The Affinity and Specificity of Ca²⁺‐Binding Sites of Cytochrome‐c Peroxidase from Paracoccus Denitrificans

The binding of Ca²⁺ to the dihaem cytochrome‐c. peroxidase from Paracoccus denitrificans was analysed by following perturbations in the visible and ¹H‐NMR spectra of both haem groups. The enzyme contains at least two types of Ca²⁺‐binding site. Site I is occupied in the isolated enzyme, binds Ca²⁺ with a redox‐state‐independent K_d of 1.2 μM and accommodates neither Mg²⁺ nor Mn²⁺. Site II is unoccupied in dilute solutions of the isolated oxidised enzyme and binds Ca²⁺ cooperatively with a K_d of 0.52 mM. In the mixed valence form, the binding affinity increases to resemble that of site I. The cooperativity was shown by ‐Ca²⁺ binding to site II, the titration of haem methyl ¹H‐NMR resonances, and a half‐of‐sites effect observed for modification of an essential histidine with diethylpyrocarbonate. These are all consistent with site II being situated at the interface between two monomers of a dimeric enzyme. Thus the equilibrium of binding to site II is a reflection of the equilibrium for dimerisation and conditions which shift that equilibrium towards the dimer, such as increased ionic strength or high protein concentration, also increase Ca²⁺ affinity. Binding of Ca²⁺ to site II is required for formation of the active high spin state at the peroxidatic haem.