When the substrate binds, the HOMO is a tyrosine orbital but the dx2-y2-orbital (see Figure ) remains the highest-lying orbital with d-character, while the LUMO is always an orbital of co-substrate (H2O2) character. Each LUMO is shown in Figure and has the shape of a σ*-orbital. This agrees well with the LUMO described by Lim et al. However, as in the Cu(I) systems, we observe that substrate binding brings the remaining orbitals of copper d-character closer to the dx2-y2-orbital, decreasing the energy difference between them to 0.8-1.8 eV. Additionally, the substrate lowers the energy difference between the dx2-y2-orbital and the H2O2 σ*-orbital (from 3.9 eV in Cu(I)…H2O2 to 3.1 eV in Cu(I)-Cell…H2O2, see Table and), making the electron transfer (reaction 6b in Figure ) more favourable with the bound substrate. This is in line with QM/MM and QM-cluster calculations that show that this reaction is overall favourable in LsAA9A with and without substrate. For comparison with older LPMO papers and to investigate if the conclusions for H2O2 were independent of the co-substrate, we additionally carried out the same experiment with O2 (see Section S1 in the SI for further details). However, since several independent studies showed that the former is not a cosubstrate of LsAA9A, we will discuss these results in the SI.