RESUMEN
The importance of energized nanoconfinement for facilitating the study and execution of enzyme cascades that feature multiple exchangeable cofactors is demonstrated by experiments with carboxylic acid reductase (CAR), an enzyme that requires both NADPH and ATP during a single catalytic cycle. Conversion of cinnamic acid to cinnamaldehyde by a package of four enzymes loaded into and trapped in the random nanopores of an indium tin oxide (ITO) electrode is driven and monitored through the simultaneous delivery of electrical and chemical energy. The electrical energy is transduced by ferredoxin NADP+ reductase, which undergoes rapid, direct electron exchange with ITO and regenerates NADP(H). The chemical energy provided by phosphoenolpyruvate, a fuel contained in the bulk solution, is cotransduced by adenylate kinase and pyruvate kinase, which efficiently convert the AMP product back into ATP that is required for the next cycle. The use of the two-kinase system allows the recycling process to be dissected to evaluate the separate roles of AMP removal and ATP supply during presteady-state and steady-state catalysis.