QM/MM calculations: All QM/MM calculations were based on equilibrated and QM/MM optimized structures of LsAA9 from Lentinus similis (5ACF ) and TaAA9A from Thermoascus aurantiacus (2YET ) from the studies ref. The second-sphere tyrosine is present in both enzymes (Tyr175 and Tyr164 in TaAA9 and LsAA9, respectively, as shown in Fig. ) and in both LPMOs a tyrosyl species was spectroscopically characterised. The protonation state of the second-coordination sphere histidine (His164) in TaAA9 was changed from a doubly protonated form (Hip164) to a singly protonated one (Hie164), to match the protonation state present in the LsAA9 structure. The employed QM regions are shown in Fig. . For LsAA9 the QM region comprised the copper ion, the oxyl/hydroxyl ligand, the complete methylated His1 residue, and the side chains of His78, Tyr164, Thr2, Gln162, Glu148 and Hie147 as well as four water molecules. This is the same QM region also employed in ref. and a slightly larger QM region (extended by Glu143 and three water molecules) as employed in ref. . For TaAA9, the QM region consists of the copper ion and all the residues of the first coordination sphere, i.e., the full His1 residue, the oxyl/hydroxyl ligand, the imidazole ring of His86, the phenol ring of Tyr175 and the axial water molecule. Additionally, the side chains of Gln173 and Hie164 were included, as well as parts of the backbone from the Gly2 residue. The latter was included up to the C α atom and capped by replacing the carboxyl C with a hydrogen atom. The same applies to the corresponding residue Thr2 in LsAA9. The side chains of all other residues were capped with a hydrogen by replacing C α . Note that TaAA9 does not have a Glu residue corresponding to Glu148 in LsAA9, and we therefore did not include the residue at this position in the QM region for TaAA9.