Cytochrome c Oxidase (CcO) is the terminal electron acceptor in aerobic respiratory chain, reducing O₂ to water. The released free energy is stored by pumping protons through the protein, maintaining the transmembrane electrochemical gradient. Protons are held transiently in a proton loading site (PLS) that binds and releases protons driven by the electron transfer reaction cycle. Multi-Conformation Continuum Electrostatics (MCCE) was applied to crystal structures and Molecular Dynamics snapshots of the B-type Thermus thermophilus CcO. Six residues are identified as the PLS, binding and releasing protons as the charges on heme b and the binuclear center are changed: the heme a₃ propionic acids, Asp287, Asp372, His376 and Glu126B. The unloaded state has one proton and the loaded state two protons on these six residues. Different input structures, modifying the PLS conformation, show different proton distributions and result in different proton pumping behaviors. One loaded and one unloaded protonation states have the loaded/unloaded states close in energy so the PLS binds and releases a proton through the reaction cycle. The alternative proton distributions have state energies too far apart to be shifted by the electron transfers so are locked in loaded or unloaded states. Here the protein can use active states to load and unload protons, but has nearby trapped states, which stabilize PLS protonation state, providing new ideas about the CcO proton pumping mechanism. The distance between the PLS residues Asp287 and His376 correlates with the energy difference between loaded and unloaded states.

细胞色素c氧化酶（CcO）是有氧呼吸链中的末端电子受体，可将O ₂还原为水。释放的自由能通过泵送质子穿过蛋白质来存储，从而维持跨膜电化学梯度。质子被短暂地保留在质子加载位点（PLS）中，该位点结合并释放由电子转移反应周期驱动的质子。将多构象连续静电学（MCCE）应用于B型嗜热栖热菌CcO的晶体结构和分子动力学快照。六个残基被识别为PLS，随着血红素b和双核中心上电荷的改变，结合和释放质子：血红素a ₃丙酸，Asp287，Asp372，His376和Glu126B。在这六个残基上，未加载状态具有一个质子，而已加载状态具有两个质子。修改PLS构型的不同输入结构显示不同的质子分布，并导致不同的质子泵送行为。一个已加载和一个未加载的质子化状态的加载/未加载状态的能量接近，因此PLS在整个反应周期中结合并释放质子。替代质子分布的状态能量相距太远，无法被电子转移移动，因此被锁定在有载或无载状态。在这里，蛋白质可以利用活跃状态来加载和卸载质子，但是具有附近的捕获状态，可以稳定PLS质子化状态，从而提供有关CcO质子泵送机制的新思路。