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Redox-active metalloproteins including metalloenzymes play important roles in denitrification, photosynthesis and respiration. To understand their enzymatic reaction mechanisms and the effects of reaction environments on protein properties, protein film electrochemistry (PFE) coupled with surface-enhanced infrared absorption (SEIRA) spectroscopy is a powerful technique. Herein, we present and discuss PFE-SEIRA spectroscopy results on the determination of reduction potential, formation processes of bilayer lipid membranes on the electrode surface, and effects of protein-protein or protein-lipid interactions on the enzymatic activities for transmembrane metalloenzymes of cytochrome c-dependent nitric oxidase and cytochrome c oxidase. [ABSTRACT FROM AUTHOR]

The electrolysis of dilute CO2 streams suffers from low concentrations of dissolved substrate and its rapid depletion at the electrolyte‐electrocatalyst interface. These limitations require first energy‐intensive CO2 capture and concentration, before electrolyzers can achieve acceptable performances. For direct electrocatalytic CO2 reduction from low‐concentration sources, we introduce a strategy that mimics the carboxysome in cyanobacteria by utilizing microcompartments with nanoconfined enzymes in a porous electrode. A carbonic anhydrase accelerates CO2 hydration kinetics and minimizes substrate depletion by making all dissolved carbon available for utilization, while a highly efficient formate dehydrogenase reduces CO2 cleanly to formate; down to even atmospheric concentrations of CO2. This bio‐inspired concept demonstrates that the carboxysome provides a viable blueprint for the reduction of low‐concentration CO2 streams to chemicals by using all forms of dissolved carbon. [ABSTRACT FROM AUTHOR]

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.

Only two enzymes are capable of directly reducing CO2: CO dehydrogenase, which produces CO at a [NiFe4S4] active site, and formate dehydrogenase, which produces formate at a mononuclear W or Mo active site. Both metalloenzymes are very rapid, energy‐efficient and specific in terms of product. They have been connected to electrodes with two different objectives. A series of studies used protein film electrochemistry to learn about different aspects of the mechanism of these enzymes (reactivity with substrates, inhibitors...). Another series focused on taking advantage of the catalytic performance of these enzymes to build biotechnological devices, from CO2‐reducing electrodes to full photochemical devices performing artificial photosynthesis. Here, we review all these works. [ABSTRACT FROM AUTHOR]

The formate dehydrogenase enzyme from Cupriavidus necator (FdsABG) carries out the two-electron oxidation of formate to CO2, but is also capable of reducing CO2 back to formate, a potential biofuel. FdsABG is a heterotrimeric enzyme that performs this transformation using nine redox-active cofactors: a bis(molybdopterin guanine dinucleotide) (bis-MGD) at the active site coupled to seven iron–sulfur clusters, and one equivalent of flavin mononucleotide (FMN). To better understand the pathway of electron flow in FdsABG, the reduction potentials of the various cofactors were examined through direct electrochemistry. Given the redundancy of cofactors, a truncated form of the FdsA subunit was developed that possesses only the bis-MGD active site and a singular [4Fe–4S] cluster. Electrochemical characterization of FdsABG compared to truncated FdsA shows that the measured reduction potentials are remarkably similar despite the truncation with two observable features at − 265 mV and − 455 mV vs SHE, indicating that the voltammetry of the truncated enzyme is representative of the reduction potentials of the intact heterotrimer. By producing truncated FdsA without the necessary maturation factors required for bis-MGD insertion, a form of the truncated FdsA that possesses only the [4Fe–4S] was produced, which gives a single voltammetric feature at − 525 mV, allowing the contributions of the molybdenum cofactor to be associated with the observed feature at − 265 mV. This method allowed for the deconvolution of reduction potentials for an enzyme with highly complex cofactor content to know more about the thermodynamic landscape of catalysis. [ABSTRACT FROM AUTHOR]

The applicability of the extremely active [FeFe] hydrogenase from Desulfovibrio desulfuricans in H2-producing devices is studied. Despite being the most active enzyme for H2 catalysis, its high sensitivity towards O2 has prevented its use in electrolytic water splitting cells. Using electrochemical methods, the catalytic activity of the enzyme at H2-producing potentials and its inactivation upon exposure to limited amounts of O2 or under illumination is analysed. This enzyme is shown to maintain H2 production activity for extended periods of time at low potentials. At such potentials, the enzyme can deliver the required electrons to fully reduce O2 to H2O, minimising the damage of the enzyme. Additionally, the robustness of this enzyme under illumination at negative applied potentials is demonstrated. These results show that the [FeFe] hydrogenase from D. desulfuricans is an excellent candidate to be used in devices to store solar energy in hydrogen. [ABSTRACT FROM AUTHOR]

The electrolysis of dilute CO 2 streams suffers from low concentrations of dissolved substrate and its rapid depletion at the electrolyte-electrocatalyst interface. These limitations require first energy-intensive CO 2 capture and concentration, before electrolyzers can achieve acceptable performances. For direct electrocatalytic CO 2 reduction from low-concentration sources, we introduce a strategy that mimics the carboxysome in cyanobacteria by utilizing microcompartments with nanoconfined enzymes in a porous electrode. A carbonic anhydrase accelerates CO 2 hydration kinetics and minimizes substrate depletion by making all dissolved carbon available for utilization, while a highly efficient formate dehydrogenase reduces CO 2 cleanly to formate; down to even atmospheric concentrations of CO 2 . This bio-inspired concept demonstrates that the carboxysome provides a viable blueprint for the reduction of low-concentration CO 2 streams to chemicals by using all forms of dissolved carbon., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Hydrogenase-1 (Hyd-1) from Escherichia coli is a membrane-bound enzyme that catalyses the reversible oxidation of molecular H2. The active site contains one Fe and one Ni atom and several conserved amino acids including an arginine (Arg509), which interacts with two conserved aspartate residues (Asp118 and Asp574) forming an outer shell canopy over the metals. There is also a highly conserved glutamate (Glu28) positioned on the opposite side of the active site to the canopy. The mechanism of hydrogen activation has been dissected by site-directed mutagenesis to identify the catalytic base responsible for splitting molecular hydrogen and possible proton transfer pathways to/from the active site. Previous reported attempts to mutate residues in the canopy were unsuccessful, leading to an assumption of a purely structural role. Recent discoveries, however, suggest a catalytic requirement, for example replacing the arginine with lysine (R509K) leaves the structure virtually unchanged, but catalytic activity falls by more than 100-fold. Variants containing amino acid substitutions at either or both, aspartates retain significant activity. We now propose a new mechanism: heterolytic H2 cleavage is via a mechanism akin to that of a frustrated Lewis pair (FLP), where H2 is polarized by simultaneous binding to the metal(s) (the acid) and a nitrogen from Arg509 (the base). [ABSTRACT FROM AUTHOR]

Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH. [ABSTRACT FROM AUTHOR]

A long-standing contradiction in the field of mononuclear Mo enzyme research is that small-molecule chemistry on active-site mimic compounds predicts ligand participation in the electron transfer reactions, but biochemical measurements only suggest metal-centered catalytic electron transfer.With the simultaneous measurement of substrate turnover and reversible electron transfer that is provided by Fourier-transformed alternating-current voltammetry, we show that Escherichia coli YedY is a mononuclear Mo enzyme that reconciles this conflict. In YedY, addition of three protons and three electrons to the well-characterized "as-isolated" Mo(V) oxidation state is needed to initiate the catalytic reduction of either dimethyl sulfoxide or trimethylamine N-oxide. Based on comparison with earlier studies and our UV-vis redox titration data, we assign the reversible one-proton and one-electron reduction process centered around +174 mV vs. standard hydrogen electrode at pH 7 to a Mo(V)-to-Mo(IV) conversion but ascribe the two-proton and two-electron transition occurring at negative potential to the organic pyranopterin ligand system. We predict that a dihydro-to-tetrahydro transition is needed to generate the catalytically active state of the enzyme. This is a previously unidentified mechanism, suggested by the structural simplicity of YedY, a protein in which Mo is the only metal site. [ABSTRACT FROM AUTHOR]

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydrogenase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe-3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygentolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observedwith oxygen-tolerant [NiFe]-hydrogenases. It is possible thatHis229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function. [ABSTRACT FROM AUTHOR]

Apoptosis is triggered by an accumulation of ROS (reactive oxygen species) produced by proteins of the mitochondrial respiratory chain. The levels of ROS are controlled by the activities of mitochondrial redox proteins such as glutaredoxin 2 that help to modulate the susceptibility of a cell to apoptosis. However, once downstream events have resulted in the release of cytochrome c to the cytosol, it is widely considered that cell death is inevitable. Cytochrome c may promote its own release from mitochondria through interactions with the mitochondrial phospholipid cardiolipin (diphosphatidylglycerol). In the present article, spectroelectrochemistry of the cardiolipin complex of cytochrome c and protein film electrochemistry of glutaredoxin 2 are reviewed to illustrate how electrochemical methods provide insight into the properties of signalling proteins. [ABSTRACT FROM AUTHOR]

Abstract: Dynamic electrochemistry of adsorbed proteins, often termed protein film electrochemistry (PFE), is widely used for the characterisation of redox proteins. The method provides a powerful alternative to spectroscopic studies that interrogate protein solutions. The reduction potential and electron stoichiometry of redox couples can be defined. The rates of catalytic redox transformations can also be quantified. Often it is considered that the behaviour of the adsorbed protein should match that displayed in solution studies if it is to be relevant to understanding the biological role of the protein. However, the environment of the protein in PFE is fundamentally different from that when it is freely diffusing in solution. As a consequence different behaviours may be expected. This raises the question, which approach is more relevant when aiming to provide insight into the cellular role of the protein? We consider this question here taking as an example the properties of a penta-heme cytochrome NrfB from Escherichia coli. The redox properties of NrfB containing solutions were presented previously (Clarke et al., Journal of Biological Chemistry (2004)). Here we present PFE of NrfB adsorbed on graphite and optically transparent mesoporous, nanocrystalline SnO2 electrodes. [Copyright &y& Elsevier]

Abstract: Indium tin oxide (ITO) is a promising material for developing spectroelectrochemical methods due to its combination of excellent transparency in the visible region and high conductivity over a broad range of potential. However, relatively few examples of immobilization of redox proteins at ITO with retention of the ability to transfer electrons with the underlying material with native characteristics have been reported. In this work, we utilize an alkylsilane functionalized ITO surface as a biocompatible interface for immobilization of the blue copper protein azurin. Adsorption of azurin at ITO as well as ITO coated with self-assembled monolayers of (3-mercaptopropyl)trimethoxysilane (MPTMS) and n-decyltrimethoxysilane (DTMS) was achieved, and immobilized protein probed using protein film electrochemistry. The native redox properties of the protein were perturbed by adsorption directly to ITO or to the MPTMS layer on an ITO surface. However, azurin adsorbed at a DTMS covered ITO surface retained native electrochemical properties (E 1/2 =122±5mV vs. Ag/AgCl) and could exchange electrons directly with the underlying ITO layer without need for an intervening chemical mediator. These results open new opportunities for immobilizing functional redox proteins at ITO and developing spectroelectrochemical methods for investigating them. [Copyright &y& Elsevier]

Abstract: Salmonella enterica serovar Typhimurium is a Gram negative bacterial pathogen and a common cause of food-borne illness. Molecular hydrogen has been shown to be a key respiratory electron donor during infection and H2 oxidation can be catalysed by three genetically-distinct [NiFe] hydrogenases. Of these, hydrogenases-1 (Hyd-1) and Hyd-2 have well-characterised homologues in Escherichia coli. The third, designated Hyd-5 here, is peculiar to Salmonella and is expressed under aerobic conditions. In this work, Salmonella was genetically modified to enable the isolation and characterisation of Hyd-5. Electrochemical analysis established that Hyd-5 is a H2-oxidising enzyme that functions in very low levels of H2 and sustains this activity in high levels of O2. In addition, electron paramagnetic resonance spectroscopy of the Hyd-5 isoenzyme reveals a complex paramagnetic FeS signal at high potentials which is comparable to that observed for other O2-tolerant respiratory [NiFe] hydrogenases. Taken altogether, Hyd-5 can be classified as an O2-tolerant hydrogenase that confers upon Salmonella the ability to use H2 as an electron donor in aerobic respiration. [Copyright &y& Elsevier]

Green algae such as Chiamydomonas reinhardtii synthesize an [FeFe] hydrogenase that is highly active in hydrogen evolution. However, the extreme sensitivity of [FeFe] hydrogenases to oxygen presents a major challenge for exploiting these organisms to achieve sustainable photosynthetic hydrogen production. In this study, the mechanism of oxygen inactivation of the [FeFe] hydrogenase CrHydAl from C. reinhardtii has been investigated. X-ray absorption spectroscopy shows that reaction with oxygen results in destruction of the [4Fe-4SJ domain of the active site H-cluster while leaving the di-iron domain (2FeH) essentially intact. By protein film electrochemistry we were able to determine the order of events leading up to this destruction. Carbon monoxide, a competitive inhibitor of CrHydAl which binds to an Fe atom of the 2FeH domain and is otherwise not known to attack FeS clusters in proteins, reacts nearly two orders of magnitude faster than oxygen and protects the enzyme against oxygen damage. These results therefore show that destruction of the [4Fe-4S] cluster is initiated by binding and reduction of oxygen at the di-iron domain-a key step that is blocked by carbon monoxide. The relatively slow attack by oxygen compared to carbon monoxide suggests that a very high level of discrimination can be achieved by subtle factors such as electronic effects (specific orbital overlap requirements) and steric constraints at the active site. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Electron paramagnetic resonance spectroscopy encompasses a versatile set of techniques that allow detailed insight into intrinsically occurring paramagnetic centers in metalloproteins and enzymes that undergo oxidation-reduction reactions. In this chapter, we discuss the process from isolating the protein to acquiring and analyzing pulse EPR spectra, adopting a practical perspective. We start with considerations when preparing the protein sample, explain techniques and procedures available for determining the reduction potential of the redox-active center of interest and provide details on methodologies to trap a given paramagnetic state for detailed pulse EPR studies, with an emphasis on biochemical and spectroscopic tools available when multiple EPR-active species are present. We elaborate on some of the most commonly used pulse EPR techniques and the choices the user has to make, considering advantages and disadvantages and how to avoid pitfalls. Examples are provided throughout., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Only two enzymes are capable of directly reducing CO 2 : CO dehydrogenase, which produces CO at a [NiFe 4 S 4 ] active site, and formate dehydrogenase, which produces formate at a mononuclear W or Mo active site. Both metalloenzymes are very rapid, energy-efficient and specific in terms of product. They have been connected to electrodes with two different objectives. A series of studies used protein film electrochemistry to learn about different aspects of the mechanism of these enzymes (reactivity with substrates, inhibitors…). Another series focused on taking advantage of the catalytic performance of these enzymes to build biotechnological devices, from CO 2 -reducing electrodes to full photochemical devices performing artificial photosynthesis. Here, we review all these works., (© 2021 Wiley-VCH GmbH.)

While protein film electrochemistry (PFE) has proven to be an effective tool in the interrogation of redox cofactors and assessing the electrocatalytic activity of many different enzymes, recently it has been proven to be useful for the study of the redox potentials of the cofactors of AdoMet radical enzymes (AREs). In this chapter, we review the challenges and opportunities of examining the redox cofactors of AREs in a high level of detail, particularly for the deconvolution of redox potentials of multiple cofactors. We comment on how to best assess the electroactive nature of any given ARE, and we see that when applied well, PFE allows for not only determining redox potentials, but also determining proton-coupling and ligand-binding phenomena in the ARE superfamily., (© 2018 Elsevier Inc. All rights reserved.)

A suite of dynamic electrochemical techniques known as protein film electrochemistry (PFE) offers important insight into the roles of active sites in enzymes, including properties of electron-transfer centers (individually or collectively), rates and dependences of catalytic electron transport, and binding and dissociation of inhibitors. In this chapter, we explain how PFE is used to investigate the properties of FeS clusters-centers lacking distinctive or convenient spectroscopic signatures that are often very sensitive to O 2 . We see that PFE allows simultaneous detection and control of the reactions of individual FeS clusters, and measurement of their relaying efficiency in long-range electron transfer., (© 2018 Elsevier Inc. All rights reserved.)

The applicability of the extremely active [FeFe] hydrogenase from Desulfovibrio desulfuricans in H 2 -producing devices is studied. Despite being the most active enzyme for H 2 catalysis, its high sensitivity towards O 2 has prevented its use in electrolytic water splitting cells. Using electrochemical methods, the catalytic activity of the enzyme at H 2 -producing potentials and its inactivation upon exposure to limited amounts of O 2 or under illumination is analysed. This enzyme is shown to maintain H 2 production activity for extended periods of time at low potentials. At such potentials, the enzyme can deliver the required electrons to fully reduce O 2 to H 2 O, minimising the damage of the enzyme. Additionally, the robustness of this enzyme under illumination at negative applied potentials is demonstrated. These results show that the [FeFe] hydrogenase from D. desulfuricans is an excellent candidate to be used in devices to store solar energy in hydrogen., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Carbon monoxide dehydrogenases (CODHs) catalyse the reversible conversion between CO and CO2 . Several small molecules or ions are inhibitors and probes for different oxidation states of the unusual [Ni-4 Fe-4 S] cluster that forms the active site. The actions of these small probes on two enzymes-CODH ICh and CODH IICh -produced by Carboxydothermus hydrogenoformans have been studied by protein film voltammetry to compare their behaviour and to establish general characteristics. Whereas CODH ICh is, so far, the better studied of the two isozymes in terms of its electrocatalytic properties, it is CODH IICh that has been characterised by X-ray crystallography. The two isozymes, which share 58.3% sequence identity and 73.9% sequence similarity, show similar patterns of behaviour with regard to selective inhibition of CO2 reduction by CO (product) and cyanate, potent and selective inhibition of CO oxidation by cyanide, and the action of sulfide, which promotes oxidative inactivation of the enzyme. For both isozymes, rates of binding of substrate analogues CN(-) (for CO) and NCO(-) (for CO2 ) are orders of magnitude lower than turnover, a feature that is clearly revealed through hysteresis of cyclic voltammetry. Inhibition by CN(-) and CO is much stronger for CODH IICh than for CODH ICh, a property that has relevance for applying these enzymes as model catalysts in solar-driven CO2 reduction., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)