A combined quantum and molecular mechanical study of the O2 reductive cleavage in the catalytic cycle of multicopper oxidases.

The four-electron reduction of dioxygen to water in multicopper oxidases takes place in a trinuclear copper cluster, which is linked to a mononuclear blue copper site, where the substrates are oxidized. Recently, several intermediates in the catalytic cycle have been spectroscopically characterized, and two possible structural models have been suggested for both the peroxy and native intermediates. In this study, these spectroscopic results are complemented by hybrid quantum and molecular mechanical (QM/MM) calculations, taking advantage of recently available crystal structures with a full complement of copper ions. Thereby, we obtain optimized molecular structures for all of the experimentally studied intermediates involved in the reductive cleavage of the O(2) molecule and energy profiles for individual reaction steps. This allows identification of the experimentally observed intermediates and further insight into the reaction mechanism that is probably relevant for the whole class of multicopper oxidases. We suggest that the peroxy intermediate contains an O(2)(2-) ion, in which one oxygen atom bridges the type 2 copper ion and one of the type 3 copper ions, whereas the other one coordinates to the other type 3 copper ion. One-electron reduction of this intermediate triggers the cleavage of the O-O bond, which involves the uptake of a proton. The product of this cleavage is the observed native intermediate, which we suggest to contain a O(2-) ion coordinated to all three of the copper ions in the center of the cluster.