Tuning O$_2$ enzymatic reduction: Roles of methionine-rich domains and electrochemical metalation of active centers
Résumé
CueOs are multicopper oxidases which feature a methionine rich domain (Met-rich) covering the first electron acceptor and proposed to play roles in enzymatic catalysis. However, how these domains impact catalysis is still a matter of debate. In this work, we compare O$_2$ reduction at gold electrodes according to the Met-rich content of two CueOs from Escherichia coli and Hafnia alvei bacteria. The extent of electrocatalysis is shown to depend on the type of CueO adsorbed on the gold surface, with activity being nearly zero when using the CueO protein containing the largest Met-rich domain. To clarify the role of Met-rich domains, CueO engineering was additionally performed by deleting Met-rich domains or exchanging them between the two proteins to create chimeric enzymes. Using a combination of electrochemistry, surface plasmon resonance and molecular dynamic simulation, we demonstrate that the Met-rich domain induces steric hindrance that affects the electron transfer process. In addition, a progressive and potential dependent increase in the catalytic currents is observed. We demonstrate that this activation process is associated with the incorporation of copper cofactors in the immobilized proteins by electrogenerated Cu$^+$ . This electrochemically induced CueO metalation paves the way for the large-scale production of multicopper oxidases (MCO) followed by in vitro maturation, as high copper concentrations are toxic in vivo.
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