In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase—The Interplay of Key Amino Acids, pH, Buffer and Glycerol

Dye-decolorizing peroxidases (DyPs) have gained interest for their ability to oxidize anthraquinone-derived dyes and lignin model compounds. Spectroscopic techniques, such as electron paramagnetic resonance and optical absorption spectroscopy, provide main tools to study how the enzymatic function i...

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Main Authors:	Kevin Nys, Vera Pfanzagl, Jeroen Roefs, Christian Obinger, Sabine Van Doorslaer
Format:	Article
Language:	English
Published:	MDPI AG 2021-09-01
Series:	International Journal of Molecular Sciences
Subjects:	heme peroxidases electron paramagnetic resonance active site structure UV-vis spectroscopy alkaline transition ligand binding
Online Access:	https://www.mdpi.com/1422-0067/22/18/9849

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spelling	doaj-7780cd970c32497b8d2ff9721d0e5b112021-09-26T00:23:14ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-09-01229849984910.3390/ijms22189849In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase—The Interplay of Key Amino Acids, pH, Buffer and GlycerolKevin Nys0Vera Pfanzagl1Jeroen Roefs2Christian Obinger3Sabine Van Doorslaer4BIMEF Laboratory, Department of Chemistry, University of Antwerp, 2610 Antwerp, BelgiumDivision of Biochemistry, Department of Chemistry, BOKU—University of Natural Resources and Life Sciences, 1190 Vienna, AustriaBIMEF Laboratory, Department of Chemistry, University of Antwerp, 2610 Antwerp, BelgiumDivision of Biochemistry, Department of Chemistry, BOKU—University of Natural Resources and Life Sciences, 1190 Vienna, AustriaBIMEF Laboratory, Department of Chemistry, University of Antwerp, 2610 Antwerp, BelgiumDye-decolorizing peroxidases (DyPs) have gained interest for their ability to oxidize anthraquinone-derived dyes and lignin model compounds. Spectroscopic techniques, such as electron paramagnetic resonance and optical absorption spectroscopy, provide main tools to study how the enzymatic function is linked to the heme-pocket architecture, provided the experimental conditions are carefully chosen. Here, these techniques are used to investigate the effect of active site perturbations on the structure of ferric P-class DyP from <i>Klebsiella pneumoniae</i> (K<i>p</i>DyP) and three variants of the main distal residues (D143A, R232A and D143A/R232A). Arg-232 is found to be important for maintaining the heme distal architecture and essential to facilitate an alkaline transition. The latter is promoted in absence of Asp-143. Furthermore, the non-innocent effect of the buffer choice and addition of the cryoprotectant glycerol is shown. However, while unavoidable or indiscriminate experimental conditions are pitfalls, careful comparison of the effects of different exogenous molecules on the electronic structure and spin state of the heme iron contains information about the inherent flexibility of the heme pocket. The interplay between structural flexibility, key amino acids, pH, temperature, buffer and glycerol during in vitro spectroscopic studies is discussed with respect to the poor peroxidase activity of bacterial P-class DyPs.https://www.mdpi.com/1422-0067/22/18/9849heme peroxidaseselectron paramagnetic resonanceactive site structureUV-vis spectroscopyalkaline transitionligand binding
collection	DOAJ
language	English
format	Article
sources	DOAJ
author	Kevin Nys Vera Pfanzagl Jeroen Roefs Christian Obinger Sabine Van Doorslaer
spellingShingle	Kevin Nys Vera Pfanzagl Jeroen Roefs Christian Obinger Sabine Van Doorslaer In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase—The Interplay of Key Amino Acids, pH, Buffer and Glycerol International Journal of Molecular Sciences heme peroxidases electron paramagnetic resonance active site structure UV-vis spectroscopy alkaline transition ligand binding
author_facet	Kevin Nys Vera Pfanzagl Jeroen Roefs Christian Obinger Sabine Van Doorslaer
author_sort	Kevin Nys
title	In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase—The Interplay of Key Amino Acids, pH, Buffer and Glycerol
title_short	In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase—The Interplay of Key Amino Acids, pH, Buffer and Glycerol
title_full	In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase—The Interplay of Key Amino Acids, pH, Buffer and Glycerol
title_fullStr	In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase—The Interplay of Key Amino Acids, pH, Buffer and Glycerol
title_full_unstemmed	In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase—The Interplay of Key Amino Acids, pH, Buffer and Glycerol
title_sort	in vitro heme coordination of a dye-decolorizing peroxidase—the interplay of key amino acids, ph, buffer and glycerol
publisher	MDPI AG
series	International Journal of Molecular Sciences
issn	1661-6596 1422-0067
publishDate	2021-09-01
description	Dye-decolorizing peroxidases (DyPs) have gained interest for their ability to oxidize anthraquinone-derived dyes and lignin model compounds. Spectroscopic techniques, such as electron paramagnetic resonance and optical absorption spectroscopy, provide main tools to study how the enzymatic function is linked to the heme-pocket architecture, provided the experimental conditions are carefully chosen. Here, these techniques are used to investigate the effect of active site perturbations on the structure of ferric P-class DyP from <i>Klebsiella pneumoniae</i> (K<i>p</i>DyP) and three variants of the main distal residues (D143A, R232A and D143A/R232A). Arg-232 is found to be important for maintaining the heme distal architecture and essential to facilitate an alkaline transition. The latter is promoted in absence of Asp-143. Furthermore, the non-innocent effect of the buffer choice and addition of the cryoprotectant glycerol is shown. However, while unavoidable or indiscriminate experimental conditions are pitfalls, careful comparison of the effects of different exogenous molecules on the electronic structure and spin state of the heme iron contains information about the inherent flexibility of the heme pocket. The interplay between structural flexibility, key amino acids, pH, temperature, buffer and glycerol during in vitro spectroscopic studies is discussed with respect to the poor peroxidase activity of bacterial P-class DyPs.
topic	heme peroxidases electron paramagnetic resonance active site structure UV-vis spectroscopy alkaline transition ligand binding
url	https://www.mdpi.com/1422-0067/22/18/9849
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In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase—The Interplay of Key Amino Acids, pH, Buffer and Glycerol

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