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

• 批准号: 10590756
• 负责人: Daniel Leif Migdow Suess
• 金额: $ 30.6万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/10590756
• 关键词: 3-Dimensional; Active Sites; Address; Air; Ammonia; Apoproteins; Articulation; Binding; Biological; Biological Availability; Carrier Proteins; Catalysis; Chemicals; Chemistry; Complex; Crystallization; Crystallography; Disease; Electron Nuclear Double Resonance; Electrons; Elements; Enzymes; Fertilizers; Generations; Health; Heart; Human; Incubated; Iron; Isotopes; Kinetics; Label; Life; Maps; Metals; Methodology; Methods; Modification; Molecular; Mononuclear; Nature; Nitrogen; Nitrogen Fixation; Nitrogenase; Nuclear; Oxidation-Reduction; Planets; Play; Population; Preparation; Property; Protocols documentation; Reaction; Reporting; Research; Rest; Role; Sampling; Signal Transduction; Site; Spectrum Analysis; Structure; Sulfur; Techniques; Testing; biophysical properties; chemical bond; cofactor; electronic structure; geometric structure; insight; iron deficiency; metalloenzyme; novel; reconstitution; spectroscopic survey; three dimensional structure; tool

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 活性部位具有复杂金属辅酶因子的酶催化与人类健康有关的无数转化和 疾病。要了解它们的反应机理，需要在分子水平上表征它们的休眠状态和 中间态和特定于金属的光谱技术在这一努力中特别有用。然而，高- 许多金属辅因的核作用可能限制这种技术的有效性；来自多种金属的信号 特别是在反应中间体的混合物中，位点的解析可能是具有挑战性的。此外，通常不可能绘制地图 丰富的光谱信息传递到几何结构上，这严重限制了我们对化学物质的理解 复合金属因子的结合--因此也就是反应性。我们建议通过以下方式应对这些挑战 修饰复杂金属辅酶，特别是固氮酶的同位素和元素组成的方法 催化辅因子。固氮酶负责提供地球上很大一部分固定氮，并且 因此，它们在维持健康和不断增长的人口方面发挥着重要作用。它们的催化辅因子是 它们是自然界中最复杂的化合物之一，因此它们的反应机理特别难表征。 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 精确改变固氮酶辅因子的同位素和元素组成的化学方法。我们的方法将 将发现温和的方案，用于去除固氮酶辅助因子中的特定铁位点，并随后将其替换为 57Fe。标记的位置选择性将允许电子结构(如光谱所阐明的)连接 几何结构(根据结晶学的定义)，并因此将提供前所未有的洞察 固氮酶辅助因子的化学键和反应性。这些辅因子在静息状态和静息状态下的研究 中间国家构成了这项提议的核心。我们还将把位点选择性57Fe标记方案扩展到 将不同的金属结合到固氮酶辅助因子的特定位置。这将产生人造金属酶，它将 作为具有潜在独特性质和/或反应性的机械探头。该项目的完成将提供 对生物固氮的前所未有的机械洞察力，并将阐明渲染的概念和协议 复杂的金属因子，作为单核活性部位，在机械上易于处理。