Time-resolved step-scan FTIR (TRS2-FTIR) and density functional theory have been applied to probe the structural dynamics of Cu-B in heme-copper oxidases at room temperature. The TRS2-FTIR data of cbb(3) from Pseudomonas stutzeri indicate a small variation in the frequency of the transient CO bound to Cu-B in the pH/pD 7-9 range. This observation in conjunction with density functional theory calculations, in which significant frequency shifts of the v(CO) are observed upon deprotonation and/or detachment of the Cu-B ligands, demonstrates that the properties of the Cu-B ligands including the cross-linked tyrosine, in contrast to previous reports, remain unchanged in the pH 7-9 range. We attribute the small variations in the v(CO) of Cu-B to protein conformational changes in the vicinity Of Cu-B. Consequently, the split of the heme Fe-CO vibrations (alpha-, beta-, and gamma-forms) is not due to changes in the ligation and/or protonation states of the Cu-B ligands or to the presence of one or more ionizable groups, as previously suggested, but the result of global protein conformational changes in the vicinity of Cu-B which, in turn, affect the position of Cu-B with respect to the heme Fe.