CYTOCHROME OXIDASE INTERMEDIATES

此应用程序的目标是确定结构和 02还原反应中间体的动力学研究 H20与细胞色素氧化酶反应，从而勾画出催化 这种酶的作用机制。在拟议的研究中， 将采取综合方法，在相同的情况下 中间体将通过几种物理技术和 将以不同的方式生成。要做到这一点，我们将结合 我们大学研究小组的资源和技能 在宾夕法尼亚州和AT&T贝尔实验室。我们计划使用 光吸收、共振拉曼散射和电子 顺磁共振用于研究反应中间体 三种不同类型的酶制剂-哈泽尔- Beinert、Yonetani和Yoshikawa-Ceh的准备工作。 为了研究我们将制造的短命中间体 流量/闪光/探头测量，时间分辨率为1微秒。 这一阶段工作的主要目标将是确定和 表征涉及02结合到的第一中间体 细胞色素氧化酶。我们希望确定属性是否为 初始结合络合物可以解释02的快速减少 这种情况就会发生。一旦最初的中间体被表征，我们 将继续描述其他早期中间体的特征。这个 较长时间的中间体(大于10毫秒)将 通过快速混合完全还原的细胞色素氧化酶进行研究 用氧饱和溶液，在反应之后 休眠状态。快速混合研究的最初目标是 来描述“脉冲”细胞色素氧化酶的特性。最新研究 没有产生一套一致的特性和 因此，没有关于属性或 这一重要中间体的结构。在其他工作中，我们 计划在放松中研究其他长寿中间体 从氧结合的还原状态到静止状态的酶 州政府。低温中间体将由 三重诱捕技术。三重诱捕的目标 研究将确定放松路径(和 中间体的性质)在低温下是相同的 因为它们是在室温下。通过追求全面的 方法，预计在许多不一致 文学将会得到解决，而彻底理解 得到了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.