Physical Studies of Xanthine Oxidase and Trimethylamine Dehydrogenase

在PI实验室过去成功的基础上，对两种氧化还原酶，黄嘌呤氧化酶和三甲胺脱氢酶的生物物理性质进行了研究。这项工作的目的是充分表征电子转移动力学在两个主题蛋白和确定其在催化中的作用。来自牛奶的黄嘌呤氧化酶催化黄嘌呤的氧化羟基化形成尿酸，这一反应发生在酶的钼中心。由于种种原因，该酶已成为酶的典型钼中心。由于种种原因，该酶已成为钼羟基化底物的原型。该酶的四个氧化还原活性中心之间的电子转移研究将沿着两条不同的路线进行：(1)在D2O中进行脉冲辐射分解研究，并作为温度的函数，目的是扩展PI过去的工作；(2)利用在黄嘌呤氧化酶的钼中心附近共价连接的钌中心，通过闪光光解非常迅速地将还原等价物引入酶中。该方案的优点是电子转移的起始是一个分子内过程，使得在低温下进行研究成为可能。其他工作将集中在三甲胺脱氢酶的几个相关方面，三甲胺脱氢酶是一种来自甲基营养细菌W3A1的酶，它催化三甲胺氧化去甲基化为二甲胺和甲醛。该酶具有一个共价连接的黄素单核苷酸辅助因子和一个4Fe/4S（铁氧化还原蛋白型）中心，因此代表了一个有用的系统，可以扩展过去对黄嘌呤氧化酶的研究。工作内容包括：(1)利用脉冲辐射法研究两个氧化还原活性中心之间的电子转移速率；(2)还原三甲胺脱氢酶与其生理氧化剂电子转移环黄蛋白的反应表征；(3)为三甲胺脱氢酶开发一种光稳定的慢底物，该底物将适用于劳伊实验，在劳伊实验中，反应在活性位点的进展是通过时间分辨晶体学来跟踪的。设计这些实验是为了实现两个目标。第一个与质子化/去质子化事件如何调节电子进入和离开具有氧化性的有机辅助因子（如黄素和醌）有关，使用黄嘌呤氧化酶和三甲胺脱氢酶作为实验系统。第二项研究涉及三甲胺脱氢酶的两个辅助因子如何相互作用，以及这种相互作用在催化周转中的作用。这项工作涉及动力学和晶体学方法的结合，旨在更全面地了解这种酶的机制功能。＊＊＊

Hille 9420185 A study of the biophysical properties of two oxidoreductases, xanthine oxidase and trimethylamine dehydrogenase, builds on past success in the PI's laboratory. The goal of this work is to fully characterize the kinetics of electron transfer within the two subject proteins and ascertain its role in catalysis. Xanthine oxidase from cow's milk catalyzes the oxidative hydroxylation of xanthine to form uric acid, a reaction that takes place at the molybdenum center of the enzyme. For a variety of reasons, the enzyme has become the prototypical molybdenum center of the enzyme. For a variety of reasons, the enzyme has become the prototypical molybdenum hydroxylation of substrate. Studies of electron transfer among the four redox-active centers of this enzyme will be pursued along two different lines: (1) pulse radiolysis studies in D2O and as a function of temperature will be performed with the aim of extending past work by the PI; and (2) use will be made of a ruthenium center that has been covalently attached near the molybdenum center of xanthine oxidase to very rapidly introduce reducing equivalents into the enzyme by flash photolysis. The advantage of this protocol is that the initiation of electron transfer is an intramolecular process, making possible studies at cryogenic temperatures. Other work will focus on several related aspects of trimethylamine dehydrogenase, an enzyme from the methylotrophic bacterium W3A1 that catalyzes the oxidative demethylation of trimethylamine to dimethylamine and formaldehyde. The enzyme possesses a covalently linked flavin mononucleotide cofactor and a 4Fe/4S (ferredoxin-type) center, and thus represents a useful system with which to extend past work on xanthine oxidase. The work includes: (1) studies of the rates of electron transfer between the two redox-active centers using pulse radiolysis; (2) characterization of the reaction of reduced trimethylamine dehydrogenase with its physiological oxidant, electron-transfe rring flavoprotein; and (3) development of a photolabile slow substrate for trimethylamine dehydrogenase that will be suitable for a Laue experiment in which the progress of the reaction at the active site is followed by timeresolved crystallography. %%% The experiments are designed to address two goals. The first has to do with how protonation/deprotonation events modulate electron transfer into and out of organic cofactors that are redoxactive (e.g., flavins and quinones), using xanthine oxidase and trimethylamine dehydrogenase as the experimental systems. The second involves a study of how the two cofactors of the enzyme trimethylamine dehydrogenase interact with one another, and the role of this interaction in catalytic turnover. This work involves a combination of kinetic and crystallographic approaches aimed at gaining a more complete understanding of how this enzyme functions mechanistically. ***