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Chemical Approaches to Studying the Mechanisms and Biophysical Properties of Complex Metallocofactors

研究复杂金属辅因子的机制和生物物理性质的化学方法

基本信息

批准号：
10798896
负责人：
Daniel Leif Migdow Suess
金额：
$ 8.29万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2026-01-31
项目状态：
未结题

项目摘要

Project Summary/Abstract Enzymes with complex metallocofactors in their active sites catalyze myriad transformations relevant to human health and disease. Understanding their reaction mechanisms requires molecular-level characterization of their resting states and intermediate states, and metal-specific spectroscopic techniques are especially useful in this endeavor. However, the high- nuclearity of many metallocofactors can limit the usefulness of such techniques; the signals arising from multiple metal sites can be challenging to resolve, especially in mixtures of reaction intermediates. Moreover, it is often impossible to map the rich spectroscopic information onto the geometric structure, and this severely limits our understanding of the chemical bonding—and therefore the reactivity—of complex metallocofactors. We propose to address these challenges by developing methods for modifying the isotopic and elemental compositions of complex metallocofactors, in particular the nitrogenase catalytic cofactors. Nitrogenases are responsible for supplying a significant portion of the fixed nitrogen on the planet, and they therefore play an important role in maintaining a healthy and growing human population. Their catalytic cofactors are among the most complex in Nature, and as a result their reaction mechanisms have been especially difficult to characterize. To overcome these challenges and gain new insights into the mechanism of biological nitrogen fixation, we will develop chemical methods for precisely altering the isotopic and elemental composition of nitrogenase cofactors. Our approach will be to discover mild protocols for removing specific Fe sites in nitrogenase cofactors and subsequently replacing them with 57Fe. The site-selectivity of the label will allow for the electronic structure (as elucidated spectroscopically) to be connected to the geometric structure (as defined crystallographically), and will thereby provide unprecedented insights into the chemical bonding and reactivity of nitrogenase cofactors. Studies of these cofactors in both their resting states and intermediate states comprise the heart of the proposal. We will also extend the site-selective 57Fe labeling protocol to incorporating different metals into specific sites of nitrogenase cofactors. This will yield artificial metalloenzymes that will serve as mechanistic probes with potentially unique properties and/or reactivity. Completion of this project will provide unprecedented mechanistic insights into biological nitrogen fixation and will articulate concepts and protocols for rendering complex metallocofactors as mechanistically tractable as mononuclear active sites.

项目概要/摘要活性位点具有复杂金属辅因子的酶可催化与人类健康和营养相关的无数转化。疾病。了解它们的反应机制需要对其静息态和分子水平进行表征中间态和金属特定光谱技术在这一努力中特别有用。然而，高许多金属辅因子的核性会限制此类技术的实用性；多种金属产生的信号位点的解析可能具有挑战性，特别是在反应中间体的混合物中。此外，通常无法绘制地图几何结构上丰富的光谱信息，这严重限制了我们对化学的理解复杂金属辅因子的键合以及反应性。我们建议通过开发来应对这些挑战修饰复杂金属辅因子，特别是固氮酶的同位素和元素组成的方法催化辅助因子。固氮酶负责提供地球上很大一部分的固定氮，并且因此，它们在维持人口健康和不断增长方面发挥着重要作用。它们的催化辅助因子是自然界中最复杂的物质之一，因此它们的反应机制特别难以表征。为了克服这些挑战并获得对生物固氮机制的新见解，我们将开发精确改变固氮酶辅助因子的同位素和元素组成的化学方法。我们的方法将是发现去除固氮酶辅因子中特定 Fe 位点并随后用它们替换的温和方案 57Fe。标签的位点选择性将允许连接电子结构（如光谱所阐明的）几何结构（如晶体学定义），从而提供前所未有的见解固氮酶辅因子的化学键和反应性。对这些辅因子在静息状态和静息状态下的研究中间状态是该提案的核心。我们还将把位点选择性 57Fe 标记方案扩展到将不同的金属掺入固氮酶辅因子的特定位点。这将产生人造金属酶，用作具有潜在独特性质和/或反应性的机械探针。该项目的完成将提供对生物固氮的前所未有的机械见解，并将阐明渲染的概念和协议复杂的金属辅因子与单核活性位点一样在机械上易于处理。