Goyal Puja, Yang Shuo, Cui Qiang
Department of Chemistry and Theoretical Chemistry Institute, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706.
Chem Sci. 2015 Jan;6(1):826-841. doi: 10.1039/C4SC01674B.
Understanding the mechanism of vectorial proton pumping in biomolecules requires establishing the microscopic basis for the regulation of both thermodynamic and kinetic features of the relevant proton transfer steps. For the proton pump cytochrome c oxidase, while the regulation of thermodynamic driving force for key proton transfers has been discussed in great detail, the microscopic basis for the control of proton transfer kinetics has been poorly understood. Here we carry out extensive QM/MM free energy simulations to probe the kinetics of relevant proton transfer steps and analyze the effects of local structure and hydration level. We show that protonation of the proton loading site (PLS, taken to be a propionate of heme a) requires a concerted process in which a key glutamic acid (Glu286H) delivers the proton to the PLS while being reprotonated by an excess proton coming from the D-channel. The concerted nature of the mechanism is a crucial feature that enables the loading of the PLS before the cavity containing Glu286 is better hydrated to lower its pK to experimentally measured range; the charged rather than dipolar nature of the process also ensures a tight coupling with heme a reduction, as emphasized by Siegbahn and Blomberg. In addition, we find that rotational flexibility of the PLS allows its protonation before that of the binuclear center (the site where oxygen gets reduced to water). Together with our recent study (P. Goyal, , 110:18886-18891, 2013) that focused on the modulation of Glu286 pK , the current work suggests a mechanism that builds in a natural sequence for the protonation of the PLS prior to that of the binuclear center. This provides microscopic support to the kinetic constraints revealed by kinetic network analysis as essential elements that ensure an efficient vectorial proton transport in cytochrome c oxidase.
要理解生物分子中矢量质子泵浦的机制，需要为相关质子转移步骤的热力学和动力学特征的调节建立微观基础。对于质子泵细胞色素c氧化酶，虽然已经详细讨论了关键质子转移的热力学驱动力的调节，但对质子转移动力学控制的微观基础却知之甚少。在此，我们进行了广泛的量子力学/分子力学自由能模拟，以探究相关质子转移步骤的动力学，并分析局部结构和水合水平的影响。我们表明，质子装载位点（PLS，被认为是血红素a的一个丙酸酯）的质子化需要一个协同过程，其中一个关键的谷氨酸（Glu286H）将质子传递给PLS，同时被来自D通道的过量质子重新质子化。该机制的协同性质是一个关键特征，它使得在含有Glu286的腔更好地水合以将其pK降低到实验测量范围之前，PLS就能够被装载；正如Siegbahn和Blomberg所强调的，该过程的带电而非偶极性质也确保了与血红素a还原的紧密耦合。此外，我们发现PLS的旋转灵活性允许其在双核中心（氧气被还原为水的位点）质子化之前就进行质子化。结合我们最近专注于调节Glu286 pK的研究（P. Goyal，，110:18886 - 18891，2013），当前的工作提出了一种机制，可以在双核中心质子化之前为PLS的质子化建立一个自然顺序。这为动力学网络分析揭示的动力学限制提供了微观支持，这些限制是确保细胞色素c氧化酶中高效矢量质子传输的基本要素。
