We use the second order spin-flip constricted variational DFT method to probe a possible multideterminantal nature of the PI and to devise a computational strategy for its treatment. In the present study, we investigate by DFT and quantum mechanics/molecular mechanics (QM/MM) the potential energy surface (PES) of the PI as a function of the orientation of O 2 within the copper cluster to examine the influence of the second coordination sphere on the structure of the PI. This orientation of the oxygen moiety has been discussed as a result of the influence from the second coordination sphere. Earlier density functional theory (DFT) studies as well as spectral and structural comparison to a fully oxidized structural analogue of the PI known as the peroxide adduct (PA) reveal that O 2 bridges all three copper atoms of the trinuclear cluster in the PI. The peroxide intermediate (PI) is obtained in the first step of the reduction of O 2 by multicopper oxidases.
