Fraser A. Armstrong

This extended essay describes the development of protein film electrochemistry (PFE) as a powerful suite of techniques for investigating how electron-transfer processes occurring in biological molecules are coupled to chemical reactions. Based on the author’s personal experiences, the article explores the wider picture through connections to related work and other scientific areas, as well as hypotheses and predictions. Extending far beyond its electrochemical origins and early expectations, PFE has reached a wide audience – across disciplines as diverse as electrochemistry, renewable energy, biological coordination chemistry, molecular catalysis, biocatalysis, molecular and cell biology, biotechnology and pharmacology. Much is owed to many researchers, from undergraduates to senior figures, with whom the author has worked alongside and been inspired by for more than 40 years.

Protein film electrochemistry has helped to unravel many complex reactivities of electron-transferring proteins and enzymes. A versatile descendant, the ‘Electrochemical Leaf’, offers new opportunities to extend electrochemical control to myriad enzymes that neither transfer electrons nor catalyse any redox reaction, including those dependent on spectroscopically limited, labile or other challenging metal ions. By embedding a cascade comprised of several enzymes—one of which electrochemically recycles NAD(P)(H), a second being a dehydrogenase—within a porous electrode formed from fused nanoparticles, the interconnected reactions are tightly channeled to transmit energy and information, rapidly and interactively. Under nanoconfinement, nicotinamide cofactors and cascade intermediates serve as specific current carriers, far beyond the electron itself. Graphical 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.

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.

An emerging concept and platform, the electrochemical Leaf (e-Leaf), offers a radical change in the way tandem (multi-step) catalysis by enzyme cascades is studied and exploited. The various enzymes are loaded into an electronically conducting porous material composed of metallic oxide nanoparticles, where they achieve high concentration and crowding – in the latter respect the environment resembles that found in living cells. By exploiting efficient electron tunneling between the nanoparticles and one of the enzymes, the e-Leaf enables the user to interact directly with complex networks, rendering simultaneous the abilities to energise, control and observe catalysis. Because dispersion of intermediates is physically suppressed, the output of the cascade – the rate of flow of chemical steps and information – is delivered in real time as electrical current. Myriad enzymes of all major classes now become effectively electroactive in a technology that offers scalability between micro-(analytical, multiplex) and macro-(synthesis) levels. This Perspective describes how the e-Leaf was discovered, the steps in its development so far, and the outlook for future research and applications.

Hydrogenases catalyze hydrogen/proton interconversion that is normally electrochemically reversible (having minimal overpotential requirement), a special property otherwise almost exclusive to platinum metals. The mechanism of [NiFe]-hydrogenases includes a long-range proton-coupled electron-transfer process involving a specific Ni-coordinated cysteine and the carboxylate of a nearby glutamate. A variant in which this cysteine has been exchanged for selenocysteine displays two distinct changes in electrocatalytic properties, as determined by protein film voltammetry. First, proton reduction, even in the presence of H₂ (a strong product inhibitor), is greatly enhanced relative to H₂ oxidation: this result parallels a characteristic of natural [NiFeSe]-hydrogenases which are superior H₂ production catalysts. Second, an inflection (an S-shaped "twist" in the trace) appears around the formal potential, the small overpotentials introduced in each direction (oxidation and reduction) signaling a departure from electrocatalytic reversibility. Concerted proton-electron transfer offers a lower energy pathway compared to stepwise transfers. Given the much lower proton affinity of Se compared to that of S, the inflection provides compelling evidence that concerted proton-electron transfer is important in determining why [NiFe]-hydrogenases are reversible electrocatalysts.

[FeFe]-hydrogenases are efficient H₂ converting biocatalysts that are inhibited by formaldehyde (HCHO). The molecular mechanism of this inhibition has so far not been experimentally solved. Here, we obtained high-resolution crystal structures of the HCHO-treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum, showing HCHO reacts with the secondary amine base of the catalytic cofactor and the cysteine C299 of the proton transfer pathway which both are very important for catalytic turnover. Kinetic assays via protein film electrochemistry show the CpI variant C299D is significantly less inhibited by HCHO, corroborating the structural results. By combining our data from protein crystallography, site-directed mutagenesis and protein film electrochemistry, a reaction mechanism involving the cofactor's amine base, the thiol group of C299 and HCHO can be deduced. In addition to the specific case of [FeFe]-hydrogenases, our study provides additional insights into the reactions between HCHO and protein molecules.

The [4Fe-3S] cluster of an O₂-tolerant [NiFe] hydrogenase was transformed into different [4Fe-4S] clusters, and structural, spectroscopic and electrochemical analyses of the enzyme variants revealed an O₂-tolerance mechanism at various levels.

The ability to control enzyme cascades entrapped in a nanoporous electrode material (the “Electrochemical Leaf”, e‐Leaf) has been exploited to gain detailed kinetic insight into the mechanism of an anti‐cancer drug. Ivosidenib, used to treat acute myeloid leukemia, acts on a common cancer‐linked variant of isocitrate dehydrogenase 1 (IDH1 R132H) inhibiting its “gain‐of‐function” activity—the undesired reduction of 2‐oxoglutarate (2OG) to the oncometabolite 2‐hydroxyglutarate (2HG). The e‐Leaf quantifies the kinetics of IDH1 R132H inhibition across a wide and continuous range of conditions, efficiently revealing factors underlying the inhibitor residence time. Selective inhibition of IDH1 R132H by Ivosidenib and another inhibitor, Novartis 224, is readily resolved as a two‐stage process whereby initial rapid non‐inhibitory binding is followed by a slower step to give the inhibitory complex. These kinetic features are likely present in other allosteric inhibitors of IDH1/2. Such details, essential for understanding inhibition mechanisms, are not readily resolved in conventional steady‐state kinetics or by techniques that rely only on measuring binding. Extending the new method and analytical framework presented here to other enzyme systems will be straightforward and should rapidly reveal insight that is difficult or often impossible to obtain using other methods.

The ability to control enzyme cascades entrapped in a nanoporous electrode material (the “Electrochemical Leaf”, e‐Leaf) has been exploited to gain detailed kinetic insight into the mechanism of an anti‐cancer drug. Ivosidenib, used to treat acute myeloid leukemia, acts on a common cancer‐linked variant of isocitrate dehydrogenase 1 (IDH1 R132H) inhibiting its “gain‐of‐function” activity—the undesired reduction of 2‐oxoglutarate (2OG) to the oncometabolite 2‐hydroxyglutarate (2HG). The e‐Leaf quantifies the kinetics of IDH1 R132H inhibition across a wide and continuous range of conditions, efficiently revealing factors underlying the inhibitor residence time. Selective inhibition of IDH1 R132H by Ivosidenib and another inhibitor, Novartis 224, is readily resolved as a two‐stage process whereby initial rapid non‐inhibitory binding is followed by a slower step to give the inhibitory complex. These kinetic features are likely present in other allosteric inhibitors of IDH1/2. Such details, essential for understanding inhibition mechanisms, are not readily resolved in conventional steady‐state kinetics or by techniques that rely only on measuring binding. Extending the new method and analytical framework presented here to other enzyme systems will be straightforward and should rapidly reveal insight that is difficult or often impossible to obtain using other methods.