Cytochrome c oxidase (CcO) is a crucial enzyme in the respiratory chain. Its function is to couple the reduction of molecular oxygen, which takes place in the Fea3−CuB binuclear center, to proton translocation across the mitochondrial membrane. Although several high-resolution structures of the enzyme are known, the molecular basis of proton pumping activation and its mechanism remain to be elucidated. We examine a recently proposed scheme (J. Am. Chem. Soc. 2004, 126, 1858; FEBS Lett. 2004, 566, 126) that involves the deprotonation of the CuB-bound imidazole ring of a histidine (H291 in mammalian CcO) as a key element in the proton pumping mechanism. The central feature of that proposed mechanism is that the pKa values of the imidazole vary significantly depending on the redox state of the metals in the binuclear center. We use density functional theory in combination with continuum electrostatics to calculate the pKa values, successively in bulk water and within the protein, of the Cu-bound imidazole in various Cu− and Cu−Fe complexes. From pKas in bulk water, we derived a value of −266.34 kcal·mol-1 for the proton solvation free energy (Δ ). This estimate is in close agreement with the experimental value of −264.61 kcal·mol-1 (J. Am. Chem. Soc. 2001, 123, 7314), which reinforces the conclusion that Δ is more negative than previous values used for pKa calculations. Our approach, on the basis of the study of increasingly more detailed models of the CcO binuclear center at different stages of the catalysis, allows us to examine successively the effect of each of the two metals' redox states and of solvation on the acidity of imidazole, whose pKa is approximately 14 in bulk water. This analysis leads to the following conclusions:  first, the effect of Cu ligation on the imidazole acidity is negligible regardless of the redox state of the metal. Second, results obtained for Cu−Fe complexes in bulk water indicate that Cu-bound imidazole pKa values lie within the range of 14.8−16.6 throughout binuclear redox states corresponding to the catalytic cycle, demonstrating that the effect of the Fe oxidation states is also negligible. Finally, the low-dielectric CcO proteic environment shifts the acid−base equilibrium toward a neutral imidazole, further increasing the corresponding pKa values. These results are inconsistent with the proposed role of the Cu-bound histidine as a key element in the pumping mechanism. Limitations of continuum solvation models in pKa calculations are discussed.