CYTOCHROME OXIDASE INTERMEDIATES

本申请的目的是确定结构和 在将O2还原为 H2O的催化作用，从而描绘出细胞色素氧化酶的催化活性。 这种酶的作用机制。 在拟议的研究中， 将采取综合办法， 中间体将通过几种物理技术进行研究， 将以不同的方式产生。 为此，我们将联合收割机 我们大学研究小组的资源和技能 和AT&T贝尔实验室的实验室。 我们计划使用 光吸收、共振拉曼散射和电子 顺磁共振研究反应中间体从 三种不同类型的酶制剂-Hartzell- Beinert、Yonetani和Yoshikawa-Caughey制剂。 为了研究我们将制造的短寿命中间体， 流量/闪光/探针测量，时间分辨率为1微秒。 这一阶段工作的主要目标是确定和 表征第一中间体，所述第一中间体涉及O2与 细胞色素氧化酶 我们希望确定， 初始结合复合物可以解释O2的快速减少 发生了什么 一旦初始中间体被表征， 将继续对其他早期中间体进行表征。 的 更长时间的中间体（大于10毫秒）将被 通过快速混合完全还原的细胞色素氧化酶 与氧饱和溶液反应，反应后， 休息状态。 快速混合研究的最初目标是 来表征“脉冲”细胞色素氧化酶。 最近的研究 没有产生一组一致的属性， 因此，没有关于财产或 这个重要的中间体的结构。 在额外的工作中，我们 计划研究弛豫过程中其他长寿命的中间体 从氧结合的还原态到静息态 状态 低温中间体将由 三重诱捕技术 三重诱捕的目的 研究将确定是否放松途径（和 中间体的性质）在低温下相同 因为它们在室温下。 通过追求这一全面 预计，在这种方法中， 文学将得到解决，一个彻底的理解， 细胞色素氧化酶的催化机理。

The objective of this application is to determine the structure and kinetics of the reaction intermediates in the reduction of 02 to H20 by cytochrome oxidase so as to delineate the catalytic mechanism of this enzyme. In the proposed research a comprehensive approach will be taken in which the same intermediates will be studied by several physical techniques and will be generated in different ways. To do this we will combine the resources and skills of our research groups at the University of Pennsylvania and at AT&T Bell Laboratories. We plan to use optical absorption, resonance Raman scattering and electron paramagnetic resonance to study reaction intermediates from three different types of enzyme preparations - the Hartzell- Beinert, Yonetani, and Yoshikawa-Caughey preparations. To study the short lived intermediates we will make flow/flash/probe measurements with a time resolution of 1 musec. The primary goal of this phase of the work will be to identify and characterize the first intermediate involving the binding of 02 to cytochrome oxidase. We wish to determine if properties of the initial bound complex can account for the rapid 02 reduction which occurs. Once the initial intermediate is characterize we will proceed to characterize other early intermediates. The longer time intermediates (is greater than 10 msec) will be studied by rapid mixing of the fully reduced cytochrome oxidase with an oxygen saturated solution and following the reaction to the resting state. The initial goal of the rapid mixing studies will be to characterize "pulsed" cytochrome oxidase. Recent studies of this species have not yielded a consistent set of properties and consequently there is no agreement on the properties or the structure of this important intermediate. In additional work we plan to study the other long-lived intermediates in the relaxation of the enzyme from the oxygen-bound reduced state to the resting state. Low temperature intermediates will be studied by the triple trapping technique. The objective of the triple trapping studies will be to determine if the relaxation pathways (and the properties of the intermediates) are the same at low temperature as they are at room temperature. By pursuing this comprehensive approach it is expected that the many inconsistencies in the literature will be resolved and that a thorough understanding of the catalytic mechanism of cytochrome oxidase will be achieved.