Functional hydration and conformational gating of proton uptake in cytochrome c oxidase.

Cytochrome c oxidase couples the reduction of dioxygen to proton pumping against an electrochemical gradient. The D-channel, a 25-A-long cavity, provides the principal pathway for the uptake of chemical and pumped protons. A water chain is thought to mediate the relay of protons via a Grotthuss mechanism through the D-channel, but it is interrupted at N139 in all available crystallographic structures. We use free-energy simulations to examine the proton uptake pathway in the wild type and in single-point mutants N139V and N139A, in which redox and pumping activities are compromised. We present a general approach for the calculation of water occupancy in protein cavities and demonstrate that combining efficient sampling algorithms with long simulation times (hundreds of nanoseconds) is required to achieve statistical convergence of equilibrium properties in the protein interior. The relative population of different conformational and hydration states of the D-channel is characterized. Results shed light on the role of N139 in the mechanism of proton uptake and clarify the physical basis for inactive phenotypes. The conformational isomerization of the N139 side chain is shown to act as a gate controlling the formation of a functional water chain or "proton wire." In the closed state of N139, the spatial distribution of water in the D-channel is consistent with available crystallographic models. However, a metastable state of N139 opens up a narrow bottleneck in which 50% occupancy by a water molecule establishes a proton pathway throughout the D-channel. Results for N139V suggest that blockage of proton uptake resulting from persistent interruption of the water pathway is the cause of this mutant's marginal oxidase activity. In contrast, results for N139A indicate that the D-channel is a continuously hydrated cavity, implying that the decoupling of oxidase activity from proton pumping measured in this mutant is not due to interruption of the proton relay chain.