In living cells, redox chains rely on nanoconfinement using tiny enclosures, such as the mitochondrial matrix or chloroplast stroma, to concentrate enzymes and limit distances that nicotinamide cofactors and other metabolites must diffuse. In a chemical analogue exploiting this principle, nicotinamide adenine dinucleotide phosphate (NADPH) and NADP + are cycled rapidly between ferredoxin–NADP + reductase and a second enzyme—the pairs being juxtaposed within the 5–100 nm scale pores of an indium tin oxide electrode. The resulting electrode material, denoted (FNR+E2)@ITO/support, can drive and exploit a potentially large number of enzyme‐catalysed reactions.

Multistep enzyme-catalyzed cascade reactions are highly efficient in nature due to the confinement and concentration of the enzymes within nanocompartments. In this way, rates are exceptionally high, and loss of intermediates minimised. Similarly, extended enzyme cascades trapped and crowded within the nanoconfined environment of a porous conducting metal oxide electrode material form the basis of a powerful way to study and exploit myriad complex biocatalytic reactions and pathways. One of the confined enzymes, ferredoxin-NADP+ reductase, serves as a transducer, rapidly and reversibly recycling nicotinamide cofactors electrochemically for immediate delivery to the next enzyme along the chain, thereby making it possible to energize, control and observe extended cascade reactions driven in either direction depending on the electrode potential that is applied. Here we show as proof of concept the synthesis of aspartic acid from pyruvic acid or its reverse oxidative decarboxylation/deamination, involving five nanoconfined enzymes.

Reduction of CO2 and its direct entry into organic chemistry is achieved efficiently and in a highly visible way using a metal oxide electrode in which two enzyme catalysts, one for electrochemically regenerating reduced nicotinamide adenine dinucleotide phosphate and the other for assimilating CO2 and converting pyruvate (C3) to malate (C4), are entrapped within its nanopores. The resulting reversible electrocatalysis is exploited to construct a solar CO2 reduction/water-splitting device producing O2 and C4 with high faradaic efficiency.

In living cells, the overall rates of catalytic reaction chains (cascades) are massively enhanced by nanoconfinement of enzymes in tiny enclosed volumes: presented in such a way, interdependent catalysts are highly concentrated, and distances (active site‐to‐active site) across which intermediates and cofactors must diffuse, may be tiny. In a parallel technology exploiting this principle, enzyme cascades are powered, amplified, and monitored in real time as they work in concert, being nanoconfined within the pores of an electrically conductive metal oxide electrode. The technology, nicknamed the electrochemical leaf, mimics chloroplast biosynthesis by exploiting nicotinamide adenine dinucleotide (NADP(H)) recycling catalysed by the flavoenzyme, ferredoxin NADP+ reductase (FNR). Adsorbed on the inner walls of the nanopores, FNR rapidly transfers electrons between the electrode and NADP(H). This activity is coupled to an oxidoreductase enzyme, also nanoconfined within the pores, which recycles the cofactor and selectively synthesises a desired product or senses an analyte. Use of catalyst and cofactor is very efficient. The technology is simple, inexpensive and adaptable, and scalable to both microscopic levels (diagnostics) and macroscopic levels (organic synthesis). Whereas native FNR is specific for NADP(H), the technology can be extended by genetic engineering to include NAD(H) as recycling cofactor.

Electron transport, mediated by NDA2 in H2-producing C. reinhardtii cells, shifts redox equilibria between the plastoquinone pool and PSII, and is observed as a transient fluorescence wave after a single flash.

In living cells,redoxchains rely on nanoconfinement using tiny enclosures,s uch as the mitochondrial matrix or chloroplast stroma, to concentrate enzymes and limit distances that nicotinamide cofactors and other metabolites must diffuse. In ac hemical analogue exploiting this principle,nicotinamide adenine dinucleotide phosphate (NADPH) and NADP + are cycled rapidly between ferredoxin-NADP + reductase and as econd enzyme-the pairs being juxtaposed within the 5-100 nm scale pores of an indium tin oxide electrode.T he resulting electrode material, denoted (FNR + E2)@ITO/support, can drive and exploit ap otentially large number of enzyme-catalysed reactions.

Hydrogenase-1 (Hyd-1) from E. coli poses a conundrum regarding the properties of electrocatalytic reversibility and associated bidirectionality now established for many redox enzymes. Its excellent H2-oxidizing activity begins only once a substantial overpotential is applied, and it cannot produce H2. A major reason for its unidirectional behavior is that the reduction potentials of its electron-relaying FeS clusters are too positive relative to the 2H+/H2 couple at neutral pH; consequently, electrons held within the enzyme lack the energy to drive H2 production. However, Hyd-1 is O2-tolerant and even functions in air. Changing a tyrosine (Y) or threonine (T), located on the protein surface within 10 Å of the distal [4Fe-4S] and medial [3Fe-4S] clusters, to cysteine (C), allows site-selective attachment of a silver nanocluster (AgNC), the reduced or photoexcited state of which is a powerful reductant. The AgNC provides a new additional redox site, capturing externally supplied electrons with sufficiently high energy to drive H2 production. Assemblies of Y’227C (or T’225C) with AgNCs/PMAA (PMAA = polymethyl acrylate templating several AgNC) are also electroactive for H2 production at a TiO2 electrode. A colloidal system for visible-light photo-H2 generation is made by building the hybrid enzyme into a heterostructure with TiO2 and graphitic carbon nitride (g-C3N4), the resulting scaffold promoting uptake of electrons excited at the AgNC. Each hydrogenase produces 40 molecules of H2 per second and sustains 20% activity in air.

Living organisms are characterized by the ability to process energy (all release heat). Redox reactions play a central role in biology, from energy transduction (photosynthesis, respiratory chains) to highly selective catalyzed transformations of complex molecules. Distance and scale are important: electrons transfer on a 1 nm scale, hydrogen nuclei transfer between molecules on a 0.1 nm scale, and extended catalytic processes (cascades) operate most efficiently when the different enzymes are under nanoconfinement (10 nm–100 nm scale). Dynamic electrochemistry experiments (defined broadly within the term “protein film electrochemistry,” PFE) reveal details that are usually hidden in conventional kinetic experiments. In PFE, the enzyme is attached to an electrode, often in an innovative way, and electron-transfer reactions, individual or within steady-state catalytic flow, can be analyzed in terms of precise potentials, proton coupling, cooperativity, driving-force dependence of rates, and reversibility (a mark of efficiency). The electrochemical experiments reveal subtle factors that would have played an essential role in molecular evolution. This article describes how PFE is used to visualize and analyze different aspects of biological redox chemistry, from long-range directional electron transfer to electron/hydride (NADPH) interconversion by a flavoenzyme and finally to NADPH recycling in a nanoconfined enzyme cascade.