Abstract
In this research, the binding mechanism of Cu2+ to bovine liver catalase (BLC) was studied by fluorescence spectroscopy, ultraviolet–visible (UV–vis) absorption spectroscopy, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC) and molecular docking methods. The cellar experiment was firstly carried out to investigate the inhibition effect of catalase. During the fluorescence quenching study, after correcting the inner filter effect (IFE), the fluorescence of BLC was found to be quenched by Cu2+. The quenching mechanism was determined by fluorescence lifetime measurement, and was confirmed to be the dynamic mode. The secondary structure content of BLC was changed by the addition of Cu2+, as revealed by UV–vis absorption and CD spectra, which further induces the decrease in BLC activity. Molecular simulation study indicates that Cu2+ is located between two β-sheets and two random coils of BLC near to the heme group, and interacts with His 74 and Ser 113 residues near a hydrophilic area. The decrease of α-helix and the binding of His 74 are considered to be the major reason for the inhibition of BLC activity caused by Cu2+. The ITC results indicate that the binding stoichiometry of Cu2+ to catalase is 11.4. Moreover, the binding of Cu2+ to BLC destroyed H-bonds, which was confirmed by the CD result.
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