Targeting the reactive intermediate in polysaccharide monooxygenases

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are copper metalloenzymes that can enhance polysaccharide depolymerization through an oxidative mechanism, making them interesting for the production of biofuel from cellulose. However, the details of this activation are unknown; in particular, the nature of the intermediate that attacks the glycoside C-H bond in the polysaccharide is not known, and a number of different species have been suggested. The homolytic bond-dissociation energy (BDE) has often been used as a descriptor for the bond-activation power, especially for inorganic model complexes. We have employed quantum-chemical cluster calculations to estimate the BDE for a number of possible LPMO intermediates to bridge the gap between model complexes and the actual LPMO active site. The calculated BDEs suggest that the reactive intermediate is either a Cu(II)-oxyl, a Cu(III)-oxyl, or a Cu(III)-hydroxide, which indicate that O-O bond breaking occurs before the C-H activation step.

Keywords: Computational chemistry; Density functional theory; Lytic polysaccharide monooxygenase; Reaction mechanism.

Scheme 1

Reaction catalyzed by LPMO

Scheme 2

Hydrogen abstraction by the LPMO active intermediate

Fig. 1

Active site of an LPMOs in the resting state, [Cu–OH${}_2$]${}^{2+}$ with a bound trisaccharide substrate (G3). Residue numbers refer to the enzyme from Lentinus similis [20]

Scheme 3

Homolytic bond cleavage reactions involving the intermediate and substrate

Fig. 2

Overview over possible intermediates responsible for the C–H activation in LPMOs and their relation in terms of oxidation/reduction and protonation/deprotonation reactions. The product of the hydrogen-abstraction reaction in Scheme 2 is equivalent with the addition of an electron and a proton to the reactant, leading to the species one step along the diagonal down and right in the figure. Intermediates in dashed boxes a–c are considered in this study. The insets show Lewis structures of two of the intermediates

Fig. 3

Left The QM systems employed in the QM/MM optimisation of [Cu–OO]${}^{+}$. Right The truncated system employed for calculations of BDEs for [Cu–OO]${}^{+}$. The other intermediates were truncated similarly, but with varying ligands (cf. Fig. 2). Atoms marked with “$\star$” were frozen in the structure optimisations. Color codes: Cu is brown, C is gray, O is red, N is blue, and H is white