Allosteric modulation of Euphorbia peroxidase by nickel ions

• Published in: The FEBS journal Vol. 275; no. 6; pp. 1201 - 1212
• Main Authors: Pintus, Francesca, Mura, Anna, Bellelli, Andrea, Arcovito, Alessandro, Spanò, Delia, Pintus, Anna, Floris, Giovanni, Medda, Rosaria
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.03.2008; Blackwell Publishing Ltd
• Subjects: Allosteric Regulation; Binding sites; Botany; calcium; Calcium - chemistry; Catalysis; Cations, Divalent - chemistry; Cations, Divalent - metabolism; Circular Dichroism; Enzymes; Euphorbia - enzymology; Flowers & plants; Fluorescence; heme proteins; hydrogen peroxide; Hydrogen Peroxide - chemistry; Ions; Kinetics; Lasers; Nickel; Nickel - chemistry; Nickel - metabolism; Oxidation-Reduction; peroxidase; Peroxidases - antagonists & inhibitors; Peroxidases - chemistry; Peroxidases - metabolism; Photolysis; Plant Proteins - chemistry; Plant Proteins - metabolism
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2008.06280.x

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 Ca²⁺ per mole of enzyme, and in the presence of excess Ca²⁺, the catalytic efficiency was enhanced by three orders of magnitude. The incubation of the native enzyme with Ni²⁺ causes reversible inhibition, whereas, in the presence of excess Ca²⁺, Ni²⁺ 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 Ni²⁺, 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 Ni²⁺. In the presence of H₂O₂ and in the absence of a reducing agent, Ni²⁺ 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 Ni²⁺ affects the H₂O₂-binding site and inhibits the formation of compound I. In the presence of excess Ca²⁺, Ni²⁺ accelerates the reduction of compound I to the native enzyme. The reported results are compatible with the hypothesis that ELP has two Ni²⁺-binding sites with opposite functional effects.