INFRARED STUDY OF OXIDASE DYNAMICS

地球上大多数有氧生物都依赖于酶的功能 已知为血红素-铜氧化酶的超家族，其包含末端 氧化酶系统的细胞呼吸在所有的植物和动物和许多 更简单的生物该提案的主要目的是应用静态 红外光谱和时间分辨红外（TRIR）技术（沿着 与互补振动拉曼方法）来研究动力学 的血红素-铜氧化酶，特别是表征特定的 这些动态的结构/功能方面。有争议的进程 包括：外源配体（如O2）的行为。其进入 蛋白质、配位和活化;金属的行为 中心，因为它们执行协调，激活和电子 转移函数;电子转移反应和现象， 决定他们的利率;质子泵的运作，取代了 “载体质子”对抗跨膜pH梯度; 质子泵到电子转移自由能; “标量质子”，其与O2结合联合收割机以形成H2O至活性位点： 以及多肽本身的结构反应， 侧链，到前述过程。 我们的具体目标是阐明以下问题： 小的外源配体进出O2结合位点的途径 和还原，金属的配位化学和动力学 中心，酶的O2激活位点的结构特征， 血红素A辅基和Cu-B中心的结构动力学， 有关残留物的特性和作用的假设检验 在质子动力过程中，蛋白质动力学的性质， 伴随（或控制）金属中心和金属中心的反应。 质子动力反应和活化势垒的性质 涉案从这一点，我们希望能够推断出关键的新特征， 功能动力学和结构-功能关系 系统，并有助于了解这些问题的分子 详细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.