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 O_Tyr ligand of Y167 and the equatorial aqueous ligand (O_eq). T74 forms a H bond with the distal aqueous ligand (O_dis). 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 O_dis 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.