title: The affinity and specificity of Ca(2+)-binding sites of cytochrome-c peroxidase from Paracoccus denitrificans.
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 Ca(2+)-binding site. Site I is occupied in the isolated enzyme, binds Ca2+ with a redox-state-independent Kd of 1.2 microM 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.
authors.affiliation: Department of Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, University of Edinburgh, UK.
authors.forename: R
authors.initials: R
authors.lastname: Gilmour
authors.forename: S
authors.initials: S
authors.lastname: Prazeres
authors.forename: D F
authors.initials: DF
authors.lastname: McGinnity
authors.forename: C F
authors.initials: CF
authors.lastname: Goodhew
authors.forename: J J
authors.initials: JJ
authors.lastname: Moura
authors.forename: I
authors.initials: I
authors.lastname: Moura
authors.forename: G W
authors.initials: GW
authors.lastname: Pettigrew
mesh_terms: D001205:Ascorbic Acid / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D013379:Substrate Specificity; D017186:Titrimetry
chemical_list: D008345:Manganese; D004533:Egtazic Acid; D004492:Edetic Acid; D003578:Cytochrome-c Peroxidase; D008274:Magnesium; D001205:Ascorbic Acid; D002118:Calcium