Spectroscopic Studies of Molybdoenzymes and Models

描述(申请人提供)：在我们对吡喃蝶呤钼酶结构和功能之间的关系的理解上存在着根本的差距。我们研究的长期目标是了解酶的作用机制，以提高人类健康和环境的质量。我们追求这些目标的目标是全面了解活性中心的几何结构和电子结构是如何对适当的酶功能做出贡献的。这将通过组合详细的光谱(电子吸收、MCD、拉曼、XAS、EPR等)来实现。以及三种吡喃蝶呤钼酶家族的成键研究。这项工作将得到小分子类似物平行研究的补充。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，我们的计算研究将根据光谱和反应性数据进行校准，以获得高水平的机理细节。中心假设是，活性中心的几何结构和电子结构之间存在复杂的相互作用，其功能是促进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有助于吡喃蝶呤钼酶的反应，将有助于更深入地了解创新药物和前药设计，了解与钼酶活性相关的疾病状态，并总体上改善人类健康和环境。我们将验证我们的中心假设，以通过成功地追求三个具体目标来实现本提案所述的目标：1)确定钼羟基酶的反应坐标，2)全面了解亚硫酸盐氧化酶家族酶YedY和Marc中活性中心对催化的贡献，以及3)确定二甲基亚砜还原酶家族酶中钼-硫共价对电子转移(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)利用光谱方法结合复杂的计算研究来探索关键的酶状态，而内源发色团的干扰最小或没有干扰；2)建议研究在人类中发现的一种新的钼酶(MARC)；3)有助于更好地理解吡喃喋呤二硫杂环烯在催化中的作用。这项研究具有重要意义，因为它将使人们更好地理解活性中心的几何结构和电子结构如何直接影响钼酶底物的专一性和反应配位的性质，催化(氧化/还原)反应的性质，以及吡喃喋呤二硫杂环戊烯辅助因子在催化中的作用。

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.