To probe the protonation states of key catalytic residues and intermediates in the NcLPMO9D active site, a neutron diffraction dataset to 2.4 Å resolution was collected at 100 K on a crystal of NcLPMO9D reduced with ascorbate at pD 6.0 (pH 5.6) in the presence of atmospheric oxygen. Structure refinement was performed solely against neutron data to circumvent potential artefacts induced by X-ray radiation damage. The axial position is vacant, supporting displacement of both the equatorial and axial water molecules upon Cu 2+ reduction. The neutron scattering length density maps at the active site of Molecule A indicate the presence a fully occupied equatorially coordinated dioxygen species in a mixed state of a two-atom superoxo species and a three-atom hydroperoxo species with occupancies of 0.30 and 0.70, respectively (Fig. and). The intermediates have η1 end-on geometry at a Cu-O1 distance of ~1.96 Å and ~1.98 Å for the superoxo and hydroperoxo species, respectively. The superoxo species is modelled with an O-O bond length of 1.28 Å while the hydroperoxo species is modelled with a bond length of 1.46 Å. The Cu-O1-O2 angles are ~147° and 151° for the superoxo and hydroperoxo species respectively. A Glu30 residue from Molecule B is modeled 2.06 Å and 2.08 Å away from the O2 of the superoxo and hydroperoxo species, respectively, however it displays a disordered conformation with limited NSLD, as has been observed for NcLPMO9D structures with the same crystal packing. The modeling of an activated dioxygen species is in good accordance with our previous high resolution X-ray structures which indicated the presence of a peroxo species with a 1.44 ± 0.06 Å bond length with a Cu-O1-O2 angle of 140.52° and 1.90 Å from the Cu following ascorbate reduction (Fig. ). ). In our previous high-resolution X-ray structure, discrimination between a putative superoxo and peroxo species was based on bond length determination due to the lack of visibility of hydrogen atoms. In addition, the occupancy of the activated dioxygen species only refined to 0.59 and an axial water remained present with an occupancy of 0.48. We reason that photoreduction due to X-ray beam exposure resulted in an intermediates with reduced occupancy. The NcLPMO9D active site presented here is absent of radiation damage since neutron protein crystallography is a non-destructive technique permitting capture of a mixed occupancy of a superoxo and hydroperoxo species. The observed hydroperoxo species may represent an intermediate in the mechanism of LPMO-catalyzed H2O2 formation, since LPMOs are known to produce H2O2 in the absence of substrate. Caldararu et al. have further shown it is more energetically favorable for H2O2 formation to proceed by dissociation of H2O2 from the copper active site than HO2. This higher dissociation energy of HO2 is supported by the observed binding stability of the hydroperoxo species in our structure.