Conformational changes and activity alterations induced by nickel ion in horseradish peroxidase

Conformational changes induced by the binding of nickel to horseradish peroxidase C (HRPC) were studied by electronic absorption spectroscopy, fluorescence spectroscopy and circular dichroism spectroscopy. Incubation of HRPC with various concentrations of Ni2+ for 5 minutes resulted in changes in the enzyme absorption spectrum, including variations in the intensities of the Soret, β and charge transfer (CT1) bands absorption, shift in the Soret, β and CT1 bands maxima and absorption increase at 275 nm. Increases in the enzyme's intrinsic fluorescence as determined by fluorescence spectroscopy, as well as changes in the α-helical content, as determined by circular dichroism spectroscopy, were also found. Correlatively, alterations of the enzymatic activity by Ni2+ were studied by following the H2O2-mediated oxidation of o-dianisidine and 2,2′-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) by HRPC. With both reducing substrates, it was found that in the presence of sufficient amount of enzyme, 1-10 mM nickel would enhance the enzymatic activity, while higher Ni2+ concentrations (20–50 mM) would inhibit it. The enzyme was completely inhibited after 5 minutes incubation in 50 mM Ni2+. Prolonged incubation would induce complete inhibition at lower Ni2+ concentrations. Spectrophotometry investigations also showed that inhibitory concentrations of Ni2+ altered compounds I and II formation, compound II being the first affected. Based on spectrophotometry, fluorescence and circular dichroism spectroscopy, and data on compounds I and II formation, a scheme is suggested for HRPC conformational changes in different Ni2+ concentrations. HRPC was found to have four potential attachment sites for Ni2+ which were sequentially occupied in a dose- and time-dependent manner by the metallic ion.