Cytochrome c oxidase couples the reduction of dioxygen to proton pumping against an electrochemical gradient. The D-channel provides the principal uptake pathway for protons. A water chain is thought to mediate the relay of protons through the D-channel, but it is interrupted at N139 in all crystallographic structures. Here, free energy simulations are used to examine the proton uptake pathway in the wild type and in single-point mutants N139V and N139A, where reduction and pumping is compromised. A general approach for the calculation of water occupancy in protein cavities is presented and demonstrates that combining efficient sampling algorithms with long simulation times is required to achieve statistical convergence of equilibrium properties in the protein interior. The relative population of conformational and hydration states of the D-channel is characterized. Results shed light onto the role of N139 in the mechanism of proton uptake and clarify the physical basis for inactive phenotypes.

Cytochrome c oxidase couples the reduction of dioxygen to proton pumping against an electrochemical gradient. The D-channel provides the principal uptake pathway for protons. A water chain is thought to mediate the relay of protons through the D-channel, but it is interrupted at N139 in all crystallographic structures. Here, free energy simulations are used to examine the proton uptake pathway in the wild type and in single-point mutants N139V and N139A, where reduction and pumping is compromised. A general approach for the calculation of water occupancy in protein cavities is presented and demonstrates that combining efficient sampling algorithms with long simulation times is required to achieve statistical convergence of equilibrium properties in the protein interior. The relative population of conformational and hydration states of the D-channel is characterized. Results shed light onto the role of N139 in the mechanism of proton uptake and clarify the physical basis for inactive phenotypes.