Biophysical Studies of Metalloenzymes

生物固氮--将氮气还原成氨--是由一种名为固氮酶的复杂金属酶催化的。这种酶的农学、经济和社会意义可以通过认识到今天约三分之二的S人口的生活依赖于涉及生物固定氮的植物生长来实现。然而，固氮酶是最复杂的金属酶，尽管经过了近40年的深入研究，但它拒绝了所有关于生物还原氮素机理的研究，而且对这一过程的中间阶段一无所知。然而，最近，该项目参与了捕获催化中间体的第一次成功，并获得了它们的结构的第一批细节。这一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)光谱仪获得的。这些技术允许核磁共振确定氮结合/还原活性中心FeMo-辅因子([7Fe-9S-Mo-X-Homocitate])的完整配位几何构型和成键，并可以提供金属结合底物、中间体和产物的详细结构。本项目有三个组成部分：(I)捕获的氮还原中间体的Endor/ESEEM表征：本研究旨在确定在固氮酶催化循环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)活性中心钼铁辅因子(FEMO-co)活性中心的电子结构。平行的目标是确定FeMo-Co在整个催化循环中的金属离子价态。(Iii)FeMo-Co的间隙原子的身份。在确定了FeMo-co的基本结构很久之后，更高分辨率的X射线结构显示其中心只有一个N、O或C原子。该项目的这一组成部分旨在确定这种原子的身份。广泛影响：该项目将涉及开创性地开发工具Endor和ESEEM，以及共同赋予这些技术力量的分析程序和算法。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分发分析程序来实现的。最重要的是，它参与了作为研究生和博士后研究员的下一代从业者的培训。通过这些贡献，它正在从根本上改变矿物生物化学/生物无机化学的学科。

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.