Cytochrome c oxidase (CcO) couples
the reduction of dioxygen to water with transmembrane proton pumping,
which leads to the generation of an electrochemical gradient. In this
study we analyze how one of the components of the electrochemical
gradient, the difference in pH across the membrane, or ΔpH,
influences the protonation states of residues in CcO. We modified our continuum electrostatics/Monte Carlo (CE/MC) method
in order to include the ΔpH and applied it to the study of CcO, in what is, to our best knowledge, the first CE/MC study
of CcO in the presence of a pH gradient. The inclusion
of a transmembrane pH gradient allows for the identification of residues
whose titration behavior depends on the pH on both sides of the membrane.
Among the several residues with unusual titration profiles, three
are well-known key residues in the proton transfer process of CcO: E286I, Y288I, and K362I. All three residues have been previously identified as being critical
for the catalytic or proton pumping functions of CcO. Our results suggest that when the pH gradient increases, these
residues may be part of a regulatory mechanism to stem the proton
flow.