Characterisation of the heme aqua-ligand coordination environment in an engineered peroxygenase cytochrome P450 variant

Date

2023

Authors

Podgorski, M.N.
Lee, J.H.Z.
Harbort, J.S.
Nguyen, G.T.H.
Doherty, D.Z.
Donald, W.A.
Harmer, J.R.
Bruning, J.B.
Bell, S.G.

Editors

Advisors

Journal Title

Journal ISSN

Volume Title

Type:

Journal article

Citation

Journal of Inorganic Biochemistry, 2023; 249:1-13

Statement of Responsibility

Matthew N. Podgorski, Joel H.Z. Lee, Joshua S. Harbort, Giang T.H. Nguyen, Daniel Z. Doherty, William A. Donald, Jeffrey R. Harmer, John B. Bruning, Stephen G. Bell

Conference Name

DOI

10.1016/j.jinorgbio.2023.112391

Abstract

The cytochrome P450 enzymes (CYPs) are heme-thiolate monooxygenases that catalyse the insertion of an oxygen atom into the C–H bonds of organic molecules. In most CYPs, the activation of dioxygen by the heme is aided by an acid-alcohol pair of residues located in the I-helix of the enzyme. Mutation of the threonine residue of this acid-alcohol pair of CYP199A4, from the bacterium Rhodospeudomonas palustris HaA2, to a glutamate residue induces peroxygenase activity. In the X-ray crystal structures of this variant an interaction of the glutamate side chain and the distal aqua ligand of the heme was observed and this results in this ligand not being readily displaced in the peroxygenase mutant on the addition of substrate. Here we use a range of bulky hydrophobic and nitrogen donor containing ligands in an attempt to displace the distal aqua ligand of the T252E mutant of CYP199A4. Ligand binding was assessed by UV–visible absorbance spectroscopy, native mass spectrometry, electron paramagnetic resonance and X-ray crystallography. None of the ligands tested, even the nitrogen donor ligands which bind directly to the iron in the wild-type enzyme, resulted in displacement of the aqua ligand. Therefore, modification of the I-helix threonine residue to a glutamate residue results in a significant strengthening of the ferric distal aqua ligand. This ligand was not displaced using any of the ligands during this study and this provides a rationale as to why this mutant can shutdown the monooxygenase pathway of this enzyme and switch to peroxygenase activity.

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Dissertation Note

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Description

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Rights

© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Grant ID

http://purl.org/au-research/grants/arc/DP140103229
 http://purl.org/au-research/grants/arc/DP200100335
 http://purl.org/au-research/grants/arc/DP160102681
 http://purl.org/au-research/grants/arc/FT140100355
 http://purl.org/au-research/grants/arc/FT120100421
 http://purl.org/au-research/grants/arc/FT200100798

Published Version

https://doi.org/10.1016/j.jinorgbio.2023.112391

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Persistent link to this record

https://hdl.handle.net/2440/140315