Resonance Raman Studies of Cytochrome Oxidase

Abstract

The resonance Raman spectra of bovine heart cytochrome oxidase were observed with excitation at 488.0 and 514.5 nm for various molecular states. The resting enzyme as purified gave two Raman lines in the Band IV region at 1370 and 1357 cm⁻¹. On ferricyanide oxidation a single line appeared at 1372 cm⁻¹, indicating simultaneous oxidation of the two heme α molecules of cytochrome oxidase by ferricyanide. The resting enzyme was reduced by laser illumination under anaerobic conditions and a single Raman line appeared at 1362 cm⁻¹, at the same frequency as Band IV of dithionite-reduced oxidase. This photoreduction was confirmed by the appearance of a 440 nm band in the absorption spectrum. In the presence of air, however, a molecular state characterized by the coexistence of two Raman lines in the Band IV region emerged under laser illumination. These two lines were also produced by oxygen bubbling of the dithionite-reduced oxidase. The heme α species yielding the lower-frequency counterpart must have ensued from some form of dimeric cytochrome oxidase, because this line was lost when the oxidase was dissociated into monomers by treatment with sodium dodecyl sulfate. Since the absorption spectra of the resting, aerobically laser-illuminated, and oxygen-bubbled oxidases apparently resemble the spectrum of the ferricyanide-oxidized oxidase, the oxidase having the two Raman lines in the Band IV region is suggested to be in the same oxidation state as, but in a different conformational state from, the ferricyanide-oxidized oxidase. The Raman spectra indicate the presence of a low-spin heme iron for both ferricyanide-oxidized and dithionite-reduced cytochrome oxidase. Addition of cyanide to reduced cytochrome oxidase resulted in a Raman spectrum quite similar to that of ferroheme α bis-imidazole cyanhydrin, suggesting the formation of cyanhydrin at the peripheral formyl group but not the replacement of axial ligands with cyanide in the oxidase thus treated.