Spectroscopic Studies of Molybdoenzymes and Models

描述（由申请人提供）：我们对pyranopterin钼酶结构与功能之间的关系的理解存在根本性的空白。我们研究的长期目标是了解酶的机制，以改善人类健康和环境的质量。我们追求这些目标的目的是全面了解活性位点的几何和电子结构如何有助于酶的正常功能。这将通过详细的光谱（电子吸收，MCD，拉曼，XAS， EPR等）和对所有三个pyranopterin Mo酶家族的酶的键合研究相结合来完成。这项工作将辅以对小分子类似物的平行研究。我们的组合光谱方法旨在提供对催化的关键电子结构贡献的详细见解，我们的计算研究将校准光谱和反应性数据，以获得高水平的机械细节。中心假设是在活性位点几何和电子结构之间存在复杂的相互作用，这些相互作用促进了这些酶催化的独特反应。这项研究的基本原理是，全面了解电子结构如何影响pyranopterin Mo酶的反应性，将有助于更深入地了解创新药物和前药物设计，了解与Mo酶活性相关的疾病状态，并从总体上改善人类健康和环境。我们将测试我们的中心假设，以便通过成功追求三个特定目标来实现本提案的既定目标：1)确定钼羟化酶的反应坐标；2)全面了解亚硫酸盐氧化酶家族酶YedY和mARC的活性位点对催化的贡献；3)确定钼硫共价对二甲基亚砜还原酶家族中电子转移（ET）和氧化还原电位调节的关键贡献。我们的研究计划是创新的，因为它1)利用结合光谱方法和复杂的计算研究，在内源性发色团最小或没有干扰的情况下探测关键酶状态，2)提出研究一种新的Mo酶（mARC）， 3)有助于更好地了解pyranopterin二硫代烯在催化中的作用。这项研究具有重要意义，因为它将使人们对活性位点几何和电子结构如何直接影响钼酶底物特异性和反应坐标的性质、催化反应的性质（氧化/还原）以及pyranopterin二硫代烯辅助因子在催化中的作用有更深入的了解。

DESCRIPTION (provided by applicant): There exists a fundamental gap in our understanding of the relationship between pyranopterin molybdenum enzyme structure and function. The long-term goals of our research are to understand enzyme mechanism in order to improve the quality of human health and the environment. Our objective in pursuit of these goals is to develop a comprehensive understanding of how active site geometric and electronic structure contributes to proper enzyme function. This will be accomplished through a combination of detailed spectroscopic (electronic absorption, MCD, Raman, XAS, EPR, etc.) and bonding studies on enzymes from all three pyranopterin Mo enzyme families. This work will be complemented by parallel studies on small molecule analogues. Our combined spectroscopic approach is designed to provide detailed insight into key electronic structure contributions to catalysis and our computational studies will be calibrated to spectroscopic and reactivity data in order to obtain a high level of mechanistic detail. The central hypothesis is that a complex interplay exists between active site geometric and electronic structure that functions to facilitate the unique reactions these enzymes catalyze. The rationale for this research is that a comprehensive understanding of how electronic structure contributes to reactivity in pyranopterin Mo enzymes will lead to greater insight into innovative drug and pro-drug design, understanding disease states related to Mo enzyme activity, and generally improving human health and the environment. We will test our central hypothesis in order to accomplish the stated objective of this proposal through the successful pursuit of the three Specific Aims 1) Determine the reaction coordinate for the molybdenum hydroxylases, 2) Develop a comprehensive understanding of active site contributions to catalysis in the sulfite oxidase family enzymes YedY and mARC, and 3) Identify key molybdenum-sulfur covalency contributions to electron transfer (ET) and redox potential modulation in dimethylsulfoxide reductase family enzymes. Our research plan is innovative because it 1) utilizes a combined spectroscopic approach coupled with sophisticated computational studies to probe key enzyme states with minimal or no interference from endogenous chromophores, 2) proposes to study a new Mo enzyme found in humans (mARC), and 3) contributes to a greater understanding of the pyranopterin dithiolene in catalysis. This proposed research is significant because it will lead to a markedly greater understanding of how active site geometric and electronic structure directly affect molybdoenzyme substrate specificity and the nature of the reaction coordinate, the nature of the reaction catalyzed (oxidation/reduction), and the role of the pyranopterin dithiolene cofactor in catalysis.