Before investigating the C-H abstraction in the next subsection, we comment on using His147 as a proton donor. This residue is highly conserved and has been discussed intensively in the literature. Mutation studies have shown that it is a crucial residue for LPMO activity, although mutations did not completely remove the activity. A study with neutron crystallography provided experimental evidence of the formation of a [CuO 2 ] n+ and a [CuOOH] n+ intermediate in the dioxygen activation process, and identified the His157 (equivalent to His147 in the investigated NcAA9D LPMO) as the proton donor. Meanwhile, an X-ray crystallographic study employing specialized refinement techniques concluded that His147 is unlikely to be protonated. These authors interpreted this result as the histidine is an unlikely proton donor during catalysis. Yet, if the HIP state was sufficiently stable to be observed during an X-ray crystallographic measurement, it may also be argued that, in this case, the proton would have been unlikely to dissociate. Hence, HIP would have been a poor proton donor. A very recent computational study on the equivalent second-sphere histidine in PcAA9D and NcAA9D found that the histidine could indeed be protonated. Still, it then rotated out of the active site, which is unlikely in the presence of a substrate. An QM/MM MD investigation on the same LsAA9 as investigated here, but without substrate, found that the second proton transfer to form 6a was significantly uphill 75 (earlier proton transfer from His147 was not investigated). This generally agrees with our results; while we did not investigate the energy associated with forming the HIP state, we can conclude that even if it was formed, it is not sufficiently reactive to form a highly reactive oxygen species for the oxidation of the substrate. We therefore conclude that the effect on the activity seen in mutation studies is rather due to the role of stabilizing the [Cu(II