Hydrogen peroxide oxidation by catalase-peroxidase follows a non-scrambling mechanism

Vlasits, Jutta; Jakopitsch, Christa; Schwanninger, Manfred; Holubar, Peter; Obinger, Christian

doi:10.1016/j.febslet.2006.12.037

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Volume 581, Issue 2 , Pages 320-324, 23 January 2007

Hydrogen peroxide oxidation by catalase-peroxidase follows a non-scrambling mechanism

Edited by Judit Ovádi

Jutta Vlasits
- Department of Chemistry at BOKU, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria
Christa Jakopitsch
- Department of Chemistry at BOKU, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria
Manfred Schwanninger
- Department of Chemistry at BOKU, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria
Peter Holubar
- Department of Biotechnology at BOKU, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria
Christian Obinger
- Department of Chemistry at BOKU, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria
- Corresponding author. Fax: +43 1 36006 6059.

Received 9 November 2006; received in revised form 6 December 2006; accepted 19 December 2006. published online 05 January 2007.

Abstract

Despite catalyzing the same reaction (2H₂O₂→2H₂O+O₂) heme-containing monofunctional catalases and bifunctional catalase-peroxidases (KatGs) do not share sequence or structural similarities raising the question of whether or not the reaction pathways are similar or different. The production of dioxygen from hydrogen peroxide by monofunctional catalases has been shown to be a two-step process involving the redox intermediate compound I which oxidizes H₂O₂ directly to O₂. In order to investigate the origin of O₂ released in KatG mediated H₂O₂ degradation we performed a gas chromatography–mass spectrometry investigation of the evolved O₂ from a 50:50 mixture of H₂¹⁸O₂/H₂¹⁶O₂ solution containing KatGs from Mycobacterium tuberculosis and Synechocystis PCC 6803. The GC–MS analysis clearly demonstrated the formation of ¹⁸O₂ (m/e=36) and ¹⁶O₂ (m/e=32) but not ¹⁶O¹⁸O (m/e=34) in the pH range 5.6–8.5 implying that O₂ is formed by two-electron oxidation without breaking the O–O bond. Also active site variants of Synechocystis KatG with very low catalase but normal or even enhanced peroxidase activity (D152S, H123E, W122F, Y249F and R439A) are shown to oxidize H₂O₂ by a non-scrambling mechanism. The results are discussed with respect to the catalatic mechanism of KatG.