Chakrabarty Suman, Namslauer Ida, Brzezinski Peter, Warshel Arieh
Department of Chemistry, University of Southern California, 418 SGM Building, 3620 McClintock Avenue, Los Angeles, CA 90089-1062, USA.
Biochim Biophys Acta. 2011 Apr;1807(4):413-26. doi: 10.1016/j.bbabio.2011.01.004. Epub 2011 Jan 10.
Gaining detailed understanding of the energetics of the proton-pumping process in cytochrome c oxidase (CcO) is a problem of great current interest. Despite promising mechanistic proposals, so far, a physically consistent model that would reproduce all the relevant barriers needed to create a working pump has not been presented. In addition, there are major problems in elucidating the origin of key mutational effects and in understanding the nature of the apparent pK(a) values associated with the pH dependencies of specific proton transfer (PT) reactions in CcO. This work takes a key step in resolving the above problems, by considering mutations, such as the Asn139Asp replacement, that blocks proton pumping without affecting PT to the catalytic site. We first introduce a formulation that makes it possible to relate the apparent pK(a) of Glu286 to different conformational states of this residue. We then use the new formulation along with the calculated pK(a) values of Glu286 at these different conformations to reproduce the experimentally observed apparent pK(a) of the residue. Next, we take the X-ray structures of the native and Asn139Asp mutant of the Paracoccus denitrificans CcO (N131D in this system) and reproduce for the first time the change in the primary PT pathways (and other key features) based on simulations that start with the observed structural changes. We also consider the competition between proton transport to the catalytic site and the pump site, as a function of the bulk pH, as well as the H/D isotope effect, and use this information to explore the relative height of the two barriers. The paper emphasizes the crucial role of energy-based considerations that include the PT process, and the delicate control of PT in CcO.
深入了解细胞色素c氧化酶（CcO）中质子泵浦过程的能量学是当前备受关注的问题。尽管有一些很有前景的机制性提议，但到目前为止，尚未提出一个能重现产生有效泵所需的所有相关势垒的物理上自洽的模型。此外，在阐明关键突变效应的起源以及理解与CcO中特定质子转移（PT）反应的pH依赖性相关的表观pK(a)值的本质方面存在重大问题。这项工作通过考虑诸如Asn139Asp替换等突变迈出了解决上述问题的关键一步，这种突变会阻断质子泵浦而不影响向催化位点的PT。我们首先引入一种表述方式，使得能够将Glu286的表观pK(a)与该残基的不同构象状态联系起来。然后，我们使用这种新的表述方式以及在这些不同构象下计算出的Glu286的pK(a)值来重现该残基实验观察到的表观pK(a)。接下来，我们获取了反硝化副球菌CcO（该系统中为N131D）的天然和Asn139Asp突变体的X射线结构，并首次基于从观察到的结构变化开始的模拟重现了主要PT途径的变化（以及其他关键特征）。我们还考虑了作为整体pH函数的向催化位点和泵浦位点的质子传输之间的竞争以及H/D同位素效应，并利用这些信息来探索两个势垒的相对高度。本文强调了包括PT过程在内的基于能量的考虑因素的关键作用，以及CcO中PT的精细控制。
