Testing of different protonation states and visual examination of the 2Fo-Fc and Fo-Fc omit neutron scattering length density maps of the Molecule A secondary coordination shell His157 suggest that this residue is best modelled by the neutral Ne-protonated form and the imidazolate form with occupancies of 0.30 and 0.70, respectively (Fig. and). Analysis of the Molecule B neutron scattering length density maps indicate that His157 is singly Ne-protonated at full occupancy (Fig. ). The Ne nitrogen of His157 in Molecule A is observed at a distance of ~3.0 Å from the distal oxygen O2 of the equatorially bound superoxo species and hydroperoxo species. These findings indicate that His157 plays a role in protonation of the activated dioxygen species observed in the barricaded active site presented here. Activity and structural studies of MtPMO3* as well as QM/MM calculations of LsLPMO9A have implicated this conserved histidine residue in proton transfer to the copper-bound superoxide during LPMO catalysis (His161 and His147 in MtPMO3* and LsLPMO9A, respectively). QM/MM studies have also indicated that the Glu201 residue near the copper active site in JdLPMO10A is capable of donating a proton to a bound superoxo to form a hydroperoxo species. Computational studies have suggested that ascorbate may function as a proton donor to the superoxo species; 26 however, the active site occlusion due to crystal packing in our structure does not permit ascorbate to access the copper active site, and there was no evidence in the NSLD maps for an ascorbate molecule near the active site. Following formation of the superoxide in NcLPMO9D, protonation by His157 may form the copper(II)-hydroperoxyl species