Laccase Employing Water-Mediated Double-Proton-Coupled Electron Transfer To Achieve the Oxygen Reduction Reaction

The enzymatic reduction of substrate O₂ to H₂O is an essential reaction in multicopper oxidases (MCOs), which has recently been intensively studied and applied to biofuel cell cathodes to achieve higher current efficiency. However, a full understanding of the catalytic mechanism of MCOs still remains elusive, especially with regard to the exact order of the electron/proton transfer in structural details. Our molecular mechanics and quantum mechanics calculations reveal that the hydroxide ligand of T2Cu favors the first protonation of O₂ at the trinuclear copper (TNC) cluster. The protonation of the T2Cu hydroxide ligand drives the cleavage of the HO-O bond for the formation of a native intermediate. In the three subsequent protonation reactions, the oxidation state of T1Cu in the reactants plays a crucial role in determining the corresponding reaction efficiency. The cuprous T1Cu facilitates the following three-step protonation reactions of O₂ derivatives with low energy barriers. In contrast, cupric T1Cu causes the corresponding reactions to encounter high energy barriers. The protonation of the O₂ derivative simultaneously drives the reduction of the four cupric ions of laccase. Therefore, the first three steps of oxygen reduction catalyzed by laccase occur via a similar water-mediated double-proton-coupled electron transfer mechanism with electron transfer from T1Cu to TNC and simultaneously indirect proton transfer from Glu451 to the O₂ derivative through a bridging H₂O at the same time. The fourth-step protonation takes place via a double proton concerned transfer mechanism. These findings have significant implications for the understanding of the elaborate proton-coupled electron transfer mechanism for the catalytic action of MCOs and inspire further work on the construction of biocathodes for the oxygen reduction reaction.