INFRARED STUDY OF OXIDASE DYNAMICS

地球上大多数的有氧生命依赖于这种酶的功能 被称为血红素-铜氧化物酶的超家族，组成末端 所有动植物和许多动物细胞呼吸的氧化酶系统 更简单的有机体。这项提议的中心目标是应用静态 红外光谱和时间分辨红外(TRIR)技术(沿 利用互补振动拉曼方法)来研究动力学 血红素-铜氧化酶的特性，特别是表征特定的 这些动态的结构/功能方面。有争议的程序 包括：外源配体的行为(例如O2)。它的入口处 蛋白质、配位和激活；金属的行为 中心进行配位、活化和电子 传递函数；电子转移反应和现象 确定它们的速率；取代质子泵的运行 与跨膜pH梯度相反的“矢量质子”； 质子泵浦到电子转移自由能；传递 “标量质子”，它与O2结合，形成H2O到活性中心： 以及多肽本身的结构反应，以及它的 对上述过程的侧链。 我们的具体目标是澄清以下问题： 小分子外源配体进出氧结合部位的途径 和还原，金属的配位化学和动力学 中心，酶的O2激活部位的结构特征， 血红素A假体基团和铜-B中心的结构动力学， 关于残基的身份和作用的假设的检验 在质子运动过程中，蛋白质动力学的本质 伴随(或控制)金属中心和 质子动力反应和活化势垒的性质 牵涉其中。从这一点，我们希望能够推断出 它们的功能动力学和结构-功能关系 系统，并有助于理解分子中的这些问题 细节。这一结果也将阐明基本的生物物理学。 原则和开发实验方法，将适用于 生物动力学中的其他问题。

Most aerobic life on earth depends upon the functions of the enzyme superfamily known as the heme-copper oxidases, which comprise the terminal oxidase systems of cellular respiration in all plants and animals and many simpler organisms. The central aim of this proposal is to apply static infrared spectroscopy and time-resolved infrared (TRIR) techniques (along with the complementary vibrational Raman approaches) to study the dynamics of the heme-copper oxidases and in particular to characterize specific structural/functional aspects of these dynamics. The processes at issue include: the behavior of the exogenous ligand (e.g. O2). its entrance into the protein, coordination, and activation; the behavior of the metal centers as they perform their coordination, activation, and electron transfer functions; the electron transfer reactions and the phenomena that determine their rates; the operation of the proton pump that displaces the "vector protons" against the transmembrane pH gradient; the coupling of proton pumping to the electron transfer free energy; the delivery of the "scalar protons", which combine with O2 to form H2O to the active site: and the structural responses of the polypeptide itself, and its sidechains, to the foregoing processes. Our specific aims are to elucidate the following issues: details of the route of small exogenous ligands into and out of the site of O2 binding and reduction, the coordination chemistry and dynamics of the metal centers, structural features of the O2 activating site of the enzyme, the structural dynamics of the heme A prosthetic group and the Cu-B center, tests of hypotheses concerning the identity and role of residues involved in protonmotive processes, the nature of the protein dynamics which accompany (or control) the reactions of the metal centers and the protonmotive reactions, and the nature of the activation barriers involved. From this we expect to be able to infer critical new features of the functional dynamics and structure-function relationships in these systems, and to contribute to understanding of these issues in molecular detail. The results will also illuminate fundamental biophysical principles and develop experimental approaches which will be applicable to other problems in biodynamics.