Spectroscopic Studies of Molybdoenzymes & Models

 描述(申请人提供)：关于钼辅助因子硫酶C末端(MOCC)结构和功能，以及复杂的吡喃蝶呤二硫杂环配体在所有吡喃喋呤钼酶的催化循环中的作用，在知识库中存在着根本的空白。我们的长期目标是开发一种了解吡喃蝶呤钼酶分子电子结构、作用机理和功能的方法，以期对人类的健康质量产生积极的影响。我们的主要目标是确定1)电子结构对吡喃喋呤二硫戊烯功能的贡献，2)几何结构和电子结构对Mosc结构域蛋白活性的贡献，以及3)为什么二甲基亚砜还原酶家族的酶需要两个吡喃喋呤二硫烯才能发挥作用。我们将通过组合光谱方法(电子吸收、MCD、拉曼、XAS、EPR等)来实现这些目标。通过振动、光谱、成键和反应配位计算来探索吡喃喋呤和催化机理。对明智地选择的小分子类似物的平行研究将补充这一方法。中心假设是钼位的几何和电子结构，再加上吡喃蝶呤二硫杂环烯的性质，有助于这些酶的独特功能。这项研究的基本原理是，全面了解MOSC家族蛋白以及钼离子和吡喃蝶呤二硫杂环化合物之间的复杂相互作用将为了解疾病状态提供新的见解，并对人类健康产生积极影响。我们将通过成功地追求三个具体目标来检验我们的中心假设，以实现本提案所述的目标：1)确定吡喃喋呤如何参与黄嘌呤氧化酶和亚硫酸盐氧化酶家族酶中的电子传递和氧化还原过程；2)了解定义其功能的Mosc结构域蛋白之间的结构和机制关系；3)了解两种不同的吡喃蝶呤类化合物之间的配位数和合作关系如何有助于二甲基亚砜还原酶家族酶的催化作用。我们的贡献有望在分子水平上详细了解MoC介导的催化作用、吡喃喋呤二硫烯氧化状态与其催化作用之间的联系，以及配位环境与二甲基亚砜还原酶家族中两种吡喃喋呤二硫烯需求之间的关系。这项研究具有重要意义，因为它将促进我们对翻译后硫化过程和钼辅助因子转运、前药激活和外源解毒机制的理解，以及吡喃喋呤二硫杂环烯在钼酶催化中的电子柔性。

 DESCRIPTION (provided by applicant): There exists a fundamental gap in the knowledge base regarding molybdenum cofactor sulfurase C-terminal (MOSC) domain protein structure and function, as well as the role of the complex pyranopterin dithiolene ligand in the catalytic cycles of all pyranopterin molybdenum enzymes. Our long-term goal is to develop a molecular electronic structure understanding of pyranopterin molybdenum enzyme mechanism and function in order to have a positive impact on the quality of human health. Our primary objectives are to determine 1) electronic structure contributions to pyranopterin dithiolene function, 2) geometric and electronic structure contributions to the reactivity of MOSC domain proteins, and 3) why dimethyl sulfoxide reductase family enzymes require two pyranopterin dithiolenes in order to function. We will accomplish these objectives by using a combined spectroscopic approach (electronic absorption, MCD, Raman, XAS, EPR, etc.) augmented by vibrational, spectroscopic, bonding, and reaction coordinate computations to probe the pyranopterin and catalytic mechanisms. Parallel studies on judiciously chosen small molecule analogs will complement this approach. The central hypothesis is that Mo site geometric and electronic structure, coupled with the nature of the pyranopterin dithiolene, contributes to the unique function of these enzymes. The rationale for this research is that a comprehensive understanding of MOSC family proteins and the complex interplay between the Mo ion and the pyranopterin dithiolene will provide new insights into disease states and have a positive impact on human health. We will test our central hypothesis in order to accomplish the stated objective of this proposal through the successful pursuit of three Specific Aims 1) Determine how the pyranopterin contributes to electron transfer and redox processes in xanthine oxidase and sulfite oxidase family enzymes, 2) Understand structural and mechanistic relationships between MOSC domain proteins that define their function, and 3) Develop an understanding of how coordination number and the cooperative relationship between two different pyranopterins contribute to catalysis in dimethyl sulfoxide reductase family enzymes. Our contribution is expected to provide a detailed understanding of MOSC mediated catalysis at the molecular level, the link between pyranopterin dithiolene oxidation state and the role of the pyranopterin dithiolene in catalysis, and the relationship between coordination environment and the need for two pyranopterin dithiolenes in dimethyl sulfoxide reductase family enzymes. The proposed research is significant since it will advance our understanding of post-translational sulfuration processes and molybdenum cofactor trafficking, mechanisms of prodrug activation and xenobiotic detoxification, and the electronic flexibility of the pyranopterin dithiolene in molybdoenzyme catalysis.