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.