Open AccessPurification and characterization of a chlorite dismutase from Pseudomonas chloritidismutans
Author(s) -
Mehboob Farrakh,
Wolterink Arthur F.M.,
Vermeulen Arjan J.,
Jiang Bo,
Hagedoorn PeterLeon,
Stams Alfons J.M.,
Kengen Servé W.M.
Publication year - 2009
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2009.01517.x
Subject(s) - chemistry , chlorite , dithionite , nuclear chemistry , inorganic chemistry , medicinal chemistry , stereochemistry , enzyme , organic chemistry , paleontology , quartz , biology
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 V max and K m 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 18 O‐labeled water did not result in the formation of oxygen with a mass of 34 ( 16 O– 18 O) or a mass of 36 ( 18 O– 18 O), indicating that water is not a substrate in the reaction and that both oxygen atoms originate from chlorite.