Moreover, mutation of these residues also leads to loss of activity. For the O 2 -driven reaction, the formation of the [CuOOH] + species was feasible, yet the transition to a reactive oxyl species was inaccessible. This contrasts our previous QM/MM calculations with small QM regions and confirms recent experiments showing that O 2 is not a suitable co-substrate for glycosidic bond cleavage for the LsAA9 LPMO. Thus, a consistent choice of QM region is pivotal to ensure that the calculated mechanism is congruent with experimental results.