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

项目总结/摘要 在其活性位点具有复杂金属辅因子的酶催化与人类健康相关的无数转化， 疾病要了解它们的反应机制，需要在分子水平上表征它们的静止状态， 中间态和金属特异性光谱技术在这奋进特别有用。然而，高- 许多金属辅因子的核性可能限制这种技术的有用性;由多种金属辅因子引起的信号可能会影响这些技术的有效性。 尤其是在反应中间体的混合物中，位点的解析可能具有挑战性。此外，通常无法绘制 丰富的光谱信息的几何结构，这严重限制了我们的理解的化学 键合--以及因此的反应性--复杂的金属辅因子。我们建议通过发展 修饰复合金属辅因子特别是固氮酶的同位素和元素组成的方法 催化辅因子固氮酶负责提供地球上很大一部分的固定氮， 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。它们的催化辅因子是 它们是自然界中最复杂的，因此它们的反应机理特别难以表征。 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 精确改变固氮酶辅因子的同位素和元素组成的化学方法。我们的方法将 发现温和的方案，用于去除固氮酶辅因子中的特定Fe位点，随后用 57Fe。标记的位点选择性将允许电子结构（如光谱所示）连接 几何结构（如晶体学定义），从而将提供前所未有的见解， 固氮酶辅因子的化学键合和反应性。对这些辅助因子在静息状态和 中间状态是该提案的核心。我们还将扩展位点选择性57 Fe标记方案， 将不同的金属结合到固氮酶辅因子的特定位点。这将产生人工金属酶， 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 前所未有的机械见解生物固氮，并将阐明概念和协议，使 复杂的金属辅因子作为单核活性位点的机械易处理性。

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.