Issue 44, 2023

New insights into controlling radical migration pathways in heme enzymes gained from the study of a dye-decolorising peroxidase

Abstract

In heme enzymes, such as members of the dye-decolorising peroxidase (DyP) family, the formation of the highly oxidising catalytic Fe(IV)-oxo intermediates following reaction with hydrogen peroxide can lead to free radical migration (hole hopping) from the heme to form cationic tyrosine and/or tryptophan radicals. These species are highly oxidising (∼1 V vs. NHE) and under certain circumstances can catalyse the oxidation of organic substrates. Factors that govern which specific tyrosine or tryptophan the free radical migrates to in heme enzymes are not well understood, although in the case of tyrosyl radical formation the nearby proximity of a proton acceptor is a recognised facilitating factor. By using an A-type member of the DyP family (DtpAa) as an exemplar, we combine protein engineering, X-ray crystallography, hole-hopping calculations, EPR spectroscopy and kinetic modelling to provide compelling new insights into the control of radical migration pathways following reaction of the heme with hydrogen peroxide. We demonstrate that the presence of a tryptophan/tyrosine dyad motif displaying a T-shaped orientation of aromatic rings on the proximal side of the heme dominates the radical migration landscape in wild-type DtpAa and continues to do so following the rational engineering into DtpAa of a previously identified radical migration pathway in an A-type homolog on the distal side of the heme. Only on disrupting the proximal dyad, through removal of an oxygen atom, does the radical migration pathway then switch to the engineered distal pathway to form the desired tyrosyl radical. Implications for protein design and biocatalysis are discussed.

Graphical abstract: New insights into controlling radical migration pathways in heme enzymes gained from the study of a dye-decolorising peroxidase

Supplementary files

Article information

Article type
Edge Article
Submitted
24 ago 2023
Accepted
06 out 2023
First published
06 out 2023
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2023,14, 12518-12534

New insights into controlling radical migration pathways in heme enzymes gained from the study of a dye-decolorising peroxidase

M. Lučić, M. T. Wilson, J. Pullin, M. A. Hough, D. A. Svistunenko and J. A. R. Worrall, Chem. Sci., 2023, 14, 12518 DOI: 10.1039/D3SC04453J

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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