Mechanism of hydrogen peroxide formation by lytic polysaccharide monooxygenase.

Octav Caldararu,Erik D. Hedegård,Ulf Ryde,Esko Oksanen

doi:10.1039/c8sc03980a

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing metalloenzymes that can cleave the glycosidic link in polysaccharides. This could become crucial for production of energy-efficient biofuels from recalcitrant polysaccharides. Although LPMOs are considered oxygenases, recent investigations have shown that H2O2 can also act as a co-substrate for LPMOs. Intriguingly, LPMOs generate H2O2 in the absence of a polysaccharide substrate. Here, we elucidate a new mechanism for H2O2 generation starting from an AA10-LPMO crystal structure with an oxygen species bound, using QM/MM calculations. The reduction level and protonation state of this oxygen-bound intermediate has been unclear. However, this information is crucial to the mechanism. We therefore investigate the oxygen-bound intermediate with quantum refinement (crystallographic refinement enhanced with QM calculations), against both X-ray and neutron data. Quantum refinement calculations suggest a Cu(ii)-O-2 system in the active site of the AA10-LPMO and a neutral protonated -NH2 state for the terminal nitrogen atom, the latter in contrast to the original interpretation. Our QM/MM calculations show that H2O2 generation is possible only from a Cu(i) center and that the most favourable reaction pathway is to involve a nearby glutamate residue, adding two electrons and two protons to the Cu(ii)-O-2 system, followed by dissociation of H2O2.

Highlights

Lytic polysaccharide monoxoygenases (LPMOs) are metalloenzymes capable of activating molecular oxygen, and thereby inserting a single oxygen atom into the C–H bonds that comprise the glycoside link in polysaccharides.[1,2] This oxidation leads to cleavage of the glycoside link, which may disrupt the surface of crystalline polysaccharides sufficiently to boost polysaccharide decomposition.[3]
We re-investigate the nature of the Cu– oxygen species observed in crystal structures, both in terms of oxidation state and protonation state of the terminal amino group
We suggest a new mechanism of H2O2 formation, based on QM/MM calculations starting from the oxygenated species we deem most probable from the quantum re nements

Summary

Introduction

Lytic polysaccharide monoxoygenases (LPMOs) are metalloenzymes capable of activating molecular oxygen, and thereby inserting a single oxygen atom into the C–H bonds that comprise the glycoside link in polysaccharides.[1,2] This oxidation leads to cleavage of the glycoside link, which may disrupt the surface of crystalline polysaccharides sufficiently to boost polysaccharide decomposition.[3]. One histidine is the amino-terminal residue that coordinates bidentately with both the N-terminus and the imidazole side chain

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