的基本结构被确定很久之后，更高分辨率的X射线结构揭示了在其中心存在单个N、O或C原子。更广泛的影响：该项目将涉及开发ENDOR和ESEEM工具以及分析程序和算法，这些工具共同赋予这些技术强大的功能。这些能力的发展是通过出版物和演示，通过正式和非正式的合作，以及通过网络的分析程序的分布diseminated。最重要的是，它参与培训下一代从业人员，作为研究生和博士后研究员。通过这些贡献，它从根本上改变了金属生物化学/生物无机化学的学科。