Mapping the Initial Stages of a Protective Pathway that Enhances Catalytic Turnover by a Lytic Polysaccharide Monooxygenase

Jingming Zhao, Ying Zhuo, Daniel E. Diaz, Muralidharan Shanmugam, Abbey J. Telfer, Peter J. Lindley, Daniel Kracher, Takahiro Hayashi, Lisa S. Seibt, Florence J. Hardy, Oliver Manners, Tobias M. Hedison, Katherine A. Hollywood, Reynard Spiess, Kathleen M. Cain, Sofia Diaz-Moreno, Nigel S. Scrutton, Morten Tovborg, Paul H. Walton, Derren J. HeyesAnthony P. Green

Research output: Contribution to journalArticlepeer-review


Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C-H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a CuII-(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) CuII-tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues "on the fly" to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.

Original languageEnglish
Pages (from-to)20672-20682
Number of pages11
JournalJournal of the American Chemical Society
Issue number37
Early online date9 Sept 2023
Publication statusPublished - 20 Sept 2023

Bibliographical note

© 2022 The Authors

Cite this