CATALYTIC MECHANISM OF HEME/COPPER OXIDASES

描述：呼吸氧化酶是一种膜结合的电子转移。 催化分子氧还原为水的络合物和 利用相关的自由能变化来产生跨膜质子 这种质子梯度是生物能源的主要来源 人体内的自由能量。最近几年，很明显，大多数人 呼吸氧化酶是一个单一家族的成员，即血红素-铜氧化酶 超级家庭。这个家庭的成员有一个独特的双金属中心 由亚铁血红素和铜组成，中心的氧是还原的和高的 亲和配基是细胞色素c氧化酶(CcO)催化的一种 细胞色素c铁的四个等价物的氧化 位于线粒体膜内侧的胞液侧，这 氧化伴随着基质中四个质子的消耗。 空间中完成两个水分子的形成。自由能 在形成水的过程中消耗的水不会消散，而是作为一种 单质子化学计量比下的跨膜质子梯度 转移的每一个电子；这第二个过程被称为 该酶的质子泵活性。氧气的完全转化 水通过特定的氧中间体进行，对应于 双核中心的离散化学态。尽管有一大堆 近几年来积累的有价值的知识 其中某些含氧中间体的性质仍然存在争议，而且非常 人们对质子泵浦机制本身以及 这些中间体在泵送过程中的参与。的目标是 该建议是为了确定选定的氧基中间体的性质， 它们的质子化状态、机理和氧化还原化学计量 相互转化，这些中间体与质子泵浦的关系 活动性，以检验电子中性性守恒的假设 在双核中心是一个基本要求，并表征 配基相互作用机理(S)。为了解决这些问题，我们计划 使用分离的线粒体，纯化的牛CcO和CcO并入 水泡。光学光谱学，电子顺磁共振与快速 猝灭动力学、磁性和自然圆二向色性、共振拉曼光谱 光谱学、停流动力学和几种生化方法 用来实现这些目标。

DESCRIPTION: Respiratory oxidases are membrane-bound electron-transfer complexes which catalyze the reduction of molecular dioxygen to water and use the associated free energy changes to generate a transmembrane proton gradient This proton gradient is the primary source of energy for biological free energ in humans. In recent years it has become apparent that most respiratory oxidases are members of a single family, the heme-copper oxidase super-family. Members of this family have a unique bimetallic center composed of heme plus copper; at this center dioxygen is reduced and high affinity ligands are bound Cytochrome c oxidase (CcO) catalyzes in one enzymatic cycle the oxidation of four equivalents of ferrocytochrome c located on the cytosolic side of the inner mitochondrial membrane and this oxidation is accompanied by the consumption of four protons from the matrix space to complete the formation of two water molecules. The free energy expended in the formation of water is not dissipated but conserved as a trans-membrane proton gradient with a stoichiometry of one proton translocated per electron transferred; this second process is called the proton pumping activity of this enzyme. The complete conversion of oxygen to water proceeds through specific oxy intermediates corresponding to discrete chemical states of the binuclear center. Despite a large body of valuable knowledge that has been accumulated in recent years the chemical nature of certain of these oxy intermediates is still controversial and very little is known about the proton pumping mechanism per se and the involvement of these intermediates in the pumping process. The objective of this proposal is to establish the nature of selected oxy intermediates, their protonation state, the mechanism and the redox stoichiometry of their interconversion, the relation of these intermediates to the proton pumping activity, to test the hypothesis that the conservation of electroneutrality at the binuclear center is a fundamental requirement and to characterize the mechanism of ligand interaction(s). To address these problems we plan to use isolated mitochondria, purified bovine CcO and CcO incorporated into vesicles. Optical spectroscopy, electon paramagnetic resonance with rapid quenching kinetics, magnetic and natural circular dichroism, resonance Raman spectroscopy, stopped-flow kinetics, and several biochemical methods will be used to accomplish these goals.