Purification and characterization of a chlorite dismutase from Pseudomonas chloritidismutans.
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 K(m) values of 443 U mg(-1) and 84 microM, 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 ((18)O-(18)O), indicating that water is not a substrate in the reaction and that both oxygen atoms originate from chlorite.
Mehboob
Farrakh
F
Laboratory of Microbiology, Wageningen University, Dreijenplein, Wageningen, The Netherlands.
Wolterink
Arthur F M
AF
Vermeulen
Arjan J
AJ
Jiang
Bo
B
Hagedoorn
Peter-Leon
PL
Stams
Alfons J M
AJ
Kengen
Servé W M
SW
D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000032:analysis; D007700:Kinetics; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019897:Periplasm / Q000201:enzymology; D011549:Pseudomonas / Q000145:classification* / Q000201:enzymology*; D012492:Salts / Q000494:pharmacology
D012492:Salts; D006418:Heme; D010088:Oxidoreductases; C402794:chlorite dismutase