Magnetic susceptibility measurements of cytochrome c peroxidase and its complexes.

A highly purified preparation of cytochrome c peroxidase was prepared by reconstituting the enzyme from the apoprotein and protohemin. Paramagnetic susceptibilities of the enzyme and of its complexes with cyanide, azide, cyanate, and fluoride were measured at temperatures from 77” K to 273” K. The susceptibilities of the cyanide and fluoride complexes obeyed Curie’s law over the entire temperature range and it was confirmed that these complexes are in purely low spin and high spin states. All of the other complexes, in addition to the free enzyme, were found to deviate from Curie’s law above certain temperatures. The effective Bohr magneton numbers (neff) of the enzyme at different pH values were explained by assuming that cytochrome c peroxidase is a mixture of acidic and alkaline forms and that both forms have low spin and high spin states in thermal equilibria. The temperature dependence of the equilibria was also characterized spectrophotometrically for the enzyme at pH 7 and its cyanate complex. The optical measurements were in good agreement with the susceptibility measurements of the corresponding samples. The temperature-dependent changes of nfrr were analyzed on the basis of a thermal equilibrium between high spin and low spin states. Several thermodynamic parameters such as the energy difIerence (e), the enthalpy difference (AHO), and the entropy difference (AS’) between the two spin states, the entropy factor (-y), and the compensation temperature (T,) were estimated from the temperature dependence of the equilibrium constants of the mixed spin states. The relation between AH0 and AS0 is discussed and compared with myoglobin and hemoglobin.