We address the molecular mechanism by which the haem-copper oxidases translocate protons. Reduction of O2 to water takes place at a haem iron-copper (CuB) centre, and protons enter from one side of the membrane through a 'channel' structure in the enzyme. Statistical-mechanical calculations predict bound water molecules within this channel, and mutagenesis experiments show that breaking this water structure impedes proton translocation. Hydrogen-bonded water molecules connect the channel further via a conserved glutamic acid residue to a histidine ligand of CuB. The glutamic acid side chain may have to move during proton transfer because proton translocation is abolished if it is forced to interact with a nearby lysine or arginine. Perturbing the CuB ligand structure shifts an infrared mode that may be ascribed to the O-H stretch of bound water. This is sensitive to mutations of the glutamic acid, supporting its connectivity to the histidine. These results suggest key roles of bound water, the glutamic acid and the histidine copper ligand in the mechanism of proton translocation.