New insights into the proton pumping mechanism of ba3 cytochrome c oxidase: the functions of key residues and water

As the terminal oxidase of cell respiration in mitochondria and aerobic bacteria, the proton pumping mechanism of ba3-type cytochrome c oxidase (CcO) of Thermus thermophiles is still not fully understood. Especially, the functions of key residues which were considered as the possible proton loading sites (PLSs) above the catalytic center, as well as water located above and within the catalytic center, remain unclear. In this work, molecular dynamic simulations were performed on a set of designed mutants of key residues (Asp287, Asp372, His376, and Glu126II). The results showed that Asp287 may not be a PLS, but it could modulate the ability of the proton transfer pathway to transfer protons through its salt bridge with Arg225. Maintaining the closed state of the water pool above the catalytic center is necessary for the participation of inside water molecules in proton transfer. Water molecules inside the water pool can form hydrogen bond chains with PLS to facilitate proton transfer. Additional quantum cluster models of the Fe-Cu metal catalytic center are established, indicating that when the proton is transferred from Tyr237, it is more likely to reach the OCu atom directly through only one water molecule. This work provides a more profound understanding of the functions of important residues and specific water molecules in the proton pumping mechanism of CcO.