Our results show that reduction from Cu(II) to Cu(I) has a significant influence on the orbital energies, bringing the occupied orbitals of copper d-character close to the frontier, where they can engage in a reaction. This is independent of the presence or absence of substrate binding and explains why the unique priming reduction is required for LPMO activity. Further, we observed a similar orbital splitting in Cu(I) as a previous study 51 in pre-bound Cu(I)…H2O2, where the highest occupied copper d-orbital (the dx2-y2) is raised by more than 1 eV in energy compared to the other occupied copper d-orbitals. Yet, this energy difference decreases by a factor of three upon substrate binding. Previous studies suggest that a relative rise in energy of the dx2-y2 orbital could be favourable for LPMO reactions, bringing the dx2-y2-orbital closer to the anti-bonding σ*-or π*-orbitals in hydrogen peroxide or dioxygen, respectively. Our observation seems to contradict this suggestion. However, the relative energy of the anti-bonding orbitals should also be considered as well as substrate binding. Therefore, we additionally investigated how pre-bound H2O2 (and O2) impact the electronic structure of substrate-free and substrate-bound LsAA9A. For the substrate-free H2O2 structure, we find that the dx2-y2-orbital is more than 1 eV higher in energy than the lower-lying copper d-orbitals, which is consistent with previous results . This energy difference is decreased by onethird upon substrate binding, which is consistent with our findings without co-substrate. However, substrate binding additionally causes the H2O2 σ*-orbital to be lower in energy, bringing it closer to the copper dx2-y2-orbital. Based on our calculations, it is this lowering of the H2O2 σ*-orbital upon substrate binding that facilitates the electron transfer from copper to H2O2. This does not occur in calculations with O2 or superoxide (see SI). The above trends are independent of the employed functionals (B3LYP and TPSS). To further generalize our findings, and to compare to EPR data of AA10 LPMOs, substrate-bound structures of this and other LPMO families would be a valuable starting point.