A - Bonding-Assisted Molecular-Wiring of Folded-Cytochrome c and Naphthoquinone and Its Electron-Relay-Based Bioelectrocatalytic H₂O₂ Reduction Reaction Visualized by Redox-Competitive Scanning Electrochemical Microscopy

Theelectron-transfer (ET) reaction of cytochrome c (Cytc)protein with biomolecules is a cutting-edge researchareaof interest in understanding the functionalities of natural systems.Several electrochemical biomimicking studies based on Cytc-protein-modifiedelectrodes prepared via electrostatic interaction and covalent bondingapproaches have been reported. Indeed, natural enzymes involve multipletypes of bonding, such as hydrogen, ionic, covalent, and & pi;-& pi;,etc. In this work, we explore a Cytc-protein chemically modified glassycarbon electrode (GCE/CB@NQ/Cytc) prepared via & pi;-& pi;bonding using graphitic carbon as an underlying surface and an aromaticorganic molecule, naphthoquinone (NQ), as a cofactor for an effectiveET reaction. A simple drop-casting technique-based preparation ofGCE/CB@NQ showed a distinct surface-confined redox peak at a standardelectrode potential (E & DEG;) = -0.2 V vsAg/AgCl (surface excess = 21.3 nmol cm(-2)) in pH7 phosphate buffer solution. A control experiment of modificationof NQ on an unmodified GCE failed to show any such unique feature.For the preparation of GCE/CB@NQ/Cytc, a dilute solution of Cytc-pH7 phosphate buffer was drop-cast on the GCE/CB@NQ surface, whereinthe protein folding and denaturalization-based complication and itsassociated ET functionalities were avoided. Molecular dynamics simulationstudies show the complexation of NQ with Cytc at the protein bindingsites. The protein-bound surface shows an efficient and selectivebioelectrocatalytic reduction performance of H2O2, as demonstrated using cyclic voltammetry and amperometric i-t techniques. Finally, the redox-competitionscanning electrochemical microscopy (RC-SECM) technique was adoptedfor in situ visualization of the electroactive adsorbed surface. TheRC-SECM images clearly show the regions of highly bioelectrocatalyticactive sites of Cytc-proteins bound to NQ molecules on a graphiticcarbon surface. The binding of Cytc with NQ has significant implicationsfor studying the biological electron transport mechanism, and theproposed method provides the requisite framework for such a study.