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.

Fig. 1. The active site of AA10-LPMOs with the histidine brace and the nearby Phe164 residue, numbering according to PDB 5VG0. The figure also shows to the employed quantum system in the quantum-refinement calculations. (A) and (B) refer to chain A and B, respectively.

Fig. 2. Structure and nuclear density maps of the active site after joint refinement. The m2F_o – DF_c maps are contoured at 1.0σ and the mF_o – DF_c maps are contoured at +3.0σ (green) and –3.0σ (red). (A) – subunit A, ND₂; (B) – subunit B, ND₂; (C) – subunit A, ND^–, (D) – subunit B, ND^–. RSZD– values for the N-terminal atom are given for the ND₂ states to highlight if there are any extra atoms in the model. RSZD+ values for the N-terminal atom are given for the ND^– states to highlight if there are any missing atoms in the model.

Fig. 3. Structure and nuclear density maps of the active site after quantum refinement. m2F_o – DF_c maps are contoured at 1.0σ and mF_o – DF_c maps are contoured at +3.0σ (green) and –3.0σ (red) (A) – subunit A, ND₂; (B) – subunit B, ND₂; (C) – subunit A, ND^–, (D) – subunit B, ND^–.

Fig. 4. Active sites of the original crystal structure (entry 5VG0) (blue), calculated/quantum-refined structures with peroxide oxygen species (red) or superoxide oxygen species (green). (A) – QM/MM structure, subunit A; (B) – QM/MM structure, subunit B; (C) – quantum-refined structure, subunit A; (D) – quantum-refined structure, subunit B.

Fig. 5. Protonation of the superoxide moiety. (A) – reactant, proton on Glu-201 (triplet); (B) – product with the superoxide protonated (open-shell singlet).

Fig. 6. Second protonation of the superoxide moiety starting from (A) – Cu(ii)–HO₂ or (B) – Cu(i)–O₂H. Relative energies of the reactants, products and transition state (for Cu(ii)–O₂H) are depicted above each structure.

Fig. 7. Release of the dioxygen moiety after reduction of the copper center to Cu(i). (A) – optimised Cu(i)–HO₂ system; (B) – optimised Cu(i)–H₂O₂ system.