Building Localized NADP(H) Recycling Circuits to Advance Enzyme Cascadetronics

Abstract The catalytic action of enzymes in a cascade trapped within a mesoporous electrode material is simultaneously energized, controlled and observed through the efficient, reversible electrochemical NAD(P)(H) recycling catalyzed by one of the enzymes. In their nanoconfined state, nicotinamide cofactors are tightly channeled current carriers, mediating multi‐step reactions in either direction (oxidation or reduction) with a rapid response time. By incorporating a hydrogen‐borrowing enzyme pair, the internal action of which opposes the external voltage bias driving oxidation or reduction, a reduction process can be performed under overall oxidizing conditions, and vice versa. The power of the method to control and resolve complex metabolic pathways is demonstrated using a non‐linear, three‐enzyme cascade extended by incorporating a fourth enzyme, urease. The latter generates in situ ammonia, which is enzymatically consumed in a reductive process, but the immediate current response to each addition of urea is observed, unusually, by applying an oxidizing potential. A practical consequence is that enzyme‐catalyzed electrochemical reduction reactions requiring anaerobic conditions (to avoid O 2 interference) can readily be studied under ambient aerobic conditions. The results illustrate how a complex enzyme cascade confined within a porous electrode and connected to an electrical power source manifests characteristics associated with electronic circuits.