Mechanism of hydrogen peroxide formation by lytic polysaccharide monooxygenase

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.