Variable-temperature magnetic-circular-dichroism spectra of cytochrome c oxidase and its derivatives

A detailed study of the effect of temperature on the m.c.d. (magnetic circular dichroism) spectra of cytochrome c oxidase and some of its derivatives was undertaken to characterize the spin states of haem a and a₃. The fully reduced enzyme contains haem a₃²⁺in its high-spin form and haem a²⁺in the low-spin state. This conclusion is reached by comparing the spectrum with that of the mixed-valence CO derivatives and its photolysis product. The cyanide derivative of the fully reduced enzyme contains both haem a and a₃ in the low-spin ferrous form. The m.c.d. spectra of the fully oxidized derivatives are consistent with the presence of one low-spin ferric haem group, assigned to a, which remains unaltered in the presence of ligands. Haem a₃ is high spin in the resting enzyme and the fluoride derivatives, and low spin in the cyanide form. The partially reduced formate and cyanide derivatives have temperature-dependent m.c.d. spectra due to the presence of high- and low-spin haem a₃³⁺respectively. Haem a is low-spin ferrous in both. A comparison of the magnitude of the temperature-dependence of haem a₃³⁺in the fully oxidized and partially reduced forms shows a marked difference which is tentatively ascribed to the presence of anti-ferromagnetic coupling in the fully oxidized form of the enzyme, and to its absence from the partially reduced derivatives, owing to the reduction of both Cu²⁺ions.