Ca²⁺ and the bacterial peroxidases: the cytochrome c peroxidase from Pseudomonas stutzeri

Abstract.

The production of cytochrome c peroxidase (CCP) from Pseudomonas (Ps.) stutzeri (ATCC 11607) was optimized by adjusting the composition of the growth medium and aeration of the culture. The protein was isolated and characterized biochemically and spectroscopically in the oxidized and mixed valence forms. The activity of Ps. stutzeri CCP was studied using two different ferrocytochromes as electron donors: Ps. stutzeri cytochrome c ₅₅₁ (the physiological electron donor) and horse heart cytochrome c. These electron donors interact differently with Ps. stutzeri CCP, exhibiting different ionic strength dependence. The CCP from Paracoccus (Pa.) denitrificans was proposed to have two different Ca²⁺ binding sites: one usually occupied (site I) and the other either empty or partially occupied in the oxidized enzyme (site II). The Ps. stutzeri enzyme was purified in a form with tightly bound Ca²⁺. The affinity for Ca²⁺ in the mixed valence enzyme is so high that Ca²⁺ returns to it from the EGTA which was added to empty the site in the oxidized enzyme. Molecular mass determination by ultracentrifugation and behavior on gel filtration chromatography have revealed that this CCP is isolated as an active dimer, in contrast to the Pa. denitrificans CCP which requires added Ca²⁺ for formation of the dimer and also for activation of the enzyme. This is consistent with the proposal that Ca²⁺ in the bacterial peroxidases influences the monomer/dimer equilibrium and the transition to the active form of the enzyme. Additional Ca²⁺ does affect both the kinetics of oxidation of horse heart cytochrome c (but not cytochrome c ₅₅₁) and higher aggregation states of the enzyme. This suggests the presence of a superficial Ca²⁺ binding site of low affinity.