In heme copper oxidases, the correlation curve between the iron CO and C-O stretching vibrational modes (nu(Fe-CO) and nu(C-O)) respectively) is anomalous as compared to the correlation in other heme proteins. To extend the correlation curve, the resonance Raman (RR) and infrared (IR) spectra of the CO adducts of cytochrome ba(3) (ba(3)) from Thermus thermophilus were measured. The RR spectrum has two strong nu(Fe-CO) lines (508 and 515 cm(-1)) and a very weak line at 526 cm-1, and the IR spectrum has three nu(C-O) lines (1966, 1973, and 1981 cm(-1)), indicating the presence of multiple conformers. Employing photodissociation methods, the nu(Fe-CO) RR and nu(C-O) IR lines were assigned to each conformer, enabling the establishment of a reliable inverse correlation curve for the nu(Fe-CO) versus the nu(C-O) stretching frequencies. To determine the molecular basis of the correlation, a series of DFT calculations on 6-coordinate porphyrin-CO compounds and a model of the binuclear center of the heme copper oxidases were carried out. The calculations demonstrated that the copper unit model caused significant mixing among porphyrin-CO molecular orbitals (MOs) that contribute to the Fe-C and C-O bonding interactions, and also indicated the presence of mixing between the d(z)(2) orbital of the copper and MOs that are responsible for the nu(Fe-CO) vs nu(C-O) inverse correlation. Together, the spectroscopic and DFT results clarify the origin of the anomaly of nu(Fe-CO) and nu(C-O) frequencies in the heme-copper oxidases, a long-standing issue.