E COLI CYTOCHROME BD OXIDASE

大肠杆菌细胞色素BD氧化酶

• 批准号: 3432754
• 负责人: ROBERT B GENNIS
• 金额: $ 2.23万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-30 至 1996-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3432754
• 关键词: Escherichia coli; acidity /alkalinity; azides; carbon monoxide; cell membrane; chemical kinetics; circular dichroism; cyanides; cytochrome oxidase; enzyme structure; heme; liposomes; membrane reconstitution /synthesis; peroxides; protein structure function

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.

该项目的主要目的是获得结构和结构的连贯视图 细胞色素BD氧还原域的作用机制 从大肠埃希菌中提取的泛喹酚氧化酶。这种酶是唯一一口井 以呼吸氧化酶为特征的，不属于 血红素-铜氧化物酶。血红素-铜氧化酶家族的所有成员，它们 包括真核细胞色素c氧化酶，具有共同的双金属 中心，由一个血红素铁和一个铜原子组成，这就是 分子氧被还原为水。相比之下，细胞色素BD含有 没有铜。该酶共含有三个血红素假体基团： 亚铁血红素b558、亚铁血红素b595和亚铁血红素d亚铁血红素b558似乎只是涉及 在电子转移到氧被还原的位置的过程中 来自众多实验室的研究表明，亚铁血红素d是五配位的 直接与氧气结合。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 外源配体，包括氧气。许多拟议的工作都是 旨在定义两种血红素b595的配基结合性质 和血红素d.光谱学方法，包括磁性圆二色谱 (MCD)和圆二向色性(CD)将被用来表征 络合物的形成及其在不同条件下的形成动力学 环境条件。初步数据已经证明了这种能力 来定义每个原子的配位和自旋态 在外源配体存在的情况下的亚铁血红素。各种不同的 配体将被用来探测还原和氧化形式的 氧化物酶，包括氰化物、叠氮化物、一氧化碳和氟化物以及其他。 氧化形式的酶将通过与氧化酶反应而产生。 用过氧化氢和分子氧。这些因素的依赖 胞质周质和胞质侧面的pH反应 重组蛋白脂质体中的膜将使用 既定的程序。通过使用这些方法，血红素b595的作用 这种血红素与血红素d之间的任何协同作用都应该变得清晰起来。 很可能，亚铁血红素b595和亚铁血红素d形成一个类似于 众所周知的血红素-铜中心存在于大多数其他氧化酶中。 与我们的外国合作者康斯坦丁诺夫博士积极合作， 在过去的两年里已经成立，并初步 结果证明了这种方法的实用性，并证明了这些 研究可以在他在莫斯科国立大学的实验室进行。