Popović Dragan M, Stuchebrukhov Alexei A
Department of Chemistry, University of California-Davis, One Shields Avenue, Davis, CA 95616, USA.
J Am Chem Soc. 2004 Feb 18;126(6):1858-71. doi: 10.1021/ja038267w.
Cytochrome c oxidase (CcO) is the terminal enzyme of the cell respiratory chain in mitochondria and aerobic bacteria. It catalyzes the reduction of oxygen to water and utilizes the free energy of the reduction reaction for proton pumping across the inner-mitochondrial membrane, a process that results in a membrane electrochemical proton gradient. Although the structure of the enzyme has been solved for several organisms, the molecular mechanism of proton pumping remains unknown. In the present paper, continuum electrostatic calculations were employed to evaluate the electrostatic potential, energies, and protonation state of bovine heart cytochrome c oxidase for different redox states of the enzyme along its catalytic cycle. Three different computational models of the enzyme were employed to test the stability of the results. The energetics and pH dependence of the P-->F, F-->O, and O-->E steps of the cycle have been investigated. On the basis of electrostatic calculations, two possible schemes of redox-linked proton pumping are discussed. The first scheme involves His291 as a pump element, whereas the second scheme involves a group linked to propionate D of heme a(3). In both schemes, loading of the pump site is coupled to ET between the two hemes of the enzyme, while transfer of a chemical proton is accompanied by ejection of the pumped H(+). The two models, as well as the energetics results are compared with recent experimental kinetic data. The proton pumping across the membrane is an endergonic process, which requires a sufficient amount of energy to be provided by the chemical reaction in the active site. In our calculations, the conversion of OH(-) to H(2)O provides 520 meV of energy to displace pump protons from a loading site and overall about 635 meV for each electron passing through the system. Assuming that the two charges are translocated per electron against the membrane potential of 200 meV, the model predicts an overall efficiency of 63%.
细胞色素c氧化酶（CcO）是线粒体和需氧细菌中细胞呼吸链的末端酶。它催化氧气还原为水，并利用还原反应的自由能将质子泵过线粒体内膜，这一过程导致膜电化学质子梯度的形成。尽管已经解析了几种生物体中该酶的结构，但质子泵浦的分子机制仍然未知。在本文中，采用连续介质静电计算来评估牛心细胞色素c氧化酶在其催化循环中不同氧化还原状态下的静电势、能量和质子化状态。使用了该酶的三种不同计算模型来测试结果的稳定性。研究了循环中P→F、F→O和O→E步骤的能量学和pH依赖性。基于静电计算，讨论了两种可能的氧化还原偶联质子泵浦方案。第一种方案涉及His291作为泵浦元件，而第二种方案涉及与血红素a3的丙酸D相连的基团。在这两种方案中，泵浦位点的加载与酶的两个血红素之间的电子转移耦合，而化学质子的转移伴随着泵浦出的H+的排出。将这两种模型以及能量学结果与最近的实验动力学数据进行了比较。跨膜质子泵浦是一个吸能过程，需要活性位点中的化学反应提供足够的能量。在我们的计算中，OH-转化为H2O提供520 meV的能量，将泵浦质子从加载位点置换出来，每个电子通过系统时总体约为635 meV。假设每个电子对抗200 meV的膜电位转移两个电荷，该模型预测总体效率为63%。
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