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Abstract

Protein-assisted transport of protons across the bioenergetic membrane is mediated by hydrogen-bonded networks. These networks involve titratable amino acid residues of membrane-spanning protein assemblies as well as internal water molecules. In cytochrome c oxidase, the so-called D-channel defines such a network for the uptake of protons from the cytoplasmic side of the membrane. It has been proposed that conformational changes of a Glu residue are required for the establishment of a proton linkage from the channel into the active site. The thermodynamic basis for the conformational isomerization of this residue is investigated using simulated annealing and free energy molecular dynamics simulations. The results support the existence of metastable conformations of the side chain, and their interchange through local structural fluctuations of neighboring residues and nearby internal chains of water molecules. The conformational isomerization of both protonated and unprotonated states of Glu, coupled with the reorganization of hydrogen bonds, suggests a kinetically competent mechanism for proton shuttling.