title: Allosteric modulation of Euphorbia peroxidase by nickel ions.
abstract: A class III peroxidase, isolated and characterized from the latex of the perennial Mediterranean shrub Euphorbia characias, contains one ferric iron-protoporphyrin IX pentacoordinated with a histidine 'proximal' ligand as heme prosthetic group. In addition, the purified peroxidase contained 1 mole of endogenous Ca(2+) per mole of enzyme, and in the presence of excess Ca(2+), the catalytic efficiency was enhanced by three orders of magnitude. The incubation of the native enzyme with Ni(2+) causes reversible inhibition, whereas, in the presence of excess Ca(2+), Ni(2+) leads to an increase of the catalytic activity of Euphorbia peroxidase. UV/visible absorption spectra show that the heme iron remains in a quantum mechanically mixed-spin state as in the native enzyme after addition of Ni(2+), and only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ni(2+). In the presence of H(2)O(2) and in the absence of a reducing agent, Ni(2+) decreases the catalase-like activity of Euphorbia peroxidase and accelerates another pathway in which the inactive stable species accumulates with a shoulder at 619 nm. Analysis of the kinetic measurements suggests that Ni(2+) affects the H(2)O(2)-binding site and inhibits the formation of compound I. In the presence of excess Ca(2+), Ni(2+) accelerates the reduction of compound I to the native enzyme. The reported results are compatible with the hypothesis that ELP has two Ni(2+)-binding sites with opposite functional effects.
authors.affiliation: Department of Applied Sciences in Biosystems, University of Cagliari, Città Universitaria, Monserrato, Cagliari, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Pintus
authors.forename: Anna
authors.initials: A
authors.lastname: Mura
authors.forename: Andrea
authors.initials: A
authors.lastname: Bellelli
authors.forename: Alessandro
authors.initials: A
authors.lastname: Arcovito
authors.forename: Delia
authors.initials: D
authors.lastname: Spanò
authors.forename: Anna
authors.initials: A
authors.lastname: Pintus
authors.forename: Giovanni
authors.initials: G
authors.lastname: Floris
authors.forename: Rosaria
authors.initials: R
authors.lastname: Medda
mesh_terms: D000494:Allosteric Regulation; D002118:Calcium / Q000737:chemistry; D002384:Catalysis; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D028481:Euphorbia / Q000201:enzymology*; D005453:Fluorescence; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D007834:Lasers; D009532:Nickel / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D010782:Photolysis; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D010940:Plant Proteins; D009532:Nickel; D006861:Hydrogen Peroxide; D010544:Peroxidases; D002118:Calcium