On the Track of Long-Range Electron Transfer in B-Type Dye-Decolorizing Peroxidases: Identification of a Tyrosyl Radical by Computational Prediction and Electron Paramagnetic Resonance Spectroscopy.

Kevin Nys,Paul Georg Furtmüller,Vera Pfanzagl,Sabine Van Doorslaer,Christian Obinger

doi:10.1021/acs.biochem.1c00129

Abstract

The catalytic activity of dye-decolorizing peroxidases (DyPs) toward bulky substrates, including anthraquinone dyes, phenolic lignin model compounds, or 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), is in strong contrast to their sterically restrictive active site. In two of the three known subfamilies (A- and C/D-type DyPs), catalytic protein radicals at surface-exposed sites, which are connected to the heme cofactor by electron transfer path(s), have been identified. So far in B-type DyPs, there has been no evidence for protein radical formation after activation by hydrogen peroxide. Interestingly, B-type Klebsiella pneumoniae dye-decolorizing peroxidase (KpDyP) displays a persistent organic radical in the resting state composed of two species that can be distinguished by W-band electron spin echo electron paramagnetic resonance (EPR) spectroscopy. Here, on the basis of a comprehensive mutational and EPR study of computationally predicted tyrosine and tryptophan variants of KpDyP, we demonstrate the formation of tyrosyl radicals (Y247 and Y92) and a radical-stabilizing Y-W dyad between Y247 and W18 in KpDyP, which are unique to enterobacterial B-type DyPs. Y247 is connected to Y92 by a hydrogen bonding network, is solvent accessible in simulations, and is involved in ABTS oxidation. This suggests the existence of long-range electron path(s) in B-type DyPs. The mechanistic and physiological relevance of the reaction mechanism of B-type DyPs is discussed.

Highlights

The catalytic activity of dye-decolorizing peroxidases (DyPs) toward bulky substrates, including anthraquinone dyes, phenolic lignin model compounds, or 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), is in strong contrast to their sterically restrictive active site
Quenching of the porphyryl radical can occur by unspecific or specific internal electron transfer, thereby generating a socalled Compound I*, which exhibits a Compound II-like ultraviolet−visible (UV−vis) spectrum but is electronically a distinct redox state [oxoiron(IV) protein radical] that can be identified by electron paramagnetic resonance (EPR) spectroscopy
We present a computational approach of long-range electron transport (LRET) prediction in Klebsiella pneumoniae dyedecolorizing peroxidase (KpDyP) by molecular dynamics (MD) simulation combined with site-directed mutagenesis (Y18F, Y82F, Y92F, Y237F, Y247F, W176F, W18F/Y92F, and Y92F/Y247F) and multifrequency electron paramagnetic resonance spectroscopies to elucidate the localization and origin of the “restingstate” radical

Summary

Introduction

The catalytic activity of dye-decolorizing peroxidases (DyPs) toward bulky substrates, including anthraquinone dyes, phenolic lignin model compounds, or 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), is in strong contrast to their sterically restrictive active site. In two of the three known subfamilies (A- and C/D-type DyPs), catalytic protein radicals at surfaceexposed sites, which are connected to the heme cofactor by electron transfer path(s), have been identified. On the basis of a comprehensive mutational and EPR study of computationally predicted tyrosine and tryptophan variants of KpDyP, we demonstrate the formation of tyrosyl radicals (Y247 and Y92) and a radical-stabilizing Y-W dyad between Y247 and W18 in KpDyP, which are unique to enterobacterial B-type DyPs. Y247 is connected to Y92 by a hydrogen bonding network, is solvent accessible in simulations, and is involved in ABTS oxidation. Heme peroxidases follow the so-called Polous and Kraut mechanism (Figure 1D).[1,9] Hydrogen peroxide mediates the oxidation of the ferric resting state to Compound I (k1) [oxoiron(IV) porphyrin π-cation radical].9. It long eluded identification due to its short half-life and subsequent formation of additional radical sites with questionable biological significance.[13−15] More recently, a tyrosyl radical was shown to play a central role during conversion of coproheme to heme b in coproheme decarboxylases (ChdC).[16−18] Crucially, the radical sites described above are located on highly conserved, catalytically active residues within the protein core and in the proximity of the heme cofactor

Methods

Results

Conclusion