Second-Sphere\nHistidine Catalytic Function in a Fungal\nPolysaccharide Monooxygenase

Fungal polysaccharide monooxygenases (PMOs) oxidatively\ndegrade\ncellulose and other carbohydrate polymers via a mononuclear copper\nactive site using either O<sub>2</sub> or H<sub>2</sub>O<sub>2</sub> as a cosubstrate. Cellulose-active fungal PMOs in the auxiliary\nactivity 9 (AA9) family have a conserved second-sphere hydrogen-bonding\nnetwork consisting of histidine, glutamine, and tyrosine residues.\nThe second-sphere histidine has been hypothesized to play a role in\nproton transfer in the O<sub>2</sub>-dependent PMO reaction. Here\nthe role of the second-sphere histidine (H157) in an AA9 PMO, <i>Mt</i>PMO9E, was investigated. This PMO is active on soluble\ncello-oligosaccharides such as cellohexaose (Glc6), thus enabling\nkinetic analysis with the point variants H157A and H157Q. The variants\nappeared to fold similarly to the wild-type (WT) enzyme and yet exhibited\nweaker affinity toward Glc6 than WT (WT <i>K</i><sub>D</sub> = 20 ± 3 μM). The variants had comparable oxidase (O<sub>2</sub> reduction to H<sub>2</sub>O<sub>2</sub>) activity to WT at\nall pH values tested. Using O<sub>2</sub> as a cosubstrate, the variants\nwere less active for Glc6 hydroxylation than WT, with H157A being\nthe least active. Similarly, H157Q was competent for Glc6 hydroxylation\nwith H<sub>2</sub>O<sub>2</sub>, but H157A was less active. Comparison\nof the crystal structures of H157Q and WT <i>Mt</i>PMO9E\nreveals that a terminal heteroatom of Q157 overlays with N<sub>ε</sub> of H157. Altogether, the data suggest that H157 is not important\nfor proton transfer, but support a role for H157 as a hydrogen-bond\ndonor to diatomic-oxygen intermediates, thus facilitating catalysis\nwith either O<sub>2</sub> or H<sub>2</sub>O<sub>2</sub>.