Allosteric modulation of Euphorbia peroxidase by nickel ions

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.