An Analysis of the Formyl Vibrational Mode in Heme A

The formyl group on the heme in cytochrome c oxidase may play a critical role in the functional properies of the enzyme. Although resonance Raman scattering has been used successully to probe the properties of the heme a macrocycle, the assignment of the modes associated with the formyl substituent has not been resolved. We report isotopic substitution studies of the formyl group in heme a(sup 16 O -> sup 18 O) which have allowed for identification of the modes present in the Raman spectrum as well as a determination of the coupling properties of the C=O stretching mode of the formyl group. We have recroded the spectra for both oxidation states in both organic and aqueous solvents. The isotopic frequency shifts were analyzed by Raman difference spectroscopy and curve fitting techniques. These procedures were necessary in analyzing the data because we discovered an unexpected degree of complexity in that the carbonyl stretching mode of the formyl group is highly coupled to porphyrin modes. Consequently, the frequency shifts of the formyl stretching mode did not follow simple theory and porphyrin modes also changed intensity and shifted. Conclusions about cytochrome c oxidase properties based on analysis of the formyl modes must be viewed cautiously.