Probing electron pathways to multicopper oxidase copper centers reveals shortcut for enhancing oxygen reduction reaction

Directly connected to an electrode, high potential MCOs catalyse the oxygen reduction reaction (ORR) at low overpotential with high efficiency. MCOs contain two redox centers, a near surface-located mononuclear copper (T1) oxidising a substrate and a buried trinuclear copper center (TNC) reducing dioxygen to water. Which of the two copper centers is directly wired to the electrode during the bioelectrocatalytic reduction of dioxygen is a challenging question to address. Beyond potentially improving the direct electron transfer process, the rational orientation of a high potential MCO should allow to bypass the rate-limiting internal electron transfer from T1 to TNC and enhance the ORR efficiency. Variants of a high potential fungal laccase (LAC3) isolated from Trametes sp. C30 were designed to target two opposite orientations in which the T1 copper center is either as close (T1-orientation) or as far (anti-T1 orientation) as possible from the MWCNT electrode. Analysis of the electrochemical response of these variants under different conditions allow to conclude: (1) the T1 center is the first electron acceptor in randomly adsorbed enzymes, (2) pyrene-enzyme hybrids allow for a selective wiring of T1 and TNC sites to MWCNTs and (3) anti-T1 oriented hybrids are three-fold more efficient for ORR.