Formation of a Copper(II)–Tyrosyl Complex at the Active Site of Lytic Polysaccharide Monooxygenases Following Oxidation by H2O2

Paradisi, Alessandro; Johnston, Esther M.; Tovborg, Morten; Nicoll, Callum R.; Ciano, Luisa; Dowle, Adam; McMaster, Jonathan; Hancock, Y.; Davies, Gideon J.; Walton, Paul H.

Abstract

Hydrogen peroxide is a cosubstrate for the oxidative cleavage of saccharidic substrates by copper-containing lytic polysaccharide monooxygenases (LPMOs). The rate of reaction of LPMOs with hydrogen peroxide is high, but it is accompanied by rapid inactivation of the enzymes, presumably through protein oxidation. Herein, we use UV− vis, CD, XAS, EPR, VT/VH-MCD, and resonance Raman spectroscopies, augmented with mass spectrometry and DFT calculations, to show that the product of reaction of an AA9 LPMO with H2O2 at higher pHs is a singlet Cu(II)−tyrosyl radical species, which is inactive for the oxidation of saccharidic substrates. The Cu(II)−tyrosyl radical center entails the formation of significant Cu(II)−(●OTyr) overlap, which in turn requires that the plane of the d(x2−y2) SOMO of the Cu(II) is orientated toward the tyrosyl radical. We propose from the Marcus cross-relation that the active site tyrosine is part of a “hole-hopping” charge-transfer mechanism formed of a pathway of conserved tyrosine and tryptophan residues, which can protect the protein active site from inactivation during uncoupled turnover.

• Journal Article Type: Article
• Acceptance Date: Nov 1, 2019
• Online Publication Date: Nov 1, 2019
• Publication Date: Nov 20, 2019
• Deposit Date: Nov 18, 2019
• Publicly Available Date: Nov 25, 2019
• Journal: Journal of the American Chemical Society
• Print ISSN: 0002-7863
• Electronic ISSN: 1520-5126
• Publisher: American Chemical Society
• Peer Reviewed: Peer Reviewed
• Volume: 141
• Issue: 46
• Pages: 18585-18599
• DOI: https://doi.org/10.1021/jacs.9b09833
• Keywords: Colloid and surface chemistry; Biochemistry; General chemistry; Catalysis
• Public URL: https://nottingham-repository.worktribe.com/output/3322229
• Publisher URL: https://pubs.acs.org/doi/abs/10.1021/jacs.9b09833