Metalloenzymes facilitate the activation of small molecules essential in biological and energy storage reactions, such as hydrogen production, carbon dioxide reduction, carbon-carbon bond formation, and nitrogen fixation. Recently, their potential for industrial applications has garnered significant attention. Nickel-containing enzymes, such as Acetyl Coenzyme A Synthase (ACS), drive the reaction for carbon-carbon bond formation from CO and CH3, which results in the formation of an acetyl group and further thioacetate synthesis for fundamental biological energy storage. To understand the reactivity and mechanistic properties of ACS, we have engineered enzyme-like active sites within the M121A Nickel Azurin (NiAz) protein framework as an electronic and reactivity model. To drive the chemical reactivity and the redox control over the NiAz (II/I) intermediates, we use an electrochemical approach to employ 1) direct electron transfer (DET) by immobilizing NiAz within a polymer matrix and 2) mediated electron transfer (MET) by using a europium–ligand redox mediator and characterize the M121A NiAz reaction intermediates through spectroelectrochemical (SEC) methods for S-methyl thioacetate synthesis. By investigating the electrocatalytic mechanisms of NiAz, we aim to provide a fundamental understanding of the native ACS reaction mechanism and illustrate M121A NiAz potential for industrial applications.
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