Purification and characterization of a chlorite dismutase from Pseudomonas chloritidismutans

Abstract The chlorite dismutase (Cld) of Pseudomonas chloritidismutans was purified from the periplasmic fraction in one step by hydroxyapatite chromatography. The enzyme has a molecular mass of 110 kDa and consists of four 31-kDa subunits. Enzyme catalysis followed Michaelis–Menten kinetics, with Vmax and Km values of 443 U mg−1 and 84 μM, respectively. A pyridine–NaOH–dithionite-reduced Cld revealed a Soret peak at 418 nm, indicative for protoheme IX. The spectral data indicate the presence of 1.5 mol protoheme IX mol−1 tetrameric enzyme while metal analysis revealed 2.2 mol iron mol−1 tetrameric enzyme. High concentrations of chlorite resulted in the disappearance of the Soret peak, which coincided with loss in activity. Electron paramagnetic resonance analyses showed an axial high-spin ferric iron signal. Cld was inhibited by cyanide, azide, but not by hydroxylamine or 3-amino-1,2,3-triazole. Remarkably, the activity was drastically enhanced by kosmotropic salts, and chaotropic salts decreased the activity, in accordance with the Hofmeister series. Chlorite conversion in the presence of 18O-labeled water did not result in the formation of oxygen with a mass of 34 (16O–18O) or a mass of 36 (18O–18O), indicating that water is not a substrate in the reaction and that both oxygen atoms originate from chlorite.