Multiscale simulations reveal key features of the proton pumping mechanism in cytochrome c oxidase
Cytochrome c oxidase (CcO) reduces oxygen to water and uses the released free energy to pump protons across the membrane, contributing to the transmembrane proton electrochemical gradient that drives ATP synthesis. We have used multiscale reactive molecular dynamics simulations to explicitly characterize (with free energy profiles and calculated rates) the internal proton transport events that enable pumping and chemistry during the A[->]PR[->]F transition in the aa3-type CcO. Our results show that proton transport from amino acid residue E286 to both the pump loading site (PLS) and to the binuclear center (BNC) are thermodynamically driven by electron transfer from heme a to the BNC, but that the former (i.e., pumping) is kinetically favored while the latter (i.e., transfer of the chemical proton) is rate-limiting. The calculated rates are in quantitative agreement with experimental measurements. The back flow of the pumped proton from the PLS to E286 and from E286 to the inner side of membrane are prevented by the fast reprotonation of E286 through the D-channel and large free energy barriers for the back flow reactions. Proton transport from E286 to the PLS through the hydrophobic cavity (HC) and from D132 to E286 through the D-channel are found to be strongly coupled to dynamical hydration changes in the corresponding pathways. This work presents a comprehensive description of the key steps in the proton pumping mechanism in CcO.\n\nSignificanceThe long studied proton pumping mechanism in cytochrome c oxidase (CcO) continues to be a source of debate. This work provides a comprehensive computational characterization of the internal proton transport dynamics, while explicitly including the role of Grotthuss proton shuttling, that lead to both pumping and catalysis. Focusing on the A to F transition, our results show that the transfer of both the pumped and chemical protons are thermodynamically driven by electron transfer, and explain how proton back leakage is avoided by kinetic gating. This work also explicitly characterizes the coupling of proton transport with hydration changes in the hydrophobic cavity and D-channel, thus advancing our understanding of proton transport in biomolecules in general.