From these possibilities, we start by investigating whether the first proton transfer can take place before the reduction of the superoxide (reaction 2 --→ 3 in Fig. ). For all used functionals, the reactant is most stable in the triplet state (see Fig. ). Starting from the lowest-energy conformer 2a HIP in the triplet spin state with a doubly protonated second-coordination-sphere histidine HIP208 (see Fig and Table ), we perform linear transit calculations, where we restrain the distance between the distal oxygen in superoxide O d and the proton H ε2 in HIP208. During the transit, a spin crossing occurs, leading to the open-shell singlet being more stable by 9.9 kJ/mol (24.7 kJ/mol with TPSS). The reaction is not favorable with a barrier of 49.6 kJ/mol and a reaction energy of 48.7 kJ/mol, although it should be noted that we found this to be highly dependent on the employed functional; TPSS yields a barrier of 3.7 kJ/mol and a reaction energy of -5.8 kJ/mol. With B3LYP the spin density is mostly on Cu and on the oxygen atoms of O 2 /OOH for the reactant/product (see Table ), whereas TPSS results in rather small spin densities for the product, indicating that the open-shell singlet is close to collapsing into a closed-shell species. Compared to the closed-shell singlet state of the product, the open-shell singlet is 10.5 kJ/mol lower in energy with B3LYP and 1.7 kJ/mol with TPSS, showing that the spin states are essentially degenerate with TPSS. Collapsing of the open-shell singlet state has been previously observed in calculations with TPSS and we thus believe that B3LYP provides more physically sound energies for this transit.