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

Raymond Gilmour, Susana Prazeres, Dermot F. Mcginnity, Celia F. Goodhew, José J. G. Moura, Isabel Moura, Graham W. Pettigrew

Research output: Contribution to journalArticlepeer-review

30 Citations (Scopus)

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 languageEnglish
Pages (from-to)878-886
Number of pages9
JournalEuropean Journal Of Biochemistry
Volume234
Issue number3
DOIs
Publication statusPublished - 1 Jan 1995

Keywords

  • calcium
  • cytochromes
  • Paracoccus
  • peroxidase

Fingerprint Dive into the research topics of 'The Affinity and Specificity of Ca<sup>2+</sup>‐Binding Sites of Cytochrome‐c Peroxidase from Paracoccus Denitrificans'. Together they form a unique fingerprint.

Cite this