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

A putative perA gene from Archaeoglobus fulgidus was cloned and expressed i n Escherichia coh BL21(DE3), and the recombinant catalase-peroxidase was pu rified to homogeneity. The enzyme is a homodimer with a subunit molecular m ass of 85 kDa. UV-visible spectroscopic analysis indicated the presence of protoheme IX as a prosthetic group (ferric heme), in a stoichiometry of 0.2 5 heme per subunit. Electron paramagnetic resonance analysis confirmed the presence of ferric heme and identified the proximal axial ligand as a histi dine. The enzyme showed both catalase and peroxidase activity with pH optim a of 6.0 and 4.5, respectively. Optimal temperatures of 70 degreesC and 80 degreesC were found for the catalase and peroxidase activity, respectively. The catalase activity strongly exceeded the peroxidase activity, with V-ma x 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 he me inhibitors such as cyanide, azide, and hydroxylamine inhibited peroxidas e activity. However, unlike all other catalase-peroxidases, the enzyme was also inhibited by 3-amino-1,2,4-triazole. Although the enzyme exhibited a h igh thermostability, rapid inactivation occurred in the presence of H2O2, w ith half-life values of less than 1 min. This is the first catalase-peroxid ase characterized from a hyperthermophilic microorganism.