et al. used to obtain the pre-bound state, and obtained a similar orientation for H2O2 (see Figure ). Yet, the Cu-H2O2 distance is smaller in our Cu(I)…H2O2 structure (2.54 Å) compared to the structure by Lim et al. (2.93 Å), which could result from the fact that we kept the protein non-hydrogen atoms fixed at their crystal structure positions, while Lim et al. optimized the complete model. Prompted by a previous investigation of the influence of the potential coupling of the substrate and the binding of O2, we further investigated the binding of O2 to substrate-free and substrate-bound structures (resulting in the structures in green boxes in the left part of Figure ). We mainly use a frontier MO approach to evaluate electronic structure changes. The characters of the MOs were estimated by Löwdin reduced orbital populations; they provide a percentage for the contribution of the atomic orbitals to each molecular orbital. From a computational perspective, Cu(I) is in a closed-shell singlet state, where all electrons are paired, i.e., α and β electrons within a given spatial orbital have the same energy. This is not the case for intermediates with Cu(II) oxidation state (or Cu(I) with O2), which are open-shell (doublet or triplet) states. In these states, α and β orbitals may differ in energy, and we refer separately to the α and β frontier orbitals for these calculations. Structures, individual orbitals and spin densities were plotted using PYMOL and the ORCA_PLOT tool. Atomic spin populations were calculated using Löwdin partitioning.