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.

项目摘要/摘要活性部位中具有复杂金属能力的酶催化与人类健康相关的无数转化疾病。了解他们的反应机制需要分子级的静止状态表征和中间状态和金属特异性光谱技术在这项工作中特别有用。但是，高许多金属生产子的核性可以限制此类技术的有用性；由多种金属产生的信号站点可以挑战解决，尤其是在反应中间体的混合物中。而且，通常不可能映射丰富的光谱信息到几何结构，这严重限制了我们对化学物质的理解键合，因此是复合金属能力的反应性。我们建议通过发展来应对这些挑战修改复杂金属生产剂的同位素和元素组成的方法，尤其是氮酶催化辅助因子。氮酶负责在行星上提供很大一部分固定氮，并且因此，它们在维持健康和增长的人口中发挥着重要作用。它们的催化辅助因子是在本质上最复杂的中，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氮固定机制的新见解，我们将发展精确改变氮酶辅因子的同位素和元素组成的化学方法。我们的做法意愿要发现温和的方案，用于去除氮酶辅助因子中的特定Fe位点，然后用 57fe。标签的位点选择性将允许连接电子结构（阐明的光谱）到几何结构（从晶体学上定义），从而为此提供前所未有的见解氮酶辅因子的化学键合和反应性。这些辅助因子在其静止状态和中间状态构成了提案的核心。我们还将将现场选择性57FE标签协议扩展到将不同的金属掺入氮酶辅因子的特定位点。这将产生人工金属酶作为具有潜在独特特性和/或反应性的机械问题。该项目的完成将提供对生物氮固定的前所未有的机械洞察力，并将阐明概念和方案复杂的金属生产者与单核活性位点一样可机械牵引。