Interactions of Cu_B with Carbon Monoxide in Cytochrome c Oxidase: Origin of the Anomalous Correlation between the Fe-CO and C-O Stretching Frequencies

In heme-copper oxidases, the correlation curve between the iron-CO and C-O stretching vibrational modes (ν_Fe-CO and ν_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 ba₃ (ba₃) from Thermus thermophilus were measured. The RR spectrum has two strong ν_Fe-CO lines (508 and 515 cm^-1) and a very weak line at 526 cm^-1, and the IR spectrum has three ν_C-O lines (1966, 1973, and 1981 cm^-1), indicating the presence of multiple conformers. Employing photodissociation methods, the ν_Fe-CO RR and ν_C-O IR lines were assigned to each conformer, enabling the establishment of a reliable inverse correlation curve for the ν_Fe-CO versus the ν_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_z² orbital of the copper and MOs that are responsible for the ν_Fe-CO vs ν_C-O inverse correlation. Together, the spectroscopic and DFT results clarify the origin of the anomaly of ν_Fe-CO and ν_C-O frequencies in the heme-copper oxidases, a long-standing issue. (Graph Presented).