Protein Film Voltammetry Research Articles

Nanoimpact Electrochemistry for Unraveling the Reactivity of Diffusion Limited Enzymes

The ultimate challenge of modern electroanalytical chemistry is to reach a single-molecule level of detection. In this framework, nanoimpact electrochemistry (NIE) has been recently developed. It allows studying at high throughput the reactivity of small entities such as nanoparticles, cells, or proteins, that collide on a biased ultramicroelectrode (UME) inside a solution or suspension.1 The collisions cause disturbances in the recorded chronoamperogram such as current spikes or current steps.2 Single enzyme electrochemistry (SEE) can contribute to uncovering effects of dynamics and non-equilibrium behavior of individual enzyme molecules on catalysis, which are hidden in measurements on ensembles due to averaging of the measured parameters over the entire molecular population. However, NIE of smaller entities such as enzymes, is very challenging due to the low sensitivity of the state-of-the-art electrochemical equipment, and most single enzyme techniques are currently based on fluorescence.3 One strategy to overcome this issue is the amplification of the current produced by a single enzyme molecule by fast catalysis: the single enzyme converts several substrate molecules per second consuming several electrons leading measurable current. Some of us reported the implementation of this strategy for the first time, designing a single-entity electrochemical experiment that allowed the detection of catalytic currents from single laccase molecules.4 Soon after the initial SEE report in 2016, a similar approach was applied for investigations of catalase,5 an enzyme that breaks down H2O2 to O2 and H2O with diffusion limited kinetics. However, the unequivocal attribution of current disturbances to the reduction of O2 (produced locally by enzyme) on the electrode during enzyme-electrode collisions remains a challenge. We will address this challenge based on the analysis of SEE experimental data under various conditions, on protein film voltammetry (PFV) studies on rotating disk electrode (RDE), and on simulations. The collected data from the SEE, that is the current spike characteristics in each set of conditions, are statistically analyzed and discussed. PFV studies are used to evaluate basic thermodynamic and kinetic parameters of the system. Finally, we have built a model using Comsol Multiphysics. To the best of our knowledge, this is the first model reported so far that can satisfactorily reproduce the experimental SEE data, using experimentally evaluated parameters as input. The developed methodology is the succesfully implemented to the study of superoxide dismutase.6

Exploring electron transfer: Bioinspired, biomimetics, and bioelectrochemical systems for sustainable energy and Value-Added compound synthesis

Electron transfer (ET) is a fundamental process that underlies various phenomena in physics, chemistry, and biology. Understanding ET mechanisms is crucial for developing sustainable energy solutions and synthesizing value-added compounds efficiently. In this context, the present review provides the fundamental aspects of ET involving bioinspired, biomimetics, and biological entities and its significance for sustainable energy and green electrosynthesis fields. Among the theoretical and experimental cornerstones, Marcus Theory, electronic conductance, computational modeling, biomolecular thermodynamics, electrochemical and kinetic theories, protein film voltammetry, and the emergence of in situ and operando techniques are explored. Theoretical modeling is vital for understanding and predicting ET processes. Additionally, the significance of experimental techniques for investigating the ET process in biological entities and interfaces is discussed. Protein film voltammetry is a valuable and consolidated technique for studying ET processes at the protein-electrode interface, whereas in situ and operando techniques for interrogating ET processes in real time provide insights into the dynamics and mechanisms of ET. The concept of quantum conductance in biological structures is addressed, evidencing a trend and power of single-entity analysis. Aspects of extracellular and interfacial ET processes are presented and discussed in the electrochemical energy conversion systems. A deep understanding of these processes can improve the design of efficient bioinspired catalysts. Therefore, this multidisciplinary work aims to fill the gaps between different scientific fields related to ET involving bioentities to develop innovative energy and value-added compound synthesis solutions.

Replacing a Cysteine Ligand by Selenocysteine in a [NiFe]-Hydrogenase Unlocks Hydrogen Production Activity and Addresses the Role of Concerted Proton-Coupled Electron Transfer in Electrocatalytic Reversibility.

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.

Combining a Commercial Mixer with a Wall-Tube Electrode Allows the Arbitrary Control of Concentrations in Protein Film Electrochemistry.

Protein film electrochemistry is a technique in which an enzyme is immobilized on an electrode in a configuration that allows following the changes in turnover frequency as a response to changes in the experimental conditions. Insights into the reactivity of the enzyme can be obtained by quantitatively modeling such responses. As a consequence, the more the technique allows flexibility in changing conditions, the more useful it becomes. The most commonly used setup, based on the rotating disc electrode, allows easy stepwise increases in the concentration of nongaseous substrates, or exposure to constant concentration of dissolved gas, but does not permit to easily decrease the concentration of nongaseous substrates, or to change the concentration of dissolved gas in a stepwise fashion. To overcome the limitation by mass transport of the substrate toward the electrode when working with fast enzymes, we have designed another kind of electrochemical cell based on the wall-tube electrode (WTE). We demonstrate here that by using a system combining two syringe pumps, a commercial mixer, and the WTE, it is possible to change the concentration of species in a stepwise fashion in all directions, opening new possibilities to study redox enzymes. As a proof of concept, this device was applied to the study of the electrochemical response of the cytochrome c nitrite reductase of Desulfovibrio desulfuricans.

Electrochemical and structural characterization of recombinant respiratory proteins of the acidophilic iron oxidizer Ferrovum sp. PN-J47-F6 suggests adaptations to the acidic pH at protein level.

The tendency of the periplasmic redox proteins in acidophiles to have more positive redox potentials (Em) than their homologous counterparts in neutrophiles suggests an adaptation to acidic pH at protein level, since thermodynamics of electron transfer processes are also affected by acidic pH. Since this conclusion is mainly based on the electrochemical characterization of redox proteins from extreme acidophiles of the genus Acidithiobacillus, we aimed to characterize three recombinant redox proteins of the more moderate acidophile Ferrovum sp. PN-J47-F6. We applied protein film voltammetry and linear sweep voltammetry coupled to UV/Vis spectroscopy to characterize the redox behavior of HiPIP-41, CytC-18, and CytC-78, respectively. The Em-values of HiPIP-41 (571 ± 16 mV), CytC-18 (276 ± 8 mV, 416 ± 2 mV), and CytC-78 (308 ± 7 mV, 399 ± 7 mV) were indeed more positive than those of homologous redox proteins in neutrophiles. Moreover, our findings suggest that the adaptation of redox proteins with respect to their Em occurs more gradually in response to the pH, since there are also differences between moderate and more extreme acidophiles. In order to address structure function correlations in these redox proteins with respect to structural features affecting the Em, we conducted a comparative structural analysis of the Ferrovum-derived redox proteins and homologs of Acidithiobacillus spp. and neutrophilic proteobacteria. Hydrophobic contacts in the redox cofactor binding pockets resulting in a low solvent accessibility appear to be the major factor contributing to the more positive Em-values in acidophile-derived redox proteins. While additional cysteines in HiPIPs of acidophiles might increase the effective shielding of the [4Fe-4S]-cofactor, the tight shielding of the heme centers in acidophile-derived cytochromes is achieved by a drastic increase in hydrophobic contacts (A.f. Cyc41), and by a larger fraction of aromatic residues in the binding pockets (CytC-18, CytC-78).

The net peak splitting phenomenon in square-wave voltammetry – A simple diagnostic tool to distinguish between surface electrode mechanisms associated with different chemical reactions

Utilizing pulse voltammetric techniques for the electrochemical analysis of lipophilic redox systems has proven to be an effective method for studying a diverse range of redox compounds, from simple molecules to intricate proteins. To extract relevant thermodynamic and kinetic data from electrochemical analysis of redox systems exhibiting strong surface activity, there's a crucial need to decipher the underlying electrochemical mechanism in the system being examined. The "split net peak" phenomenon, a defining characteristic observed in all surface-active redox systems featuring fast electron transfer under square-wave voltammetry conditions, has been investigated in this study to establish diagnostic criteria for identifying surface electrochemical mechanisms associated with preceding, subsequent, regenerative, and reactant-inactivating chemical reactions. This understanding can be achieved by tracking the influence of the chemical reaction rate on the split square-wave voltammetric peaks in a particular electrochemical mechanism. The approach reported in the current work enables a very simple and precise differentiation between the elaborated mechanisms frequently encountered in protein-film voltammetry methodologies.

Combining Electrochemistry and Protein Engineering to Elucidate Outer-Sphere Effects in Hydrogenase Catalysis

Hydrogenases, the bacterial enzymes that oxidize and produce H2, come in two flavors, “NiFe hydrogenases” and “FeFe hydrogenases”, depending on the structure of their active site.In each of these two families of enzymes, the active site and its immediate environment are fully conserved, and yet the investigations of these enzymes using direct electrochemistry have shown that their catalytic properties vary greatly: this is true regarding catalytic bias and reversibility1, resistance to oxygen, and other properties which define whether or not a particular hydrogenase could be used under the operating conditions of a particular device.These functional variations within each family also tell us that structural features that are remote from the active site, the so-called “outer-sphere” effects, define, at least in part, the catalytic properties.2 In the FeFe hydrogenase from Clostridium beijerinckii, we have shown that oxygen resistance is related to a conformational change that depends on non-conserved residues that are up to 18 Å away from the active site. This conformational change occurs in two steps that can be resolved by combining site-directed mutagenesis and detailed protein film voltammetry experiments.3 These remote residues define an emerging family of O2-resistant FeFe hydrogenases. (a) A. Fasano, H. Land, V. Fourmond, G. Berggren, and C. Léger, « Reversible or irreversible catalysis of H+/H2 conversion by FeFe hydrogenases », J. Am. Chem. Soc. 143, 48, 20320-20325 (2021) (b) V. Fourmond, N. Plumeré, C. Léger, « Reversible catalysis », Nature Reviews Chemistry, 5, 348-360 (2021)S. Stripp, B. Duffus, V. Fourmond, C. Léger, S. Leimkühler, S. Hirota, Y. Hu, A. Jasniewski, H. Ogata, M. Ribbe, « Second and Outer Coordination Sphere Effects in Low Valent Metalloenzymes », Chemical Reviews, 122 11900 (2022) (a) M. Winkler, J. Duan, A. Rutz, C. Felbek, L. Scholtysek, O. Lampret, J. Jaenecke, U.-P.r Apfel, G. Gilardi, F. Valetti, V. Fourmond, E. Hofmann, C. Léger, T. Happe « A safety cap protects hydrogenase from oxygen attack » Nature Communications 12, 756 (2021) (b) A. Rutz, C. Das, A. Fasano, J. Jaenecke, S. Yadav, U.P. Apfel, V. Engelbrecht, V. Fourmond, C. Léger, L. Schäfer, T. Happe « Increasing the O2 resistance of the [FeFe]-hydrogenase CbA5H through enhanced protein flexibility », ACS Catalysis 13, 2, 856–865 (2023) Figure 1

Insights into the Molecular Mechanism of Formaldehyde Inhibition of [FeFe]-Hydrogenases.

[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.

A Dynamic Water Channel Affects O2 Stability in [FeFe]-Hydrogenases.

[FeFe]-hydrogenases are capable of reducing protons at a high rate. However, molecular oxygen (O2 ) induces the degradation of their catalytic cofactor, the H-cluster, which consists of a cubane [4Fe4S] subcluster (4FeH ) and a unique diiron moiety (2FeH ). Previous attempts to prevent O2 -induced damage have focused on enhancing the protein's sieving effect for O2 by blocking the hydrophobic gas channels that connect the protein surface and the 2FeH . In this study, we aimed to block an O2 diffusion pathway and shield 4FeH instead. Molecular dynamics (MD) simulations identified a novel water channel (WH ) surrounding the H-cluster. As this hydrophilic path may be accessible for O2 molecules we applied site-directed mutagenesis targeting amino acids along WH in proximity to 4FeH to block O2 diffusion. Protein film electrochemistry experiments demonstrate increased O2 stabilities for variants G302S and S357T, and MD simulations based on high-resolution crystal structures confirmed an enhanced local sieving effect for O2 in the environment of the 4FeH in both cases. The results strongly suggest that, in wild type proteins, O2 diffuses from the 4FeH to the 2FeH . These results reveal new strategies for improving the O2 stability of [FeFe]-hydrogenases by focusing on the O2 diffusion network near the active site.

Improving the Efficiency of Electrocatalysis of Cytochrome P450 3A4 by Modifying the Electrode with Membrane Protein Streptolysin O for Studying the Metabolic Transformations of Drugs.

In the present work, screen-printed electrodes (SPE) modified with a synthetic surfactant, didodecyldimethylammonium bromide (DDAB) and streptolysin O (SLO) were prepared for cytochrome P450 3A4 (CYP3A4) immobilization, direct non-catalytic and catalytic electrochemistry. The immobilized CYP3A4 demonstrated a pair of redox peaks with a formal potential of -0.325 ± 0.024 V (vs. the Ag/AgCl reference electrode). The electron transfer process showed a surface-controlled mechanism ("protein film voltammetry") with an electron transfer rate constant (ks) of 0.203 ± 0.038 s-1. Electrochemical CYP3A4-mediated reaction of N-demethylation of erythromycin was explored with the following parameters: an applied potential of -0.5 V and a duration time of 20 min. The system with DDAB/SLO as the electrode modifier showed conversion of erythromycin with an efficiency higher than the electrode modified with DDAB only. Confining CYP3A4 inside the protein frame of SLO accelerated the enzymatic reaction. The increases in product formation in the reaction of the electrochemical N-demethylation of erythromycin for SPE/DDAB/CYP3A4 and SPE/DDAB/SLO/CYP3A4 were equal to 100 ± 22% and 297 ± 7%, respectively. As revealed by AFM images, the SPE/DDAB/SLO possessed a more developed surface with protein cavities in comparison with SPE/DDAB for the effective immobilization of the CYP3A4 enzyme.

Recovering biological electron transfer reaction parameters from multiple protein film voltammetric techniques informed by Bayesian inference

Deciphering the mechanism, kinetics and energetics of biological electron-transfer reactions requires a robust, rapid and reproducible protein-film voltammetry information recovery process. Here we describe a semi-automated computational approach for inferring the chemical reaction parameters for a simple protein system, a bacterial cytochrome domain from Cellvibrio japonicus that displays reversible one-electron Fe2+/3+ redox chemistry. Despite the relative simplicity of the experimental system, developing a robust data analysis approach to find the global optimum in 13-dimensional parameter space is a challenging task because the Faradaic-to-background current ratio in such experiments is often low. We describe how a multiple-technique approach, whereby data from three voltammetry techniques (direct-current, pure sinusoidal and Fourier transform alternating current voltammetry) is combined, ultimately enables the automatic extraction of both (i) quantitative “best-fit” redox reaction parameter point values that are robust across multiple experiments performed on different protein-electrode films, and (ii) a statistical description of parameter correlation relationships, along with uncertainty in the individual parameter values, obtained using Bayesian inference. It is the latter achievement which is particularly important as it represents a method for visualising the possible limitations in the mathematical model of the experimental system. Our multi-voltammetry analysis approach enables such powerful insight because of the complementarity between the information content, simulation-speed and parameter sensitivity of the current–time data generated by the different techniques, illustrating the value of adding purely sinusoidal voltammetry to the bioelectrochemistry measurement toolkit.

Transport limited adsorption experiments give a new lower estimate of the turnover frequency of Escherichia coli hydrogenase 1

Protein Film Electrochemistry is a technique in which a redox enzyme is directly wired to an electrode, which substitutes for the natural redox partner. In this technique, the electrical current flowing through the electrode is proportional to the catalytic activity of the enzyme. However, in most cases, the amount of enzyme molecules contributing to the current is unknown and the absolute turnover frequency cannot be determined. Here, we observe the formation of electrocatalytically active films of E. coli hydrogenase 1 by rotating an electrode in a sub-nanomolar solution of enzyme. This process is slow, and we show that it is mass-transport limited. Measuring the rate of the immobilization allows the determination of an estimation of the turnover rate of the enzyme, which appears to be much greater than that deduced from solution assays under the same conditions.

Comprehensive structural, infrared spectroscopic and kinetic investigations of the roles of the active-site arginine in bidirectional hydrogen activation by the [NiFe]-hydrogenase 'Hyd-2' from Escherichia coli.

The active site of [NiFe]-hydrogenases contains a strictly-conserved pendant arginine, the guanidine head group of which is suspended immediately above the Ni and Fe atoms. Replacement of this arginine (R479) in hydrogenase-2 from E. coli results in an enzyme that is isolated with a very tightly-bound diatomic ligand attached end-on to the Ni and stabilised by hydrogen bonding to the Nζ atom of the pendant lysine and one of the three additional water molecules located in the active site of the variant. The diatomic ligand is bound under oxidising conditions and is removed only after a prolonged period of reduction with H2 and reduced methyl viologen. Once freed of the diatomic ligand, the R479K variant catalyses both H2 oxidation and evolution but with greatly decreased rates compared to the native enzyme. Key kinetic characteristics are revealed by protein film electrochemistry: most importantly, a very low activation energy for H2 oxidation that is not linked to an increased H/D isotope effect. Native electrocatalytic reversibility is retained. The results show that the sluggish kinetics observed for the lysine variant arise most obviously because the advantage of a more favourable low-energy pathway is massively offset by an extremely unfavourable activation entropy. Extensive efforts to establish the identity of the diatomic ligand, the tight binding of which is an unexpected further consequence of replacing the pendant arginine, prove inconclusive.

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

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.

Distinction between film loss and enzyme inactivation in protein-film voltammetry: a theoretical study in cyclic staircase voltammetry

Inactivation of redox enzymes and the loss of enzyme’s material from its adsorbed film on electrode surface are events that have similar outputs when studied under conditions of protein-film voltammetry. In the current work, we present a series of novel theoretical results under conditions of protein-film cyclic staircase voltammetry that clearly reveal how to distinguish between these two events. Theoretical model elaborated in this work comprises a surface electrode mechanism in which the initial form of redox enzyme is involved in same time in an electrode transformation and in an irreversible reaction of deactivation. While a large set of theoretical voltammetric curves simulated at different conditions are discussed, we also give a tabular overview of some characteristic cyclic voltammetric patterns of this electrode mechanism. The simulated voltammograms can help the experimentalists to distinguish the phenomenon of loss of protein film from the electrode mechanism associated with enzyme deactivation. In addition, we give hints to apply suitable methodologies to get access to the kinetics of both steps involved in this electrochemical mechanism.Graphical abstract

Effects of Interfacial Interactions on Electrocatalytic Activity of Cytochrome c Oxidase in Biomimetic Lipid Membranes on Gold Electrodes.

Effects of interfacial interactions on the electrocatalytic activity of protein-tethered bilayer lipid membranes (ptBLMs) containing cytochrome c oxidase (CcO) for the oxygen reduction reaction are studied by using protein film electrochemistry and surface-enhanced infrared absorption (SEIRA) spectroscopy. Mammalian CcO was immobilized on a gold electrode via self-assembled monolayers (SAMs) of mixed alkanethiols. The protein orientation on the electrode is controlled by SAM-CcO interactions and is critical to the cytochrome c (cyt c) binding. The CcO-phospholipid and CcO-cyt c interactions modulate the electrocatalytic activity of CcO, and more densely packed ptBLMs show higher electrocatalytic activity. Our study indicates that spectroscopic and electrochemical studies of ptBLMs can provide insights into the effects of relatively weak protein-protein and protein-lipid interactions on the enzymatic activity of transmembrane enzymes.

Characterization of an Unusual Metalloenzyme Containing an [FeFe] Hydrogenase and a Rubrerythrin Domain

[FeFe] hydrogenases are enzymes that catalyze the reversible reduction of protons to dihydrogen with staggering efficiency. As dihydrogen is a promising alternative energy carrier for renewable energy systems, these enzymatic systems are of interest to biotechnological applications. Members of the [FeFe] hydrogenase class are very diverse in the primary structure, especially in the metallocofactor composition, and their physiological roles. Understanding the structure‐function relationships in these enzymes provides an opportunity to engineer enzymes with uniquely modified characteristics. In this work, we aim to understand the interplay between structural elements and the phenotype of an unusual metalloprotein from Clostridium perfringens (CpHydR) containing two catalytic domains, [FeFe] hydrogenase and rubrerythrin, “fused” together into one protein. Although [FeFe] hydrogenases are typically biased toward H2 production, the postulated presence of rubrerythrin makes us hypothesize that CpHydR is biased toward H2 oxidation. Rubrerythrin is known to reduce H2O2 as a part of an oxidative stress response process; it requires the donation of two electrons and two protons, which can be supplied by the [FeFe] hydrogenase. Hydrogenases are notoriously oxygen‐sensitive enzymes inhibiting their industrial application. The postulated use of this metalloprotein in the oxidative stress response in Clostridium perfringens would suggest tolerance to O2. Here we present experiments confirming the presence of each subsystem and the postulated functionality. We utilize Fourier Transformed Infrared (FTIR) and Electron Paramagnetic Resonance (EPR) spectroscopies to elucidate the structure of the active cofactors. To understand the function of CpHydR, we employ electrochemical methods, including protein film voltammetry. Results indicate an intriguing deviation of the spectroscopic signatures of the [FeFe] hydrogenase subdomain from the commonly observed phenotypes of virtually any other known enzyme of this class. This divergence in the structural characteristics also coincides with an unexpected strong bias of this enzyme towards hydrogen oxidation.

Insights into the Thermodynamics and Kinetics of Amino-Acid Radicals in Proteins.

Some oxidoreductase enzymes use redox-active tyrosine, tryptophan, cysteine, and/or glycine residues as one-electron, high-potential redox (radical) cofactors. Amino-acid radical cofactors typically perform one of four tasks-they work in concert with a metallocofactor to carry out a multielectron redox process, serve as storage sites for oxidizing equivalents, activate the substrate molecules, or move oxidizing equivalents over long distances. It is challenging to experimentally resolve the thermodynamic and kinetic redox properties of a single-amino-acid residue. The inherently reactive and highly oxidizing properties of amino-acid radicals increase the experimental barriers further still. This review describes a family of stable and well-structured model proteins that was made specifically to study tyrosine and tryptophan oxidation-reduction. The so-called α3X model protein system was combined with very-high-potential protein film voltammetry, transient absorption spectroscopy, and theoretical methods to gain a comprehensive description of the thermodynamic and kinetic properties of protein tyrosine and tryptophan radicals.

Elucidating Film Loss and the Role of Hydrogen Bonding of Adsorbed Redox Enzymes by Electrochemical Quartz Crystal Microbalance Analysis.

The immobilization of redox enzymes on electrodes enables the efficient and selective electrocatalysis of useful reactions such as the reversible interconversion of dihydrogen (H2) to protons (H+) and formate to carbon dioxide (CO2) with hydrogenase (H2ase) and formate dehydrogenase (FDH), respectively. However, their immobilization on electrodes to produce electroactive protein films for direct electron transfer (DET) at the protein–electrode interface is not well understood, and the reasons for their activity loss remain vague, limiting their performance often to hour timescales. Here, we report the immobilization of [NiFeSe]-H2ase and [W]-FDH from Desulfovibrio vulgaris Hildenborough on a range of charged and neutral self-assembled monolayer (SAM)-modified gold electrodes with varying hydrogen bond (H-bond) donor capabilities. The key factors dominating the activity and stability of the immobilized enzymes are determined using protein film voltammetry (PFV), chronoamperometry (CA), and electrochemical quartz crystal microbalance (E-QCM) analysis. Electrostatic and H-bonding interactions are resolved, with electrostatic interactions responsible for enzyme orientation while enzyme desorption is strongly limited when H-bonding is present at the enzyme–electrode interface. Conversely, enzyme stability is drastically reduced in the absence of H-bonding, and desorptive enzyme loss is confirmed as the main reason for activity decay by E-QCM during CA. This study provides insights into the possible reasons for the reduced activity of immobilized redox enzymes and the role of film loss, particularly H-bonding, in stabilizing bioelectrode performance, promoting avenues for future improvements in bioelectrocatalysis.

Optimizing the mass transport of wall-tube electrodes for protein film electrochemistry

Protein Film Electrochemistry (PFE) is a technique in which an enzyme is directly wired to an electrode and its catalytic turnover rate is measured under the form of an electrical current. This technique has proved useful for the study of a number of enzymes, but requires fast transport of the enzymatic substrate towards the electrode. In a previous work (Fadel et al, Phys. Chem. Chem. Phys., 2019, 21, 12360), we have proposed a new design based on the wall-tube electrode that provides better transport than the rotating disc electrode, which is usually employed for PFE studies. In the present work, we use computational fluid dynamics to explore the effects of the various parameters of the cell, and propose a semi-empirical formula suitable to predict the mass-transport coefficient and the wall shear stress on the electrode. We use a 3D-printed cell to experimentally validate our predictions.