Second-Sphere Histidine Catalytic Function in a Fungal Polysaccharide Monooxygenase

Fungal polysaccharide monooxygenases (PMOs) oxidatively degrade cellulose and other carbohydrate polymers via a mononuclear copper active site using either O₂ or H₂O₂ as a cosubstrate. Cellulose-active fungal PMOs in the auxiliary activity 9 (AA9) family have a conserved second-sphere hydrogen-bonding network consisting of histidine, glutamine, and tyrosine residues. The second-sphere histidine has been hypothesized to play a role in proton transfer in the O₂-dependent PMO reaction. Here the role of the second-sphere histidine (H157) in an AA9 PMO, <i>Mt</i>PMO9E, was investigated. This PMO is active on soluble cello-oligosaccharides such as cellohexaose (Glc6), thus enabling kinetic analysis with the point variants H157A and H157Q. The variants appeared to fold similarly to the wild-type (WT) enzyme and yet exhibited weaker affinity toward Glc6 than WT (WT <i>K</i>_D = 20 ± 3 μM). The variants had comparable oxidase (O₂ reduction to H₂O₂) activity to WT at all pH values tested. Using O₂ as a cosubstrate, the variants were less active for Glc6 hydroxylation than WT, with H157A being the least active. Similarly, H157Q was competent for Glc6 hydroxylation with H₂O₂, but H157A was less active. Comparison of the crystal structures of H157Q and WT <i>Mt</i>PMO9E reveals that a terminal heteroatom of Q157 overlays with N_ε of H157. Altogether, the data suggest that H157 is not important for proton transfer, but support a role for H157 as a hydrogen-bond donor to diatomic-oxygen intermediates, thus facilitating catalysis with either O₂ or H₂O₂.