Photodissociated cytochrome oxidase: Cryotrapped metastable intermediates.

By freezing CO-bound cytochrome c- oxidase at cryogenic temperatures, we have been able to cryotrap metastable intermediates. The differences in the resonance Raman spectrum between these intermediates and ligand-free reduced cytochrome oxidase at cryogenic temperatures are the same as those between the photo- transient and the fully reduced preparation detected with 10 ns excitation at room temperature. The largest difference occurs in the iron-histidine stretching mode of cytochrome a- sub 3 which shifts by up to 8 cm sup (-1) to higher frequency in the photoproduct. At 4K the iron-histidine mode displays two unrelaxed frequencies in the photoproduct which we attribute to two different unrelaxed structures of the heme pocket. The frequencies and intensities of the lines in the resonance Raman spectrum are sensitive to the incident laser power density in both the ligand-free fully reduced preparation and the photoproduct even at 4K.