Resonance raman spectra of CN--bound cytochrome oxidase: spectral isolation of cytochromes a2+, a3(2+), and a3(2+)(CN-).

Reduced cyanide-bound cytochrome oxidase in the absence of any oxygen gives a resonance Raman spectrum consistent with that expected for low-spin heme a. Thus, in contrast to prior reports, ligand binding of cytochrome a3 to form a six-coordinate low-spin ferrous heme does not result in any unusual electronic structure, hydrogen bonding, environment, or conformation of the formyl group. It appears unlikely that there are any changes in this group in cytochrome a3 that control the ligand affinity or redox potential in physiological forms of the ferrous enzyme. With the use of our difference spectrometer and by appropriately selecting the laser excitation frequency, we are able to isolate spectrally cytochromes a2+, a3(2+), and a3(2+)(CN-). The addition of a small amount of oxygen to a preparation of the cyanide-bound reduced enzyme results in a complex with the same Raman spectrum as that previously reported to originate from the cyanide-bound reduced complex. Any oxygen present in the sample leads to enzyme turnover resulting in a mixed valence state [a2+a3(3+)(CN-)]. The comparison between the data on the cyanide-bound reduced enzyme and the data on the CO-bound reduced enzyme illustrates that cyanide binding affects only the modes that respond to the spin state of the ferrous iron, while CO binding affects vibrational modes that respond to a pi-electron density change as well.