From Protein Film Electrochemistry to Nanoconfined Enzyme Cascades and the Electrochemical Leaf

[Image: see text] Protein film electrochemistry (PFE) has given unrivalled insight into the properties of redox proteins and many electron-transferring enzymes, allowing investigations of otherwise ill-defined or intractable topics such as unstable Fe–S centers and the catalytic bias of enzymes. Many enzymes have been established to be reversible electrocatalysts when attached to an electrode, and further investigations have revealed how unusual dependences of catalytic rates on electrode potential have stark similarities with electronics. A special case, the reversible electrochemistry of a photosynthetic enzyme, ferredoxin-NADP(+) reductase (FNR), loaded at very high concentrations in the 3D nanopores of a conducting metal oxide layer, is leading to a new technology that brings PFE to myriad enzymes of other classes, the activities of which become controlled by the primary electron exchange. This extension is possible because FNR-based recycling of NADP(H) can be coupled to a dehydrogenase, and thence to other enzymes linked in tandem by the tight channelling of cofactors and intermediates within the nanopores of the material. The earlier interpretations of catalytic wave-shapes and various analogies with electronics are thus extended to initiate a field perhaps aptly named “cascade-tronics”, in which the flow of reactions along an enzyme cascade is monitored and controlled through an electrochemical analyzer. Unlike in photosynthesis where FNR transduces electron transfer and hydride transfer through the unidirectional recycling of NADPH, the “electrochemical leaf” (e-Leaf) can be used to drive reactions in both oxidizing and reducing directions. The e-Leaf offers a natural way to study how enzymes are affected by nanoconfinement and crowding, mimicking the physical conditions under which enzyme cascades operate in living cells. The reactions of the trapped enzymes, often at very high local concentration, are thus studied electrochemically, exploiting the potential domain to control rates and direction and the current–rate analogy to derive kinetic data. Localized NADP(H) recycling is very efficient, resulting in very high cofactor turnover numbers and new opportunities for controlling and exploiting biocatalysis.