Reductive cleavage of the O-O bond in multicopper oxidases: a QM/MM and QM study.

The key step in the reaction mechanism of multicopper oxidases (MCOs)--the cleavage of the O-O bond in O2--has been investigated using combined quantum mechanical and molecular mechanical (QM/MM) methods. This process represents a reaction pathway from the peroxy intermediate after it accepts one electron from the nearby type-1 Cu site to the experimentally-observed native intermediate, which is the only fully oxidised catalytically relevant state in MCOs. Scans of the QM(DFT)/MM potential energy surface have allowed us to obtain estimates of the activation energies. Furthermore, vacuum calculations on a smaller model of the active site have allowed us to estimate the entropy contributions to the barrier height and to obtain further insight into the reaction by comparing the small cluster model with the QM/MM model, which includes the entire protein. Owing to the complicated electronic structure of these low-spin exchange coupled systems, multireference quantum chemical calculations at the complete-active space second-order perturbation theory (CASPT2) were used in an attempt to benchmark the barrier heights obtained at the DFT(B3LYP) level. Our best estimate of the activation barrier is deltaG = 60-65 kJ mol(-1), in good agreement with the experimental barrier of approximately 55 kJ mol(-1), which can be inferred from the experimental rate constant of k > 350 s(-1). It has also been shown that the reaction involves protonation of the O2 moiety before bond cleavage. The proton likely comes from a nearby carboxylate residue which was recently suggested by the experiments.