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Abstract

In many cytochrome c oxidases glutamic acid 242 is required for proton transfer to the binuclear heme a(3)/Cu(B) site, and for proton pumping. When present, the side chain of Glu-242 is orientated "down" towards the proton-transferring D-pathway in all available crystal structures. A nonpolar cavity "above" Glu-242 is empty in these structures. Yet, proton transfer from Glu-242 to the binuclear site, and for proton-pumping, is well established, and the cavity has been proposed to at least transiently contain water molecules that would mediate proton transfer. Such proton transfer has been proposed to require isomerisation of the Glu-242 side chain into an "up" position pointing towards the cavity. Here, we have explored the molecular dynamics of the protonated Glu-242 side chain. We find that the "up" position is preferred energetically when the cavity contains four water molecules, but the "down" position is favoured with less water. We conclude that the cavity might be deficient in water in the crystal structures, possibly reflecting the "resting" state of the enzyme, and that the "up/down" equilibrium of Glu-242 may be coupled to the presence of active-site water molecules produced by O(2) reduction.