Electron Transfer to the Trinuclear Copper Cluster in Electrocatalysis by the Multicopper Oxidases

High-potential multicopper oxidases (MCOs) are excellent catalysts able to perform the oxygen reduction reaction (ORR) at remarkably low overpotentials. Moreover, MCOs are able to interact directly with the electrode surfaces via direct electron transfer (DET), that makes them the most commonly used electrocatalysts for oxygen reduction in biofuel cells. The central question in MCO electrocatalysis is whether the type 1 (T1) Cu is the primary electron acceptor site from the electrode, or whether electrons can be transferred directly to the trinuclear copper cluster (TNC), bypassing the rate-limiting intramolecular electron transfer step from the T1 site. Here, using site-directed mutagenesis and electrochemical methods combined with data modeling of electrode kinetics, we have found that there is no preferential superexchange pathway for DET to the T1 site. However, due to the high reorganization energy of the fully oxidized TNC, electron transfer from the electrode to the TNC does occur primarily through the T1 site. We have further demonstrated that the lower reorganization energy of the TNC in its two-electron reduced, alternative resting, form enables DET to the TNC, but this only occurs in the first turnover. This study provides insight into the factors that control the kinetics of electrocatalysis by the MCOs and a guide for the design of more efficient biocathodes for the ORR.