Abstract
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 Kd 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 Kd 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.
Original language | English |
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Pages (from-to) | 878-886 |
Number of pages | 9 |
Journal | European Journal of Biochemistry |
Volume | 234 |
Issue number | 3 |
DOIs | |
Publication status | Published - 1 Jan 1995 |
Keywords
- calcium
- cytochromes
- Paracoccus
- peroxidase