E COLI CYTOCHROME BD OXIDASE

该项目的主要目的是获得结构的连贯视图， 细胞色素bd的氧还原结构域的功能机制 来自大肠杆菌的泛醇氧化酶。这种酶是唯一 特征呼吸氧化酶不属于家庭 血红素铜氧化酶血红素铜氧化酶家族的所有成员， 包括真核细胞色素c氧化酶，其共同点是 中心，由血红素铁和铜原子组成，这是其中 分子氧被还原成水。相反，细胞色素bd含有 没有铜。这种酶总共含有三个血红素辅基： 血红素B558、血红素B595和血红素D。血红素b558似乎只是 在电子转移到氧被还原的地方过去的工作 许多实验室的研究表明血红素D是五配位的， 直接与氧结合。然而，血红素b595的作用尚不清楚。 血红素b595也是五配位的，因此，原则上，也可以结合 包括氧在内的外源配体。大部分拟议的工作是 目的在于确定血红素B595和B596的配体结合特性 和血红素D。光谱方法，包括磁性圆二色性 (MCD)和圆二色性（CD）将被用来表征这两个 形成的复合物及其在各种条件下形成的动力学 环境条件初步数据显示 这些技术来定义的协调和自旋状态的每个 血红素在外源性配体存在下。各种不同的 配体将用于探测还原和氧化形式的 氧化酶，包括氰化物、叠氮化物、CO和氟化物等。 氧化形式的酶将通过使氧化酶 过氧化物和分子氧。这些依赖 反应的pH值的周质和细胞质侧的 将在复溶的蛋白脂质体中使用 既定程序。通过使用这些方法，血红素b595的作用， 并且该血红素与血红素D的任何合作功能应该变得清楚。 很可能，血红素b595和血红素d形成类似于 在大多数其他氧化酶中发现的众所周知的血红素-铜中心。 与我们的外国合作者康斯坦丁诺夫博士积极合作， 在过去两年中已经建立， 结果证明了这种方法的实用性， 研究可以在他在莫斯科州立大学的实验室进行。

This project aims mainly at obtaining a coherent view of the structure and functional mechanism of the oxygen-reducing domain of the cytochrome bd ubiquinol oxidase from Escherichia coil. This enzyme is the only well characterized respiratory oxidase which does not belong to the family of heme-copper oxidases. All members of the heme-copper oxidase family, which includes the eukaryotic cytochrome c oxidase, have in common a bimetallic center, consisting of a heme iron and a copper atom, which is where molecular oxygen is reduced to water. Cytochrome bd, in contrast, contains no copper. This enzyme contains a total of three heme prosthetic groups: heme b558, heme b595, and heme d. Heme b558 appears to be simply involved in the transfer of electrons to the site where oxygen is reduced Past work from numerous laboratories has shown that heme d is five-coordinate and binds to oxygen directly. However, the role of heme b595 is not known. Heme b595 is also five-coordinate and, thus, in principle, Could also bind to exogenous ligands, including oxygen. Much of the proposed work is directed towards defining the ligand binding properties of both heme b595 and heme d. Spectroscopic methods, including magnetic circular dichroism (MCD) and circular dichroism (CD) will be used to characterize both the complexes that form and the kinetics of their formation under a variety of environmental conditions. Preliminary data have demonstrated the ability of these techniques to define the coordination and spin-state of each of the hemes in the presence of exogenous ligands. A variety of different ligands will be used to probe both the reduced and oxidized forms of the oxidase, including cyanide, azide, CO, and fluoride as well as others. Oxygenated forms of the enzyme will be generated by reacting the oxidase with peroxides and with molecular oxygen. The dependence of these reactions on the pH of the periplasmic and of the cytoplasmic sides of the membrane will be examined in reconstituted proteoliposomes using established procedures. By using these approaches, the role of heme b595 and any cooperative function of this heme with heme d should become clear. Quite possibly, heme b595 and heme d form a bimetallic center analogous to the well known heme-copper center found in most other oxidases. An active collaboration with Dr. Konstantinov, our foreign collaborator, has already been established over the past two years, and preliminary results have demonstrated both the utility of this approach and that these studies can be performed in his laboratory at Moscow State University.