Protein Film Voltammetry Research Articles

Electrochemical Studies of CO2 -Reducing Metalloenzymes.

Only two enzymes are capable of directly reducing CO2 : CO dehydrogenase, which produces CO at a [NiFe4 S4 ] 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.

The Nonphysiological Reductant Sodium Dithionite and [FeFe] Hydrogenase: Influence on the Enzyme Mechanism.

[FeFe] hydrogenases are highly active enzymes for interconverting protons and electrons with hydrogen (H2). Their active site H-cluster is formed of a canonical [4Fe-4S] cluster ([4Fe-4S]H) covalently attached to a unique [2Fe] subcluster ([2Fe]H), where both sites are redox active. Heterolytic splitting and formation of H2 takes place at [2Fe]H, while [4Fe-4S]H stores electrons. The detailed catalytic mechanism of these enzymes is under intense investigation, with two dominant models existing in the literature. In one model, an alternative form of the active oxidized state Hox, named HoxH, which forms at low pH in the presence of the nonphysiological reductant sodium dithionite (NaDT), is believed to play a crucial role. HoxH was previously suggested to have a protonated [4Fe-4S]H. Here, we show that HoxH forms by simple addition of sodium sulfite (Na2SO3, the dominant oxidation product of NaDT) at low pH. The low pH requirement indicates that sulfur dioxide (SO2) is the species involved. Spectroscopy supports binding at or near [4Fe-4S]H, causing its redox potential to increase by ∼60 mV. This potential shift detunes the redox potentials of the subclusters of the H-cluster, lowering activity, as shown in protein film electrochemistry (PFE). Together, these results indicate that HoxH and its one-electron reduced counterpart Hred′H are artifacts of using a nonphysiological reductant, and not crucial catalytic intermediates. We propose renaming these states as the “dithionite (DT) inhibited” states Hox-DTi and Hred-DTi. The broader potential implications of using a nonphysiological reductant in spectroscopic and mechanistic studies of enzymes are highlighted.

De Novo Design of a Self-Assembled Artificial Copper Peptide that Activates and Reduces Peroxide.

Copper-containing metalloenzymes constitute a major class of proteins which catalyze a myriad of reactions in nature. Inspired by the structural and functional characteristics of this unique class of metalloenzymes, we report the conception, design, characterization, and functional studies of a de novo artificial copper peptide (ArCuP) within a trimeric self-assembled polypeptide scaffold that activates and reduces peroxide. Using a first principles approach, the ArCuP was designed to coordinate one Cu via three His residues introduced at an a site of the peptide scaffold. X-ray crystallographic, UV-vis and EPR data demonstrate that Cu binds via the Nε atoms of His forming a T2Cu environment. When reacted with hydrogen peroxide, the putative copper-hydroperoxo species is formed where a reductive priming step accelerates the rate of its formation and reduction. Mass spectrometry was used to identify specific residues undergoing oxidative modification, which showed His oxidation only in the reduced state. The redox behavior of the ArCuP was elucidated by protein film voltammetry. Detailed characterization of the electrocatalytic behavior of the ArCuP led us to determine the catalytic parameters (KM, kcat), which established the peroxidase activity of the ArCuP. Combined spectroscopic and electrochemical data showed a pH-dependence on the reactivity, which was optimum at pH 7.5.

Evaluation of cell wall-associated direct extracellular electron transfer in thermophilic Geobacillus sp.

The online version contains supplementary material available at 10.1007/s13205-021-02917-2.

Selective cysteine-to-selenocysteine changes in a [NiFe]-hydrogenase confirm a special position for catalysis and oxygen tolerance

In [NiFe]-hydrogenases, the active-site Ni is coordinated by four cysteine-S ligands (Cys; C), two of which are bridging to the Fe(CO)(CN)2 fragment. Substitution of a single Cys residue by selenocysteine (Sec; U) occurs occasionally in nature. Using a recent method for site-specific Sec incorporation into proteins, each of the four Ni-coordinating cysteine residues in the oxygen-tolerant Escherichia coli [NiFe]-hydrogenase-1 (Hyd-1) has been replaced by U to identify its importance for enzyme function. Steady-state solution activity of each Sec-substituted enzyme (on a per-milligram basis) is lowered, although this may reflect the unquantified presence of recalcitrant inactive/immature/misfolded forms. Protein film electrochemistry, however, reveals detailed kinetic data that are independent of absolute activities. Like native Hyd-1, the variants have low apparent KMH2 values, do not produce H2 at pH 6, and display the same onset overpotential for H2 oxidation. Mechanistically important differences were identified for the C576U variant bearing the equivalent replacement found in native [NiFeSe]-hydrogenases, its extreme O2 tolerance (apparent KMH2 and Vmax [solution] values relative to native Hyd-1 of 0.13 and 0.04, respectively) implying the importance of a selenium atom in the position cis to the site where exogenous ligands (H-, H2, O2) bind. Observation of the same unusual electrocatalytic signature seen earlier for the proton transfer-defective E28Q variant highlights the direct role of the chalcogen atom (S/Se) at position 576 close to E28, with the caveat that Se is less effective than S in facilitating proton transfer away from the Ni during H2 oxidation by this enzyme.

Impact of membrane protein-lipid interactions on formation of bilayer lipid membranes on SAM-modified gold electrode

In protein film electrochemistry for redox-active membrane proteins, the reconstitution of protein-tethered bilayer lipid membranes (ptBLMs) can maximize their functionality and stability at electrolyte/electrode interfaces. To understand the impact of protein–lipid interactions on the ptBLM formation, we tracked the reconstitution process of a lipid bilayer in the presence or the absence of a transmembrane enzyme of cytochrome c-dependent nitric oxide reductase (cNOR) on a mixed self-assembled monolayer (SAM)/Au electrode. Time-resolved surface-enhanced infrared absorption (SEIRA) spectroscopy and electrochemical measurements revealed that the protein–lipid interaction affected the formation kinetics for the phospholipid bilayer and the electrocatalytic stability of the cNOR-modified electrode but not the fluidity of the phospholipid bilayer.

Nanocluster-assisted protein-film voltammetry for direct electrochemical signal acquisition.

Acquisition of the direct electrochemical response of protein is the cornerstone for the development of the third generation of electrochemical biosensors. In this work, we developed a nanocluster-assisted protein-film voltammetry technique (NCA-PFV) which can achieve the acquisition of the electrochemical signal and maintain the activity without affecting of the protein's structure. With this strategy, a lipid bilayer membrane is used to immobilize the membrane protein so as to maintain its natural state. Copper nanoclusters with a size smaller than most proteins are then used to function at sub-protein scale and to mediate the electron hopping from the electroactive center of the electrode. As a model, the direct electrochemical signal of cyclooxygenase (COX) is successfully obtained, with a pair of well-defined redox peaks located at -0.39mV and -0.31mV, which characterize the heme center of the enzyme. Its catalytic activity towards the substrate arachidonic acid (AA) is also retained. The detection range for AA is 10-1000μM and the detection limit is 2.4μM. Electrochemical monitoring of the regulation of the catalytic activity by an inhibitor DuP-697 is also achieved. This work provides a powerful tool for the fabrication of enzyme-based electrochemical biosensors, and is also of great significance for promoting the development and application of next-generation electrochemical biosensors.

Electrochemical Characterization of a Complex FeFe Hydrogenase, the Electron-Bifurcating Hnd From Desulfovibrio fructosovorans.

Hnd, an FeFe hydrogenase from Desulfovibrio fructosovorans, is a tetrameric enzyme that can perform flavin-based electron bifurcation. It couples the oxidation of H2 to both the exergonic reduction of NAD+ and the endergonic reduction of a ferredoxin. We previously showed that Hnd retains activity even when purified aerobically unlike other electron-bifurcating hydrogenases. In this study, we describe the purification of the enzyme under O2-free atmosphere and its biochemical and electrochemical characterization. Despite its complexity due to its multimeric composition, Hnd can catalytically and directly exchange electrons with an electrode. We characterized the catalytic and inhibition properties of this electron-bifurcating hydrogenase using protein film electrochemistry of Hnd by purifying Hnd aerobically or anaerobically, then comparing the electrochemical properties of the enzyme purified under the two conditions via protein film electrochemistry. Hydrogenases are usually inactivated under oxidizing conditions in the absence of dioxygen and can then be reactivated, to some extent, under reducing conditions. We demonstrate that the kinetics of this high potential inactivation/reactivation for Hnd show original properties: it depends on the enzyme purification conditions and varies with time, suggesting the coexistence and the interconversion of two forms of the enzyme. We also show that Hnd catalytic properties (Km for H2, diffusion and reaction at the active site of CO and O2) are comparable to those of standard hydrogenases (those which cannot catalyze electron bifurcation). These results suggest that the presence of the additional subunits, needed for electron bifurcation, changes neither the catalytic behavior at the active site, nor the gas diffusion kinetics but induces unusual rates of high potential inactivation/reactivation.

One isoform for one task? The second hydrogenase of Chlamydomonas reinhardtii prefers hydrogen uptake

Gaining knowledge about the algal hydrogen metabolism is prerequisite for the biotechnological exploitation of photosynthetic H2 production. Model organism Chlamydomonas reinhardtii encodes two [FeFe]-hydrogenases, which are individually capable of catalysing the reversible reduction of protons to molecular hydrogen. While physiological results indicated that HYDA1, connected to the photosynthetic electron transfer pathway via plant-type ferredoxin PETF, is accountable for a major part of algal H2 evolution, the role of HYDA2 in the algal metabolism is not understood yet. Herein, a comprehensive screening of enzymatic attributes was conducted, revealing that the two enzymes differ in their affinity to oxidised PETF. Notably, utilising protein film voltammetry, a higher catalytic preference of HYDA2 to consume H2 was observed. Site directed exchange mutagenesis revealed the contribution of a hydroxyl group in place of threonine 226, present in HYDA1, but not in HYDA2, potentially influencing the electronic properties of the active site, thereby fine-tuning catalytic function.

Electron flow between the worlds of Marcus and Warburg.

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.

Application of voltammetry in biomedicine - Recent achievements in enzymatic voltammetry

Protein-film voltammetry (PFV) is considered the simplest methodology to study the electrochemistry of lipophilic redox enzymes in an aqueous environment. By anchoring particular redox enzymes on the working electrode surface, it is possible to get an insight into the mechanism of enzyme action. The PFV methodology enables access to the relevant thermodynamic and kinetic parameters of the enzyme-electrode reaction and enzyme-substrate interactions, which is important to better understand many metabolic pathways in living systems and to delineate the physiological role of enzymes. PFV additionally provides important information which is useful for designing specific biosensors, simple medical devices and bio-fuel cells. In the current review, we focus on some recent achievements of PFV, while presenting some novel protocols that contribute to a better communication between redox enzymes and the working electrode. Insights to several new theoretical models that provide a simple strategy for studying electrode reactions of immobilized enzymes and that enable both kinetic and thermodynamic characterization of enzyme-substrate interactions are also provided. In addition, we give a short overview to several novel voltammetric techniques, derived from the perspective of square-wave voltammetry, which seem to be promising tools for application in PFV.

The roles of long-range proton-coupled electron transfer in the directionality and efficiency of [FeFe]-hydrogenases

As paradigms for proton-coupled electron transfer in enzymes and benchmarks for a fully renewable H2 technology, [FeFe]-hydrogenases behave as highly reversible electrocatalysts when immobilized on an electrode, operating in both catalytic directions with minimal overpotential requirement. Using the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1) we have conducted site-directed mutagenesis and protein film electrochemistry to determine how efficient catalysis depends on the long-range coupling of electron and proton transfer steps. Importantly, the electron and proton transfer pathways in [FeFe]-hydrogenases are well separated from each other in space. Variants with conservative substitutions (glutamate to aspartate) in either of two positions in the proton-transfer pathway retain significant activity and reveal the consequences of slowing down proton transfer for both catalytic directions over a wide range of pH and potential values. Proton reduction in the variants is impaired mainly by limiting the turnover rate, which drops sharply as the pH is raised, showing that proton capture from bulk solvent becomes critical. In contrast, hydrogen oxidation is affected in two ways: by limiting the turnover rate and by a large overpotential requirement that increases as the pH is raised, consistent with the accumulation of a reduced and protonated intermediate. A unique observation having fundamental significance is made under conditions where the variants still retain sufficient catalytic activity in both directions: An inflection appears as the catalytic current switches direction at the 2H+/H2 thermodynamic potential, clearly signaling a departure from electrocatalytic reversibility as electron and proton transfers begin to be decoupled.

Structural Properties and Catalytic Implications of the SPASM Domain Iron-Sulfur Clusters in Methylorubrum extorquens PqqE.

Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical S-adenosylmethionine enzyme in pyrroloquinoline quinone (PQQ) biosynthesis, contains three iron-sulfur cluster binding sites. Two auxiliary iron-sulfur cluster binding sites, designated as AuxI and AuxII, use distinctive ligands compared to other proteins in the family while their functions remain unclear. Here, we investigate the electronic properties of these iron-sulfur clusters and compare the catalytic efficiency of wild-type (WT) Methylorubrum extorquens AM1 PqqE to a range of mutated constructs. Using native mass spectrometry, protein film electrochemistry, and electron paramagnetic resonance spectroscopy, we confirm the previously proposed incorporation of a mixture of [2Fe-2S] and [4Fe-4S] clusters at the AuxI site and are able to assign redox potentials to each of the three iron-sulfur clusters. Significantly, a conservative mutation at AuxI, C268H, shown to selectively incorporate a [4Fe-4S] cluster, catalyzes an enhancement of uncoupled S-adenosylmethionine cleavage relative to WT, together with the elimination of detectable peptide cross-linked product. While a [4Fe-4S] cluster can be tolerated at the AuxI site, the aggregate findings suggest a functional [2Fe-2S] configuration within the AuxI site. PqqE variants with nondestructive ligand replacements at AuxII also show that the reduction potential at this site can be manipulated by changing the electronegativity of the unique aspartate ligand. A number of novel mechanistic features are proposed based on the kinetic and spectroscopic data. Additionally, bioinformatic analyses suggest that the unique ligand environment of PqqE may be relevant to its role in PQQ biosynthesis within an oxygen-dependent biosynthetic pathway.

Cytochrome P450 3A4 as a Drug Metabolizing Enzyme: the Role of Sensor System Modifications in Electocatalysis and Electroanalysis

The electroanalytical characteristics of the recombinant cytochrome P450 3A4 (CYP3A4) immobilized on the surface of screen-printed graphite electrodes modified by multi-walled carbon nanotubes (MWCNTs) have been investigated. The role and the influence of the modification of a graphite working electrode by carbon nanotubes on the electroanalytical parameters of this cytochrome have been demonstrated. The conditions for its immobilization on the obtained screen-printed graphite electrodes modified by MWCNTs have been optimized. The electrochemical parameters of the oxidation and reduction of the enzyme heme iron ion were determined: these included midpoint-potential (E0') of –0.35 ± 0.01 V (relative to the silver/silver chloride reference electrode, Ag/AgCl); the electrochemical rate constant of heterogeneous electron transfer (ks) of 0.57 ± 0.04 s–1, the amount of electroactive CYP3A4 on the modified electrode (Γ0) of (2.6 ± 0.6) × 10–10 mol/cm2. The functioning mechanism of this electrochemical sensor followed the principle of “protein film voltammetry” (i.e. voltammetry of a protein film on the electrode surface). In order to develop medical biosensors using immobilized CYP3A4 for electroanalysis of drug substances, the substrates of this hemoprotein, we performed a voltammetric study of the catalytic activity of immobilized CYP3A4. We investigated electrocatalytic properties of the recombinant CYP3A4 immobilized on modified screen-printed graphite electrode using macrolide antibiotic erythromycin as a substrate. The modification of electrodes has been shown to play a decisive role for the study of the properties of cytochromes P450 in electrochemical studies. Smart electrodes can serve as electroanalyzers for analytical purposes, as well as electrocatalysts for the study of biotransformation and metabolic processes. Electrodes modified with carbon nanomaterials are applicable for analytical purposes in the registration of hemoproteins. Electrodes modified with synthetic membrane-like compounds (for example, didodecyldimethylammonium bromide) are effective in enzyme-dependent electrocatalysis.

Understanding How the Rate of C-H Bond Cleavage Affects Formate Oxidation Catalysis by a Mo-Dependent Formate Dehydrogenase.

Metal-dependent formate dehydrogenases (FDHs) catalyze the reversible conversion of formate into CO2, a proton, and two electrons. Kinetic studies of FDHs provide key insights into their mechanism of catalysis, relevant as a guide for the development of efficient electrocatalysts for formate oxidation as well as for CO2 capture and utilization. Here, we identify and explain the kinetic isotope effect (KIE) observed for the oxidation of formate and deuterioformate by the Mo-containing FDH from Escherichia coli using three different techniques: steady-state solution kinetic assays, protein film electrochemistry (PFE), and pre-steady-state stopped-flow methods. For each technique, the Mo center of FDH is reoxidized at a different rate following formate oxidation, significantly affecting the observed kinetic behavior and providing three different viewpoints on the KIE. Steady-state turnover in solution, using an artificial electron acceptor, is kinetically limited by diffusional intermolecular electron transfer, masking the KIE. In contrast, interfacial electron transfer in PFE is fast, lifting the electron-transfer rate limitation and manifesting a KIE of 2.44. Pre-steady-state analyses using stopped-flow spectroscopy revealed a KIE of 3 that can be assigned to the C–H bond cleavage step during formate oxidation. We formalize our understanding of FDH catalysis by fitting all the data to a single kinetic model, recreating the condition-dependent shift in rate-limitation of FDH catalysis between active-site chemical catalysis and regenerative electron transfer. Furthermore, our model predicts the steady-state and time-dependent concentrations of catalytic intermediates, providing a valuable framework for the design of future mechanistic experiments.

Analysis of enzyme-substrate interactions from square-wave protein-film voltammetry of complex electrochemical-catalytic mechanism associated with reversible regenerative reaction

The surface catalytic EC′ mechanism is considered as one of the most relevant models to describe electrochemical behavior of many enzyme-substrate systems under voltammetric conditions. In this work, we elaborate theoretically a specific surface catalytic EC′ mechanism that features reversible regenerative reaction under conditions of square-wave voltammetry. The features of calculated voltammograms strongly depend on the parameters related to the thermodynamics and kinetics of regenerative chemical step involved in this mechanism. We present a series of simulated square-wave voltammograms that can be used to establish diagnostic criteria for recognizing surface EC′rev mechanism. In addition, we propose simple methodologies that can be explored to evaluate the kinetics and thermodynamics parameters relevant to chemical catalytic reaction. In a comprehensive study, we elaborate the effect of parameters of regenerative reaction to the features of phenomena known as “quasireversible maximum” and “split net peaks of square-wave voltammograms”. The results reported in this work can be of significance to many experimentalists working in the field of enzymatic voltammetry and drug-drug interactions.

Simple voltammetric approach for characterization of two-step surface electrode mechanism in protein-film voltammetry

Many enzymes embedding multivalent metal ions or quinone moieties as redox-active centres undergo electrochemical transformation via two successive electron transfer steps. If electrochemical features of such redox enzymes are analyzed with “protein-film voltammetry”, one frequently meets a challenging reaction scenario where the two electron transfers take place at the same formal potential. Under such conditions, one observes voltammogram with a single oxidation-reduction pattern hiding voltammetric features of both redox reactions. By exploring some aspects of the two-step surface EECrev mechanism one can develop simple methodology under conditions of square-wave voltammetry to enable recognizing and characterizing each electron transfer step. The method relies on the voltammetric features of the second electron transfer, which is coupled to a follow-up chemical reaction. The response of the second electron transfer step shifts to more positive potentials by increasing the rate of the chemical reaction. The proposed methodology can be experimentally applied by modifying the concentration of an electrochemically inactive substrate, which affects the rate of the follow-up chemical reaction. The final voltammetric output is represented by two well-separated square-wave voltammetric peaks that can be further exploited for complete thermodynamic and kinetic analysis of the EECrev mechanism.

Interaction of Isatin with Cytochrome P450 Isoenzymes: Investigation by Means of Spectral and Electrochemical Methods The role of Isatin in Cytochromes P450 Ligand-Protein Binding Events

In the present study, the effects of endogenous non-peptide bioregulator isatin (indole-2,3-dione) cytochrome P450 isoenzymes 3A5, 3A4, 2C19, 2E1, and 5A1 were investigated for the first time by means of electrochemical methods. Absorption spectroscopy and electrochemical methods have shown that isatin does not interact with heme as the active center of cytochrome P450 isoenzymes. Isatin has no significant effect on the electrocatalysis of cytochrome P450 3A4 (CYP3A4) in the presence of testosterone chosen as the substrate. Direct electrochemistry of thromboxane synthase (CYP5A1 or TBXAS1) has been investigated using protein film voltammetry. Reversible electrochemical signals of CYP5A1 were observed with midpoint potential of − 0.294 ± 0.012 V (vs Ag/AgCl). The results obtained indicate the existence of the binding sites for isatin on the protein surface of the investigated cytochromes P450, which differ from the active substrate-binding center. For CYP3A4 Arg375, Arg105, and Arg440; for CYP3A5 Arg375 and Arg105; and for CYP2C19 Arg433 were revealed as conserved “hot” amino acid residues involved in the binding of isatin while aligning CYP’s sequences.

Electroanalytical and electrocatalytical characteristics of cytochrome P450 3A4 using electrodes modified with nanocomposite carbon nanomaterials

The electroanalytical characteristics of recombinant cytochrome P450 3A4 (P450 3A4) immobilized on the surface of screen-printed graphite electrodes modified with multi-walled carbon nanotubes have been studied. The role and the influence of graphite working electrode modification with carbon nanotubes on electroanalytical characteristics of cytochrome P450 3A4 have been demonstrated. The conditions for the immobilization of cytochrome P450 3A4 on the obtained screen-printed graphite electrodes modified with carbon multi-walled nanotubes have been optimized. The electrochemical parameters of the oxidation and reduction of the heme iron of the enzyme have been estimated. The midpoint potential E0' was -0.35±0.01 V vs Ag/AgCl; the calculated heterogeneous electron transfer rate constant ks, was 0.57±0.04 s-1; the amount of electroactive cytochrome P450 3A4 on the electrode Г0, was determined as (2.6±0.6)⋅10-10 mol/cm2. The functioning mechanism of P450 3A4-based electrochemical sensor followed the "protein film voltammetry". In order to develop electrochemical analysis of drugs being substrates of that hemoprotein and respective medical biosensors the voltammetric study of catalytic activity of immobilized cytochrome P450 3A4 was carried out. Electrocatalytic properties of cytochrome P450 3A4, immobilized on modified screen-printed graphite electrodes, has been investigated using erythromycin (macrolide antibiotics). It has been shown that the modification of electrodes plays a decisive role for the study of the properties of cytochromes P450 in electrochemical investigations. Smart electrodes can serve as sensors for analytical purposes, as well as electrocatalysts for the study of biotransformation processes and metabolic processes. Electrodes modified with carbon nanomaterials are applicable for analytical purposes in the registration of hemoproteins. Electrodes modified with synthetic membrane-like compounds (e.g. didodecyldimethylammonium bromide) are effective in enzyme-dependent electrocatalysis.

Heme ligation and redox chemistry in two bacterial thiosulfate dehydrogenase (TsdA) enzymes

Thiosulfate dehydrogenases (TsdAs) are bidirectional bacterial di-heme enzymes that catalyze the interconversion of tetrathionate and thiosulfate at measurable rates in both directions. In contrast to our knowledge of TsdA activities, information on the redox properties in the absence of substrates is rather scant. To address this deficit, we combined magnetic CD (MCD) spectroscopy and protein film electrochemistry (PFE) in a study to resolve heme ligation and redox chemistry in two representative TsdAs. We examined the TsdAs from Campylobacter jejuni, a microaerobic human pathogen, and from the purple sulfur bacterium Allochromatium vinosum. In these organisms, the enzyme functions as a tetrathionate reductase and a thiosulfate oxidase, respectively. The active site Heme 1 in both enzymes has His/Cys ligation in the ferric and ferrous states and the midpoint potentials (Em) of the corresponding redox transformations are similar, −185 mV versus standard hydrogen electrode (SHE). However, fundamental differences are observed in the properties of the second, electron transferring, Heme 2. In C. jejuni, TsdA Heme 2 has His/Met ligation and an Em of +172 mV. In A. vinosum TsdA, Heme 2 reduction triggers a switch from His/Lys ligation (Em, −129 mV) to His/Met (Em, +266 mV), but the rates of interconversion are such that His/Lys ligation would be retained during turnover. In summary, our findings have unambiguously assigned Em values to defined axial ligand sets in TsdAs, specified the rates of Heme 2 ligand exchange in the A. vinosum enzyme, and provided information relevant to describing their catalytic mechanism(s).