During LPMO catalysis, a second shell histidine has been proposed to be involved in protonation of active site intermediates with QM/MM studies by Hedegård and Ryde on LsAA9A suggesting that His147 in a doubly protonated state can act as a proton donor. Additionally, Span et al. have proposed that His 161 in MtPMO3 plays a role as a proton donor based on structural, mutagenesis and spectroscopic studies as well as activity assays. LPMOs are expected to catalyze oxygen insertion under acidic conditions since they function synergistically alongside cellulases which show optimum activity at pH 3.5 -5.5. Given the surprising role of a neutral His157 as a proton donor to the activated dioxygen species in our structure, we sought to probe the protonation state of His157 when exposed to acidic buffer conditions. We collected a 2.14 Å room-temperature neutron diffraction dataset under low pH conditions (pH 4.4/pD 4.8) achieved by vapor exchange of the crystal with acidified buffer. Analysis of the nuclear scattering length density maps indicate that the His157 residue remains singly protonated at the Neposition at this pH, which is supported by Fo-Fc omit difference maps in both Molecule A and B (Fig. ). To further support our acidic pH structure obtained by vapor exchange, a 1.5 Å X-ray dataset was collected on a crystal directly soaked in acidic buffer. The position of His157 at pH 4.4 superimposes to its position at pH 6.0. In this position, Nd-protonation would sterically clash with the backbone amide proton, further supporting the single Ne-protonation state observed in the neutron structure (Fig. and). The barricaded active site observed in crystallo and electropositive environment of the copper potentially play a role in maintaining the observed single histidine protonation state and prevent formation of protonation pathways to the active site. In both the neutron and X-ray low pH structures, the copper remains coordinated in the histidine brace, contrary to the disordered His78 observed in LsLPMO9A at pH 3.5 (PDB 5N04). The low pH structure of LsLPMO9A was obtained using high NaCl concentrations, however,