Distal Hydrophobic Loop Modulates the Copper Active Site and Reaction of AA13 Polysaccharide Monooxygenases.

Polysaccharide monooxygenases (PMOs) use a type-2 copper center to activate O2 for the selective hydroxylation of one of the two C-H bonds of glycosidic linkages. Our electron paramagnetic resonance (EPR) analysis and molecular dynamics (MD) simulations suggest the unprecedented dynamic roles of the loop containing the residue G89 (G89 loop) on the active site structure and reaction cycle of starch-active PMOs (AA13 PMOs). In the Cu(II) state, the G89 loop could switch between an "open" and "closed" conformation, which is associated with the binding and dissociation of an aqueous ligand in the distal site, respectively. The conformation of the G89 loop influences the positioning of the copper center on the preferred substrate of AA13 PMOs. The dissociation of the distal ligand results in the bending of the T-shaped core of the Cu(II) active site, which could help facilitate its reduction to the active Cu(I) state. In the Cu(I) state, the G89 loop is in the "closed" conformation with a confined copper center, which could allow for efficient O2 binding. In addition, the G89 loop remains in the "closed" conformation in the Cu(II)-superoxo intermediate, which could prevent off-pathway superoxide release via exchange with the distal aqueous ligand. Finally, at the end of the reaction cycle, aqueous ligand binding to the distal site could switch the G89 loop to the "open" conformation and facilitate product release.

Fine Tuning of the Copper Active Site in Polysaccharide Monooxygenases.

Type 2 copper active sites, one of the several important copper active sites in biology, were recently found in the novel superfamily of polysaccharide monooxygenases (PMOs) that cleave recalcitrant polysaccharides via an unprecedented oxidative mechanism. The copper center in PMOs is ligated by the bidentate N-terminal histidine residue and another conserved histidine residue, forming a unique T-shaped core termed as Histidine brace. This core serves as the foundation for diverse structures and electronic properties among PMO families and subfamilies. Understanding of the copper active site in PMOs is limited to the static solid structures obtained with X-ray diffraction (XRD), whereas in several families, the copper center exists as a mixture of species in solution as indicated by electron paramagnetic resonance (EPR) spectroscopy. To obtain further details on the copper active sites in PMOs, we carried out density functional theory calculations and molecular dynamics simulations on MtPMO3* that were previously studied with XRD, EPR, mutagenesis, and activity assays. The results reveal the fine-tuning of the binding of the distal ligands by both proximal and distal H-bond-forming residues. Q167 forms H bonds with the proximal OTyr ligand of Y167 and the equatorial aqueous ligand (Oeq). T74 forms a H bond with the distal aqueous ligand (Odis). Removing these H bonds by mutating Q167 or T74 to alanine results in great fluctuations of the axial ligands. Strengthening the proximal H bonds by mutating Q167 to glutamate confines Y167 to the copper centers. In all mutants, the residence time of Odis is significantly reduced. Q167A, Q167E, and T74A mutants were previously shown to have a significantly reduced activity. Our results indicate that well-tuned H bonds are required for the activity of PMOs.