Characterization of a catalase-peroxidase from the hyperthermophilic archaeon Archaeoglobus fulgidus

Abstract.

A putative perA gene from Archaeoglobus fulgidus was cloned and expressed in Escherichia coli BL21(DE3), and the recombinant catalase-peroxidase was purified to homogeneity. The enzyme is a homodimer with a subunit molecular mass of 85 kDa. UV–visible spectroscopic analysis indicated the presence of protoheme IX as a prosthetic group (ferric heme), in a stoichiometry of 0.25 heme per subunit. Electron paramagnetic resonance analysis confirmed the presence of ferric heme and identified the proximal axial ligand as a histidine. The enzyme showed both catalase and peroxidase activity with pH optima of 6.0 and 4.5, respectively. Optimal temperatures of 70°C and 80°C were found for the catalase and peroxidase activity, respectively. The catalase activity strongly exceeded the peroxidase activity, with V _max values of 9600 and 36 U mg^–1, respectively. K _m values for H₂O₂ of 8.6 and 0.85 mM were found for catalase and peroxidase, respectively. Common heme inhibitors such as cyanide, azide, and hydroxylamine inhibited peroxidase activity. However, unlike all other catalase-peroxidases, the enzyme was also inhibited by 3-amino-1,2,4-triazole. Although the enzyme exhibited a high thermostability, rapid inactivation occurred in the presence of H₂O₂, with half-life values of less than 1 min. This is the first catalase-peroxidase characterized from a hyperthermophilic microorganism.