Correlating Experiment and Theory for Electron Paramagnetic Resonance and Substrate Binding in a Lytic Polysaccharide Monooxygenase

Abstract

<div>Lytic polysaccharide monooxygenases (LPMOs) are enzymes that binds polysaccharides followed by an (oxidative) disruption of the polysaccharide surface, thereby boosting depolymerization. The binding process between LPMO and polysaccharide is key to the mechanism and recent investigations have established structure-function relationships for this binding, employing hyperfine coupling constants (HFCs) from EPR spectroscopy. Unfortunately, EPR does not provide direct structural data and therefore the experimental EPR parameters have been supported with parameters cal-</div><div>culated with density functional theory. Yet, calculated HFCs are extremely sensitive</div><div>to the employed computational setup. Using the LPMO Ls(AA9)A, we here quantify</div><div>the importance of several choices in the computational setup, ranging from the use</div><div>of specialized basis, the underlying structures, and the employed exchange–correlation</div><div>functional. We compare our results to both X-ray structures and experiment (EPR spectra) for Ls(AA9)A as well as to recent experimental/theoretical results for another</div><div>(AA10) family of LPMOs.</div>

Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding

“…S15). The reduction in distance between Tyr-O and Cu is also reproduced qualitatively in the QM/MM-optimized structures of LsAA9_A, where the distance was found to decrease by 0.2-0.3 A ˚upon LsAA9_A binding the substrate (Theibich et al, 2021). While the role of tyrosine remains unclear, the shortening of the Tyr-O to Cu bond has been shown here to be reproducible for LsAA9_A.…”

“…In the lowest-dose substrate-free structures of fungal/bacterial LsAA9_A, the distance to the equatorial water ligand is in the range 2.2-2.3 A ˚, while that to the axial ligand is in the range 2.6-2.7 A ˚. For comparison, QM/MM models have shown distances of 2.16/2.25, 2.08/2.53 and 2.21/2.34 A ˚for the equatorial/axial water molecule, depending on the parameters of the calculation (Theibich et al, 2021), which could indicate that even at the lowest X-ray dose possible, a small amount of photoreduction has occurred.…”

“…5(a) and 5(c)], the equatorial solvent-facing ligand, modelled as a Cl À ion, is 2.3 A ˚from the Cu atom, while in the highest-dose structures, the distance has increased to 3.9 and 3.8 A ˚for LsAA9_A(f)-Cell 4 and LsAA9_A(Ec)-Cell 3 , respectively. For comparison, the average distance to the equatorial ligand is 2.1 A ˚between different QM/MM models in a recent theoretical study (Theibich et al, 2021). In several intermediate X-ray doses of LsAA9_A(f)-Cell 4 , the Cl À ion was modelled in a double conformation, with 90% occupancy for the conformation furthest from the Cu atom and 10% occupancy for the conformation closest to the Cu atom, indicating a mixed state between Cu 2+ and Cu + .…”

Molecular Mechanism of Substrate Oxidation in Lytic Polysaccharide Monooxygenases: Insight from Theoretical Investigations

“…For instance, the axial water displacement upon substrate binding seen in crystallographic work [27] was confirmed (along with other modifications of the active site) since it was reflected in the obtained spin‐Hamiltonian parameters [27,42,105] . In recent papers, calculated spin‐Hamiltonian parameters were compared with the experimental ones [74,100,106,107] . A qualitative agreement was achieved, but the EPR parameters are infamous for being very dependent on the computational setup.…”

“…This can be compared to the QM‐cluster and QM/MM structure optimizations, which show that the reduction is associated with dissociation of the equatorial water molecule [66,71] . A recent paper by Theibich et al [100] . compared QM/MM and X‐ray structures, which showed that QM/MM qualitatively reproduces changes induced by substrate binding (unfortunately, no RMSD values were reported).…”