Transiently Produced Hypochlorite Is Responsible for the Irreversible Inhibition of Chlorite Dismutase

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• 作者：Stefan Hofbauer ; Clemens Gruber ; Katharina F. Pirker ; Axel S眉ndermann ; Irene Schaffner ; Christa Jakopitsch ; Chris Oostenbrink ; Paul G. Furtm眉ller ; Christian Obinger
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：May 20, 2014
• 年：2014
• 卷：53
• 期：19
• 页码：3145-3157
• 全文大小：822K
• 年卷期：v.53,no.19(May 20, 2014)
• ISSN：1520-4995

文摘

Chlorite dismutases (Clds) are heme b-containing prokaryotic oxidoreductases that catalyze the reduction of chlorite to chloride with the concomitant release of molecular oxygen. Over time, they are irreversibly inactivated. To elucidate the mechanism of inactivation and investigate the role of the postulated intermediate hypochlorite, the pentameric chlorite dismutase of 鈥?i>Candidatus Nitrospira defluvii鈥?(NdCld) and two variants (having the conserved distal arginine 173 exchanged with alanine and lysine) were recombinantly produced in Escherichia coli. Exchange of the distal arginine boosts the extent of irreversible inactivation. In the presence of the hypochlorite traps methionine, monochlorodimedone, and 2-[6-(4-aminophenoxy)-3-oxo-3H-xanthen-9-yl]benzoic acid, the extent of chlorite degradation and release of molecular oxygen is significantly increased, whereas heme bleaching and oxidative modifications of the protein are suppressed. Among other modifications, hypochlorite-mediated formation of chlorinated tyrosines is demonstrated by mass spectrometry. The data obtained were analyzed with respect to the proposed reaction mechanism for chlorite degradation and its dependence on pH. We discuss the role of distal Arg173 by keeping hypochlorite in the reaction sphere for O鈥揙 bond formation.