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 mol endogenous calcium 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+ ions causes a reversible inhibition, whereas, in the presence of excess Ca2+, nickel ions lead to an increase of the catalytic activity of Euphorbia peroxidase. UV/vis absorption spectra show that the heme iron remains in a quantum mechanically mixedspin state as in native enzyme after addition of Ni2+ ions, 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+ ions. In the presence of hydrogen peroxide and in the absence of a reducing agents, Ni2+ ions decrease the catalase like activity of Euphorbia peroxidase and accelerate another pathway in which the inactive stable species accumulates with a shoulder at 619 nm. Analysis of the kinetic measurements suggests that nickel ions affect the H2O2 binding site and inhibits the formation of Compound I. In the presence of excess Ca2+, nickel ions accelerate the reduction of Compound I to the native enzyme. The reported results are compatible with the hypothesis that ELP has two Ni binding sites with opposite functional effects.