Lytic polysaccharide monooxygenases (LPMOs; also designated PMOs) are copper metalloenzymes that perform unique redox chemistry resulting in disruption of polysaccharide chains by oxygen atom insertion at the glycosidic bond. The LPMO active site contains a mononuclear Cu 2+ center within a "histidine brace" motif in which the copper is coordinated by the N-terminal histidine amino group, the Nd atom of His1 and the Ne atom of a second conserved histidine. Of particular commercial and biotechnological interest is the ability of fungal LPMOs to catalyze the oxidative cleavage of recalcitrant crystalline cellulose at the C1 and/or C4 position, thereby enhancing polysaccharide depolymerization by increasing glucan access to hydrolytic enzymes via the overall reaction shown in Scheme 1. Scheme 1 Oxidation of the C1/C4 carbon in the glycosidic bond catalyzed by LPMOs with oxygen as the co-substrate. H atoms to be abstracted by the reactive oxygen species shown in green. While H2O2 has also been shown to be a LPMO co-substrate, the work presented here focuses on the activation of O2, the first characterized co-substrate in polysaccharide oxidation and the substrate in the synthesis of H2O2 in absence of substrate. One-electron reduction of the copper active site to Cu and binding of the molecular oxygen co-substrate initiates the reaction cycle with the generally accepted formation of a Cu 2+ superoxide, [Cu-O2] + . In the current consensus for the O2-based mechanism, following the rapid reduction of molecular dioxygen to the superoxide, the LPMO reaction mechanism requires further delivery of electrons and protons to form a reactive oxygen intermediate which oxidizes the polysaccharide substrate. The identity of the activated oxygen species responsible for hydrogen atom abstraction (HAA) from the polysaccharide substrate leading to subsequent substrate oxidation, is however still a matter of ongoing research. Intensive studies encompassing X-ray crystallographic, computational and spectroscopic examinations of mononuclear copper monooxygenases as well as biomimetic copper complexes have proposed dioxygen intermediates such as the superoxide [Cu-O2] + and hydroperoxo [CuOOH] + species where the O-O bond remains intact. Furthermore, formation of the hydroxide [CuOH] 2+ and oxyl [CuO] + intermediates after O-O bond scission, with subsequent involvement in HAA, have been proposed. Formation of a hydroperoxo, hydroxy or oxyl HAA species would require further protonation and reduction of the superoxide however the identity of the proton donor is still debated. Several second-shell residues including histidine (His147 in LsLPMO9A/His 161 in MtPMO3*) as well as glutamine (Gln167 in MtPMO3* and glutamate (Glu201 in JdLPMO10A) have been proposed to be involved in intermediate protonation. Quantum mechanics/molecular mechanics (QM/MM) calculations have also shown that a hydrogen atom could be abstracted by the superoxide species from an exogenous reducing agent such as ascorbic acid. Neutron protein crystallography is particularly well suited to the determination of protonation states within a protein due to the technique's unique sensitivity to hydrogen atoms and protons often invisible to X-ray diffraction. Neutron protein crystallography is also a non-destructive technique ideally suited for the study of radiation-sensitive metalloproteins. We report here two neutron models of a carbohydrate-free C4-hydroxylating Neurospora crassa LPMO9D: a structure of the activated, ascorbate-reduced form and a structure of the resting state at pH 4.4 (pD 4.8) to further address open questions regarding initial molecular oxygen activation. The neutron crystallographic structure of the reduced NcLPMO9D shows cryo-capture of activated dioxygen at the copper active site modelled as a superoxo and protonated hydroperoxo species with a population ratio of 0.30:0.70. The neutron scattering length density maps of the conserved secondary coordination shell His157 is best represented as 70% imidazolate and a 30% singly protonated form. The copper coordinating N-terminal amino group His1 remains in a doubly protonated ND2 state following copper reduction and molecular oxygen activation. Mining minima energy calculations reveal His157 flexibility in solution which is absent in the crystal structure due to binding-face to binding-face crystal packing. Furthermore, the neutron and X-ray crystal structures of the resting state NcLPMO9D at low pH show that His157 remains singly protonated when exposed to acidic conditions.