Physical Studies of Xanthine Oxidase and Trimethylamine Dehydrogenase

两种配合物的生物物理性质研究 氧化还原酶，黄嘌呤氧化酶和三甲胺脱氢酶， 本文提出 这项工作的目标是充分表征 蛋白质中的几个氧化还原活性中心， 观点，特别是关于他们的光谱和 电子转移性质黄嘌呤氧化酶催化 黄嘌呤氧化羟基化形成尿酸的反应， 发生在酶的钼中心。 用于 由于种种原因，酶已成为 钼羟化酶，一个小的，但非常重要的一组， 利用来自水的氧而不是分子氧的酶 在底物的羟基化中。 其中两个具体方面 酶的工作重点是：（1）测定 四个氧化还原活性中心之间的电子转移速率 的酶和控制观察到的速率的因素（如 研究脉冲辐解和pH跳跃技术）;和（2） 钼中心的结构形式多种多样，包括 催化中间体，通过圆二色性进行检测， 共振拉曼光谱，其目的是表征 中心的物理和电子结构。 三甲胺 脱氢酶催化氧化去甲基化， 三甲胺转化为二甲胺和甲醛。 酶 具有共价连接的黄素单甘肽辅因子和 铁氧还蛋白型中心，因此代表了一个有用的系统， 调查《公约》各项原则的普遍适用性， 黄嘌呤氧化酶的行为。 拟议的研究包括： (1)电子分布的pH依赖性的研究 部分还原的酶，以及pH跃变和脉冲辐解 研究了两个中心之间的电子转移;（2）a 光谱贡献的确定和减少 黄素和铁硫中心的电位：（3）快速， 催化循环的稳态动力学研究， 阐明了电子转移在催化循环中的作用， 三甲基胺脱氢酶。

A study of the biophysical properties of two complex oxidoreductases, xanthine oxidase and trimethylamine dehydrogenase, is proposed. The goal of this work is to fully characterize the several redox-active centers in the proteins from a physical standpoint, with particular regard to their spectroscopic and electron-transfer properties. Xanthine oxidase 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 hydroxylase, a small but extremely important group of enzymes that utilize oxygen derived from water rather than dioxygen in the hydroxylation of substrate. Two specific aspects of this enzyme are the focus of the proposed work: (1) the determination of rates of electron transfer among the four redox-active centers of the enzyme and the factors that govern the observed rates (as studied by pulse radiolysis and pH-jump techniques); and (2) the structure of the molybdenum center in a variety of forms, including catalytic intermediates, to be examined by circular dichroism and resonance Raman spectroscopy, with the aim of characterizing the physical and electronic structure of the center. Trimethylamine dehydrogenase catalyzes the oxidative demethylation of trimethylamine to dimethylamine and formaldehyde. The enzyme possesses a covalently linked flavin mononucleotide cofactor and a ferredoxin-type center, and thus represents a useful system to investigate the general applicability of the principles governing the behavior of xanthine oxidase. The proposed studies include: (1) studies of the pH-dependence of electron distribution within partially reduced enzyme, and both pH-jump and pulse radiolysis studies of electron transfer between the two centers; (2) a determination of the spectral contributions and reduction potentials of the flavin and iron-sulfur centers: and (3) rapid and steady-state kinetic studies of the catalytic cycle aimed at elucidating the role of electron transfer in the catalytic cycle of trimethylamine dehydrogenase.