Molecular mechanism of lytic polysaccharide monooxygenases

Abstract

The lytic polysaccharide monooxygenases (LPMOs) are copper metalloenzymes that can enhance polysaccharide depolymerization through an oxidative mechanism and hence boost generation of biofuel from e.g. cellulose. By employing density functional theory in a combination of quantum mechanics and molecular mechanics (QM/MM), we report a complete description of the molecular mechanism of LPMOs. The QM/MM scheme allows us to describe all reaction steps with a detailed protein environment and we show that this is necessary. Several active species capable of abstracting a hydrogen from the substrate have been proposed previously and starting from recent crystallographic work on a substrate-LPMO complex, we investigate previously suggested paths as well as new ones. We describe the generation of the reactive intermediates, the abstraction of a hydrogen atom from the polysaccharide substrate, as well as the final recombination step in which OH is transferred back to the substrate. We show that a superoxo [CuO₂]⁺ complex can be protonated by a nearby histidine residue (suggested by recent mutagenesis studies and crystallographic work) and, provided an electron source is available, leads to formation of an oxyl-complex after cleavage of the O-O bond and dissociation of water. The oxyl complex either reacts with the substrate or is further protonated to a hydroxyl complex. Both the oxyl and hydroxyl complexes are also readily generated from a reaction with H₂O₂, which was recently suggested to be the true co-substrate, rather than O₂. The C-H abstraction by the oxyl and hydroxy complexes is overall favorable with activation barriers of 69 and 94 kJ mol^-1, compared to the much higher barrier (156 kJ mol^-1) obtained for the copper-superoxo species. We obtain good structural agreement for intermediates for which structural data are available and the estimated reaction energies agree with experimental rate constants. Thus, our suggested mechanism is the most complete to date and concur with available experimental evidence.

Fig. 1. Fungal LPMO active site. Residue numbers refer to the enzyme from Lentinus similis (5ACF).

Fig. 2. QM/MM optimised [Cu(H₂O)]²⁺ (1, upper) and [Cu(H₂O)]⁺ (2, lower) states. The optimisations were carried out with TPSS-D3/def2-SV(P) and system 2 fixed.

Fig. 3. QM/MM optimised [CuO₂]⁺ (3) without (upper) and with (lower) His147 included in the QM region. Both optimisations were carried out with TPSS-D3/def2-SV(P) and system 2 fixed.

Fig. 4. Reactant 3 (left), transition state (middle) and product 4a (right) for the reaction 3 → 4a. All structures were optimised with TPSS/def2-SV(P) and system 2 fixed.

Fig. 5. Reactant 4b (left), transition state (middle) and product 6a (right) for reaction 4b → 6a. Structures were optimised with TPSS/def2-SV(P) and energies were obtained with TPSS-D3/def2-TZVPP.

Fig. 6. Optimised structures of intermediates 5 (upper) and 6b (lower) in the HIE147 state. Structures were optimised with TPSS/def2-SV(P) and energies were obtained with TPSS-D3/def2-TZVPP.

Fig. 7. Optimised structures of intermediate 6c in the singlet (upper) and triplet states (lower) with HIP147. Structures were optimised with TPSS/def2-SV(P) and energies were obtained with TPSS-D3/def2-TZVPP.

Fig. 8. Reactant (6b, left), transition state (middle) and product (7a, right) for the 6b → 7a reaction with His147 in the HID state. Structures were optimised with TPSS/def2-SV(P) and energies were obtained with TPSS-D3/def2-TZVPP.

Fig. 9. Reactant (6b, left), transition state (middle) and product (7a, right) for the 6b → 7a reaction with His147 in the HIE state. Structures were optimised with TPSS/def2-SV(P) and energies were obtained with TPSS-D3/def2-TZVPP.

Fig. 10. Reactant 6c (left), transition state (middle) and product 7b (right) for the 6c → 7b reaction with His147 in the HID state. Structures were optimised with TPSS/def2-SV(P) and energies were obtained with TPSS-D3/def2-TZVPP.

Fig. 11. Reactant 6c (left), transition state (middle) and product 7b (right) for the 6c → 7b reaction with His147 in the HIE state. Structures were optimised with TPSS/def2-SV(P) and energies were obtained with TPSS-D3/def2-TZVPP.

Fig. 12. Reactant 7a (left), transition state (middle) and product 8a (right) for the 7a → 8a reaction with His147 is in the HIE state. The structures were optimised with TPSS/def2-SV(P) and energies were obtained with TPSS-D3/def2-TZVPP.

Fig. 13. Reactant 7a (left), transition state (middle) and product 8a (right) for the 7a → 8a reaction with His147 is in the HIP state. The structures were optimised with TPSS/def2-SV(P) and energies were obtained with TPSS-D3/def2-TZVPP.

Fig. 14. Final, calculated mechanism for LPMO C–H activation. Activation energies are given as E_TS and individual reaction energies are given below products, using the reactant as reference (all energies are in kJ mol^–1). The energies are from TPSS with B3LYP in parentheses (always with def2-TZVPP basis sets). Protons are in all cases from His147 (in the HIP form). A few reactions are calculated with His147 in HIE/HIP or HIE/HID forms, as indicated with a subscript. Energies where not obtained for cases with different number of particles between reactant and product (reductions), and in case of 5 → 6c, since 6c was formed spontaneously.