Finally, the structures with H 2 O 2 dissociated from 4 red are on average 8.4 ± 6.8 kJ/mol (7.8 ± 8.1 kJ/mol for TPSS, n = 8 for both functionals) higher in energy than the starting structure (4f red ). The spin densities of all dissociated structures are similar: most spin density is on Cu and the remaining spin density is on the coordinating nitrogen atoms of the histidine brace and the oxygen of water. Thus, the dissociation leads to H 2 O 2 and Cu(II), and is generally feasible. This result differs again from the QM/MM calculations by Caldararu et al., who find that releasing the restraint of 4.6 Å between Cu and O p resulted in H 2 O 2 binding back to Cu. When the system is further reduced, they find a dissociation of H 2 O 2 to be favorable with a reaction barrier of 4 kJ/mol and a reaction energy of 3 kJ/mol. From the above results we conclude that, dissociation of superoxide is feasible, depending on the micro-solvation. Dissociation of OOH • before reduction could be feasible, but this result is highly functional dependent. After reduction, dissociation of OOH • /OOH -is either uphill or leads to spontaneous formation of H 2 O 2 . Only the dissociation of H 2 O 2 seems consistently favorable. Thus, we conclude that it is most favorable to dissociate OOH • /OOH -or H 2 O 2 and below we investigate if these can be formed at the active-site.