Tungsten-containing Formate Dehydrogenase Research Articles

Enzymatic conversion of CO2 to formate: The potential of tungsten-containing formate dehydrogenase in flow reactor system

This study introduces a novel approach for CO2 reduction to formate using the recombinant formate dehydrogenase 1 (MeFDH1) from Methylorubrum extorquens AM1 as biocatalyst, addressing challenges in activity, productivity, and long-term stability of enzyme. We demonstrate that immobilized MeFDH1 supported by electrochemical reaction system enhances formate production and stability, achieving over 1.7 M concentration with an initial rate of 20 mM/h and near-unity Faradaic efficiency for over 200 hours. In further, the reusability of immobilized MeFDH1 was obtained without significant declination of productivity and selectivity. The electrochemical study of MeFDH1 found the product inhibition in continuous CO2 conversion. To overcome this challenge and build efficient process, the integration of a flow reactor system and in-situ separation unit further improved the system's performance and scalability. This advancement in enzymatic CO2 conversion suggests the potential of biocatalysis towards addressing global warming through sustainable chemical synthesis.

Multiple electron transfer pathways of tungsten-containing formate dehydrogenase in direct electron transfer-type bioelectrocatalysis.

Tungsten-containing formate dehydrogenase from Methylorubrum extroquens AM1 (FoDH1)-a promising biocatalyst for the interconversion of carbon dioxide/formate and nicotine adenine dinucleotide (NAD+)/NADH redox couples-was investigated using structural biology and bioelectrochemistry. FoDH1 is reported to be an enzyme that can realize "direct electron transfer (DET)-type bioelectrocatalysis." However, its 3-D structure, electrode-active sites, and electron transfer (ET) pathways remain unclear. The ET pathways were investigated using structural information, electrostatic interactions between the electrode and the enzyme, and the differences in the substrates. Two electrode-active sites and multiple ET pathways in FoDH1 were discovered.

Construction of a Bioelectrochemical Dihydrogen/Formate Interconversion System and a Bio-Solar Cell

Modern societies have energy and environmental issues such as fossil fuel depletion and excessive carbon dioxide (CO2) emissions. Therefore, an effective use of renewable energy resources instead of fossil fuels is essential for the development of a sustainable energy economy. Particularly, dihydrogen (H2), formic acid (HCOOH) and sunlight are expected to be new energy sources. These advantages are as follows; H2 emits no harmful substances in use; HCOOH has a high energy density and is a liquid fuel that can be easily stored and transported; sunlight is universally available on the ground. However, the electrochemical conversion of the above energy has the following problems; a catalyst for H2 oxidation, platinum, is expensive; byproducts such as carbon monoxide are generated in CO2 reduction at usual electrodes; solar cell materials are sometimes expensive and environmentally harmful. In order to overcome these problems, we focused on biocatalysts, which are ecofriendly, and have high catalytic performance and selectivity under mild conditions. Furthermore, we constructed two electro-enzymatic energy conversion devices: an H2/HCOO- interconversion system and a bio-solar cell.In an H2/HCOO- interconversion system, we applied two enzymes which catalyze bidirectional redox reactions: [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F (H2ase; EC 1.12.2.1) and tungsten-containing formate dehydrogenase from Methylobacterium extorquens AM1 (FoDH1; EC 1.2.1.2). H2ase and FoDH1 catalyze the H+/H2 and CO2/HCOO- interconversion, respectively, and we attempted to generate HCOO- without external power supply by the coupling of two bioelectrocatalytic reactions: direct electron transfer (DET)-type H2 oxidation catalyzed by H2ase and mediated electron transfer (MET)-type CO2 reduction catalyzed by FoDH1 using benzyl viologen (BV) as a mediator (H2 + CO2 → HCOO- + H+). H2ase was adsorbed on a bioanode functionalized with p-phenylenediamine, which leads to orientations suitable for DET-type bioelectrocatalysis of H2ase, while FoDH1 and BV were adsorbed on a biocathode. In addition, gas-diffusion-type electrodes were employed for high-speed supply of gaseous substrates. The two electrodes were short-circuited in a cell, and CO2 and H2 gasses were spontaneously supplied from the outside of the biocathode and the bioanode, respectively. The amount of formate in the electrolyte solution increased almost linearly with the reaction time. On the other hand, when the feed gasses were replaced with Ar gas to remove CO2 and H2 from the system, the amount of formate decreased with the time. This is due to the reversed reaction: (HCOO- + H+ → H2 + CO2). These results mean that the difference in equilibrium potentials of the H+/H2 and CO2/HCOO- couples determines the driving force of the reaction, which is reversed depending on conditions, and the interconversion between H2 and HCOO- was experimentally demonstrated.A bio-solar cell is an electric energy device mimicking natural photosynthesis and extracts excited electrons outside the photosynthetic system. In this study, we constructed a bio-solar cell using the thylakoid membrane from spinach as an anodic photo-bioelectrocatalyst which oxidizes water (H2O) to dioxygen (O2) and the recombinant bilirubin oxidase (BOD; EC 1.3.3.5) produced by expressing the gene of BOD from Myrothecium verucarria in Pichia pastoris as a cathodic bioelectrocatalyst which reduces O2 to H2O. In addition, we investigated the characteristics of a thylakoid membrane-functionalized bioanode with a mediator of hexaammineruthenium(III/II) ion ([Ru(NH3)6]3+/2+), which has the following characteristics: fast electrode kinetics, high stability and solubility, and somewhat low redox potential. The photo-driven bioanode prepared by applying the mixture of thylakoid membranes and water-dispersed multi-walled carbon nanotubes on a gold-spattered indium tin oxide (ITO) electrode showed the photocurrent caused by MET-type photo-bioelectrocatalytic H2O oxidation in a buffer (pH 7.0) containing 5 mM Ru(NH3)6Cl3 under the light condition. The performance of the bioanode (0.2 mA cm- 2 at 0 V vs. Ag|AgCl|sat. KCl) is better than that in the previous report using 1,2-naphthoquinone as a mediator. Then, a bio-solar cell was constructed by combining the bioanode with the BOD-modified gas-diffusion-type biocathode, and O2 was spontaneously supplied from the outside of the biocathode. The cell exhibited an open-circuit voltage of 0.61 V and a maximum power density of 50 μW cm- 2 at a cell voltage of 0.42 V under quiescent conditions. To the best of our knowledge, the power density of our cell unit is the highest among those reported for the bio-solar cells to date.These works lead to the artificial photosynthetic system, which generates H2 or HCOO- from sunlight and H2O (and CO2), and they might be a breakthrough for the sustainable energy economy.

Construction of a bioelectrochemical formate generating system from carbon dioxide and dihydrogen

We constructed a bioelectrochemical formate generating system without external power supply. CO2 was reduced with tungsten-containing formate dehydrogenase adsorbed on a biocathode on which benzyl viologen was adsorbed as a mediator, while the electron for the CO2 reduction was supplied by the oxidation of H2 with [NiFe]‑hydrogenase adsorbed on a bioanode functionalized with p-phenylenediamine (PDA2+) to adsorb H2ase in orientations suitable for direct communication with the electrode. Gas-diffusion-system was employed for high-speed supply of both of the gaseous substrates. The two electrodes were short-circuited through an ammeter and a switch. In this system, the formate generation rate of the biocathode was 290 ± 20 pmol cm−2 s−1 at pH 6.5 and 25 °C. On the other hand, the formate oxidation by H+ as the reversed reaction was also confirmed in the cell.

Assembly of direct-electron-transfer-type bioelectrodes with high performance

Direct electron transfer bioelectrocatalysis is an essential type of reaction for the development of bioelectrochemical devices such as biosensors, biofuel cells, and bioreactors. In this work, we performed several modifications of mesoporous electrodes to improve the heterogeneous electron transfer kinetics and the orientation of three different enzymes: bilirubin oxidase from Myrothecium verrucaria, hydrogenase from Desulfovibrio vulgaris Miyazaki F, and tungsten-containing formate dehydrogenase from Methylobacterium extorquens AM1. The results are discussed based on the curvature effects of mesoporous structures, the edge effect of the diffuse double layer around microporous structures, and the electrostatic interactions between enzymes and electrodes.

Direct electron transfer-type four-way bioelectrocatalysis of CO2/formate and NAD+/NADH redox couples by tungsten-containing formate dehydrogenase adsorbed on gold nanoparticle-embedded mesoporous carbon electrodes modified with 4-mercaptopyridine

Tungsten-containing formate dehydrogenase from Methylobacterium extorquens AM1 (FoDH1) catalyzes formate oxidation with NAD+. FoDH1 shows little direct communication with carbon electrodes, including mesoporous Ketjen Black-modified glassy carbon electrode (KB/GCE); however, it shows well-defined direct electron transfer (DET)-type bioelectrocatalysis of carbon dioxide reduction, formate oxidation, NAD+ reduction, and NADH oxidation on gold nanoparticle (AuNP)-embedded KB/GCE treated with 4-mercaptopyridine. Microscopic measurements reveal that the AuNPs (d=5nm) embedded on the KB surface are uniformly dispersed. Electrochemical data indicate that the pyridine moiety on the AuNPs plays important roles in facilitating the interfacial electron transfer kinetics and increasing the probability of productive orientation of FoDH1. The formal potential of the electrochemical communication site, which is most probably an ion‑sulfur cluster, is evaluated as −0.591±0.005V vs. Ag|AgCl|sat. KCl from Nernst analysis of the steady-state catalytic waves.

Efficient reduction of CO2 by the molybdenum-containing formate dehydrogenase from Cupriavidus necator (Ralstonia eutropha)

The ability of the FdsABG formate dehydrogenase from Cupriavidus necator (formerly known as Ralstonia eutropha) to catalyze the reverse of the physiological reaction, the reduction of CO2 to formate utilizing NADH as electron donor, has been investigated. Contrary to previous studies of this enzyme, we demonstrate that it is in fact effective in catalyzing the reverse reaction with a kcat of 11 ± 0.4 s-1 We also quantify the stoichiometric accumulation of formic acid as the product of the reaction and demonstrate that the observed kinetic parameters for catalysis in the forward and reverse reactions are thermodynamically consistent, complying with the expected Haldane relationships. Finally, we demonstrate the reaction conditions necessary for gauging the ability of a given formate dehydrogenase or other CO2-utilizing enzyme to catalyze the reverse direction to avoid false negative results. In conjunction with our earlier studies on the reaction mechanism of this enzyme and on the basis of the present work, we conclude that all molybdenum- and tungsten-containing formate dehydrogenases and related enzymes likely operate via a simple hydride transfer mechanism and are effective in catalyzing the reversible interconversion of CO2 and formate under the appropriate experimental conditions.

High-Power Formate/Dioxygen Biofuel Cell Based on Mediated Electron Transfer Type Bioelectrocatalysis

A high-power mediated electron transfer type formate (HCOO–)/dioxygen (O2) biofuel cell is reported herein. The cell utilizes a Ketjen Black modified waterproof carbon cloth as the electrode material. The bioanode comprises tungsten-containing formate dehydrogenase and a viologen-functionalized polymer, whereas the biocathode comprises bilirubin oxidase and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate). In addition, a gas diffusion type system was employed for the biocathode to realize a high-speed O2 supply. These electrodes exhibited a large current density of 20 mA cm–2 in the quiescent steady state for both HCOO– oxidation and O2 reduction. Finally, these electrodes were coupled to construct an HCOO–/O2 biofuel cell without a separator. The cell exhibited a maximum power density of 12 mW cm–2 at a cell voltage of 0.78 V under quiescent conditions and an open-circuit voltage of 1.2 V. We show the great potential of HCOO– for the fuel of biofuel cells.

High Performance Bioelectrocatalytic Interconversion Between Formate and Carbon Dioxide

An interconversion of HCOO-/CO2 is one of essential requirements for carbon-based sustainable energy economy. However, when CO2 is reduced and HCOO- is oxidized directly on electrodes, carbon monoxide is generated and quite high overpotential is required. One of the most promising strategies for solving these issues is the utilization of enzymes as catalysts. Enzymes allow the system to function in a specific biological reaction under mild conditions. The electro-enzymatic devices can be used as energy conversion system such as HCOO-/O2 biofuel cells and an efficient bioelectrochemical system of the CO2 reduction. We focused on the catalytic properties of tungsten-containing formate dehydrogenase (FoDH1; EC 1.2.1.2) from Methylobacterium extorquens AM1 to construct a bioelectrochemical interconversion system of HCOO-/CO2. FoDH1 is a heterodimeric soluble enzyme and catalyzes the oxidation of HCOO- to CO2 in coupled reduction of NAD+ to NADH. In this study, we have found that FoDH1 catalyzes both of the HCOO- oxidation and the HCO3 - reduction with several artificial redox partners (mediators). The bi-molecular reaction rate constants between FoDH1 and mediators and show a linear free energy relationship. The reversible reaction between HCOO- and CO2 through FoDH1 has been realized on cyclic voltammetry by using methyl viologen (MV) as a mediator and by adjusting pH from the thermodynamic viewpoint. The steady-state voltammograms with two-way bioelectrocatalytic properties are interpreted on a simple model by considering the solution equilibrium. Furthermore, we have constructed a light driven HCOO- production system using a spinach thylakoid membrane, MV, and FoDH1. When the interconversion between HCOO- and CO2 is applied to the construction of efficient bioelectrochemical devices, a large current density should be realized at potentials close to the formal potential of the HCOO-/CO2 couple (E°'CO2). We show a great possibility of MET-type bioelectrocatalysis in the HCOO- oxidation and the CO2 reduction at high current densities and low overpotentials. For the HCOO- oxidation, a high limiting current density (j lim) of about 24 mA cm- 2 was realized with a half wave potential (E 1/2) of only 0.12 V more positive than E°'CO2 at 30 °C in the presence of MV2+ as a mediator. When a viologen-functionalized polymer was co-immobilized with FoDH1 on the porous electrode, j lim of about 30 mA cm- 2 was attained at 60 °C with E 1/2 = E°'CO2 + 0.13 V. On the other hand, the CO2 reduction was also realized at j lim of about 15 mA cm- 2 with E 1/2 = E°'CO2 – 0.04 V at pH 6.6 and at 60 °C in the presence of MV. This is the first report of the enzyme-based bioelectrocatalytic CO2 reduction at such low overpotential and a high j lim. The present results are very useful to construct an effective bioelectrochemical reaction for the CO2 reduction and HCOO-/O2 biofuel cells as effective energy conversion systems.

Direct electron transfer-type bioelectrocatalytic interconversion of carbon dioxide/formate and NAD+/NADH redox couples with tungsten-containing formate dehydrogenase

Tungsten-containing formate dehydrogenase (FoDH1) with a molecular mass of 170kDa from Methylobacterium extorquens AM1 catalyzes the oxidation of formate (HCOO−) to carbon dioxide (CO2) with NAD+ as a natural electron acceptor in solution. FoDH1 does not produce any direct electron transfer (DET)-type bioelectrocatalytic wave at planar electrodes, but can adsorb on and communicate with mesoporous carbon electrodes. The curvature effect of mesoporous structures seems to increase the number of enzymes with orientations suitable for electrochemical communication. However, adsorption proceeds slowly on Ketjen Black-modified electrode and the catalytic current density remains low. Most probably, the size of the mesopores is too small to effectively trap FoDH1. The adsorbed FoDH1 catalyzes DET-type bioelectrocatalytic interconversion of the CO2/HCOO− and NAD+/NADH redox couples. Most probably, one of the iron–sulfur clusters located near the enzyme surface communicates with mesoporous electrodes. When the communication proceeds effectively, FoDH1 behaves as a novel bidirectional catalyst for the substrates, since FoDH1 can realize fast uphill intramolecular electron transfer. The non-covalently bound flavin mononucleotide (FMN) cofactor in FoDH1 is dissociated from some FoDH1 molecules and adsorbs on the mesoporous electrode to give a symmetrical surface-confined redox wave. Although adsorbed FMN cannot participate in mediated electron transfer (MET)-type bioelectrocatalysis, dissociated FMN in solution works as a mediator for MET-type bioelectrocatalysis of the HCOO− oxidation at planar electrodes.

Efficient bioelectrocatalytic CO2 reduction on gas-diffusion-type biocathode with tungsten-containing formate dehydrogenase

A new gas-diffusion-type biocathode was constructed for carbon dioxide (CO2) reduction. In this work, tungsten-containing formate dehydrogenase (FoDH1), which is a promising enzyme for interconversion of formate and CO2, was used as a catalyst and was absorbed on a Ketjen Black (KB)-modified electrode. We used 1,1′-trimethylene-2,2′-bipyridinium dibromide as a mediator, and the hydrophobicity of the FoDH1-absorbed electrode was optimized according to the weight ratio of the polytetrafluoroethylene binder to KB. We achieved cathodic current densities of about 20mAcm−2 under mild and quiescent conditions (neutral pH, atmospheric pressure, and room temperature).

Bioelectrocatalytic Interconversion Between Formate and Carbon Dioxide By Tungsten-Containing Formate Dehydrogenase

The reduction of carbon dioxide (CO2) to generate reduced carbon compounds for use as fuels and chemical feedstocks is an essential requirement for carbon-based sustainable energy economy. An interconversion system of formate (HCOO-)/CO2 is one of the answers for the purpose. HCOO- is the first stable intermediate during the reduction of CO2 to methanol or methane and is increasingly recognized as a new energy source. In addition, it can be easily be handled, stored, and transported. However, when CO2 is reduced and HCOO- is oxidized directly on electrodes, carbon monoxide is generated and quite high overpotential is required. One of the most promising strategies for solving these issues is the utilization of enzymes as catalysts. Enzymes allow the system to function in a specific biological reaction under mild conditions. The electro-enzymatic devices can be used as energy conversion system such as HCOO-/O2 biofuel cells and an efficient bioelectrochemical system of the CO2 reduction. Here, we have focused on the catalytic properties of tungsten-containing formate dehydrogenase (FoDH1; EC 1.2.1.2) from Methylobacterium extorquens AM1 to construct a bioelectrochemical interconversion system of HCOO-/CO2. FoDH1 is a heterodimeric soluble enzyme and catalyzes the oxidation of HCOO- to CO2 in coupled reduction of NAD+ to NADH. In this study, we have found that FoDH1 catalyzes both of the HCOO- oxidation and the CO2 reduction with several artificial redox partners (mediators). Mediators enable the enzymatic reaction to couple with an electrode reaction by shuttling electrons between enzymes and electrodes. This reaction is called mediated electron transfer (MET)-type bioelectrocatalysis. We have evaluated the bi-molecular reaction rate constants between FoDH1 and mediators and NAD+. They show the property called a linear free energy relationship (LFER), indicating that FoDH1 would have no specificity to NAD+. Similar LFER is also observed for the catalytic reduction of CO2. The reversible reaction between HCOO- and CO2 through FoDH1 has been realized on cyclic voltammetry by using methyl viologen (MV) as a mediator and by adjusting pH from the thermodynamic viewpoint (Fig.1). Potentiometric measurements have revealed that the three redox couples, MV2+/MV· +, HCOO-/ CO2, FoDH1 (ox/red), reach an equilibrium in the bulk solution when the two-way bioelectrocatalysis proceeds in the presence of FoDH1 and MV. The steady-state voltammograms with two-way bioelectrocatalytic properties are interpreted on a simple model by considering the solution equilibrium1. Furthermore, we have constructed a light driven HCOO- production system using a spinach thylakoid membrane, MV, and FoDH1. When the interconversion between HCOO- and CO2 is applied to the construction of efficient bioelectrochemical devices, a large current density should be realized at potentials close to the formal potential of the HCOO-/CO2 couple (E°′CO2). We show a great possibility of MET-type bioelectrocatalysis in the HCOO- oxidation and the CO2 reduction at high current densities and low overpotentials2. FoDH1 was used as a catalyst and immobilized on a Ketjen Black-modified glassy carbon electrode (FoDH1/KB/GCE). For the HCOO- oxidation, a high limiting current density (j lim) of about 24 mA cm- 2 was realized with a half wave potential (E 1/2) of only 0.12 V more positive than E°′CO2 at 30 °C in the presence of MV as a mediator. When the solution temperature was increased up to 60 °C to improve the enzymatic kinetics, j lim showed a clear dependence on the rotation rate (ω) and was enhanced to 145 ± 6 mA cm- 2 at ω = 6,000 rpm (Fig.2). The characteristics can be expressed as Koutecký-Levich equation and the equation provided a result that the enzymatic kinetic-controlled current density was 290 ± 50 mA cm- 2. The large value indicate that this FoDH1-based system is very useful for the HCOO- oxidation. When a viologen-functionalized polymer was co-immobilized with FoDH1 on the porous electrode, j lim of about 30 mA cm- 2 was attained at 60 °C with E 1/2 = E°′CO2 + 0.13 V. On the other hand, the CO2 reduction was also realized at j lim of about 15 mA cm- 2 with E 1/2 = E°′CO2 – 0.04 V at pH 6.6 and at 60 °C in the presence of MV. This is the first report of the enzyme-based bioelectrocatalytic CO2 reduction at such low overpotential and a high j lim. The present results are very useful to construct an effective bioelectrochemical reaction for the CO2 reduction and HCOO-/O2 biofuel cells as effective energy conversion systems. Reference (1) K. Sakai, et al., Sens. Biosens. Res., 5, 90 (2015) (2) K. Sakai, et al., Electrochem. Commun., 65, 31 (2016) Figure 1

Effect of tungstate on acetate and ethanol production by the electrosynthetic bacterium Sporomusa ovata.

BackgroundMicrobial electrosynthesis (MES) and gas fermentation are bioenergy technologies in which a microbial catalyst reduces CO2 into organic carbon molecules with electrons from the cathode of a bioelectrochemical system or from gases such as H2. The acetogen Sporomusa ovata has the capacity of reducing CO2 into commodity chemicals by both gas fermentation and MES. Acetate is often the only product generated by S. ovata during autotrophic growth.ResultsIn this study, trace elements in S. ovata growth medium were optimized to improve MES and gas fermentation productivity. Augmenting tungstate concentration resulted in a 2.9-fold increase in ethanol production by S. ovata during H2:CO2-dependent growth. It also promoted electrosynthesis of ethanol in a S. ovata-driven MES reactor and increased acetate production 4.4-fold compared to unmodified medium. Furthermore, fatty acids propionate and butyrate were successfully converted to their corresponding alcohols 1-propanol and 1-butanol by S. ovata during gas fermentation. Increasing tungstate concentration enhanced conversion efficiency for both propionate and butyrate. Gene expression analysis suggested that tungsten-containing aldehyde ferredoxin oxidoreductases (AORs) and a tungsten-containing formate dehydrogenase (FDH) were involved in the improved biosynthesis of acetate, ethanol, 1-propanol, and 1-butanol. AORs and FDH contribute to the fatty acids re-assimilation pathway and the Wood–Ljungdahl pathway, respectively.ConclusionsThis study presented here shows that optimization of microbial catalyst growth medium can improve productivity and lead to the biosynthesis of different products by gas fermentation and MES. It also provides insights on the metabolism of biofuels production in acetogens and demonstrates that S. ovata has an important untapped metabolic potential for the production of other chemicals than acetate via CO2-converting bioprocesses including MES.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0576-0) contains supplementary material, which is available to authorized users.

Bioelectrocatalytic formate oxidation and carbon dioxide reduction at high current density and low overpotential with tungsten-containing formate dehydrogenase and mediators

We show a great possibility of mediated enzymatic bioelectrocatalysis in the formate oxidation and the carbon dioxide (CO2) reduction at high current densities and low overpotentials. Tungsten-containing formate dehydrogenase (FoDH1) from Methylobacterium extorquens AM1 was used as a catalyst and immobilized on a Ketjen Black-modified electrode. For the formate oxidation, a high limiting current density (jlim) of ca. 24mAcm−2 was realized with a half wave potential (E1/2) of only 0.12V more positive than the formal potential of the formate/CO2 couple (E°′CO2) at 30°C in the presence of methyl viologen (MV2+) as a mediator, and jlim reached ca. 145mAcm−2 at 60°C. Even when a viologen-functionalized polymer was co-immobilized with FoDH1 on the porous electrode, jlim of ca. 30mAcm−2 was attained at 60°C with E1/2=E°′CO2+0.13V. On the other hand, the CO2 reduction was also realized with jlim≈15mAcm−2 and E1/2=E°′CO2−0.04V at pH 6.6 and 60°C in the presence of MV2+.

Interconversion between formate and hydrogen carbonate by tungsten-containing formate dehydrogenase-catalyzed mediated bioelectrocatalysis

We have focused on the catalytic properties of tungsten-containing formate dehydrogenase (FoDH1) from Methylobacterium extorquens AM1 to construct a bioelectrochemical interconversion system between formate (HCOO−) and hydrogen carbonate (HCO3−). FoDH1 catalyzes both of the HCOO oxidation and the HCO3− reduction with several artificial dyes. The bi-molecular reaction rate constants between FoDH1 and the artificial electron acceptors and NAD+ (as the natural electron acceptor) show the property called a linear free energy relationship (LFER), indicating that FoDH1 would have no specificity to NAD+. Similar LFER is also observed for the catalytic reduction of HCO3−. The reversible reaction between HCOO− and HCO3− through FoDH1 has been realized on cyclic voltammetry by using methyl viologen (MV) as a mediator and by adjusting pH from the thermodynamic viewpoint. Potentiometric measurements have revealed that the three redox couples, MV2+/MV·+, HCOO−/HCO3−, FoDH1 (ox/red), reach an equilibrium in the bulk solution when the two-way bioelectrocatalysis proceeds in the presence of FoDH1 and MV. The steady-state voltammograms with two-way bioelectrocatalytic properties are interpreted on a simple model by considering the solution equilibrium.

Reversible Interconversion of CO2 and Formate by a Molybdenum-Containing Formate Dehydrogenase

CO2 and formate are rapidly, selectively, and efficiently interconverted by tungsten-containing formate dehydrogenases that surpass current synthetic catalysts. However, their mechanism of catalysis is unknown, and no tractable system is available for study. Here, we describe the catalytic properties of the molybdenum-containing formate dehydrogenase H from the model organism Escherichia coli (EcFDH-H). We use protein film voltammetry to demonstrate that EcFDH-H is a highly active, reversible electrocatalyst. In each voltammogram a single point of zero net current denotes the CO2 reduction potential that varies with pH according to the Nernst equation. By quantifying formate production we show that electrocatalytic CO2 reduction is specific. Our results reveal the capabilities of a Mo-containing catalyst for reversible CO2 reduction and establish EcFDH-H as an attractive model system for mechanistic investigations and a template for the development of synthetic catalysts.

NADP-Specific Electron-Bifurcating [FeFe]-Hydrogenase in a Functional Complex with Formate Dehydrogenase in Clostridium autoethanogenum Grown on CO

Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP(+) with H2 or formate and the reversible formation of H2 and CO2 from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO2 reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H2 formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E0' = -520 mV).

Tungsten and Molybdenum Regulation of Formate Dehydrogenase Expression in Desulfovibrio vulgaris Hildenborough

Formate is an important energy substrate for sulfate-reducing bacteria in natural environments, and both molybdenum- and tungsten-containing formate dehydrogenases have been reported in these organisms. In this work, we studied the effect of both metals on the levels of the three formate dehydrogenases encoded in the genome of Desulfovibrio vulgaris Hildenborough, with lactate, formate, or hydrogen as electron donors. Using Western blot analysis, quantitative real-time PCR, activity-stained gels, and protein purification, we show that a metal-dependent regulatory mechanism is present, resulting in the dimeric FdhAB protein being the main enzyme present in cells grown in the presence of tungsten and the trimeric FdhABC₃ protein being the main enzyme in cells grown in the presence of molybdenum. The putatively membrane-associated formate dehydrogenase is detected only at low levels after growth with tungsten. Purification of the three enzymes and metal analysis shows that FdhABC₃ specifically incorporates Mo, whereas FdhAB can incorporate both metals. The FdhAB enzyme has a much higher catalytic efficiency than the other two. Since sulfate reducers are likely to experience high sulfide concentrations that may result in low Mo bioavailability, the ability to use W is likely to constitute a selective advantage.

Reversible interconversion of carbon dioxide and formate by an electroactive enzyme

Carbon dioxide (CO(2)) is a kinetically and thermodynamically stable molecule. It is easily formed by the oxidation of organic molecules, during combustion or respiration, but is difficult to reduce. The production of reduced carbon compounds from CO(2) is an attractive proposition, because carbon-neutral energy sources could be used to generate fuel resources and sequester CO(2) from the atmosphere. However, available methods for the electrochemical reduction of CO(2) require excessive overpotentials (are energetically wasteful) and produce mixtures of products. Here, we show that a tungsten-containing formate dehydrogenase enzyme (FDH1) adsorbed to an electrode surface catalyzes the efficient electrochemical reduction of CO(2) to formate. Electrocatalysis by FDH1 is thermodynamically reversible--only small overpotentials are required, and the point of zero net catalytic current defines the reduction potential. It occurs under thoroughly mild conditions, and formate is the only product. Both as a homogeneous catalyst and on the electrode, FDH1 catalyzes CO(2) reduction with a rate more than two orders of magnitude faster than that of any known catalyst for the same reaction. Formate oxidation is more than five times faster than CO(2) reduction. Thermodynamically, formate and hydrogen are oxidized at similar potentials, so formate is a viable energy source in its own right as well as an industrially important feedstock and a stable intermediate in the conversion of CO(2) to methanol and methane. FDH1 demonstrates the feasibility of interconverting CO(2) and formate electrochemically, and it is a template for the development of robust synthetic catalysts suitable for practical applications.

Gene Sequence and the 1.8 Å Crystal Structure of the Tungsten-Containing Formate Dehydrogenase from Desulfovibrio gigas

Desulfovibrio gigas formate dehydrogenase is the first representative of a tungsten-containing enzyme from a mesophile that has been structurally characterized. It is a heterodimer of 110 and 24 kDa subunits. The large subunit, homologous to E. coli FDH-H and to D. desulfuricans nitrate reductase, harbors the W site and one [4Fe-4S] center. No small subunit ortholog containing three [4Fe-4S] clusters has been reported. The structural homology with E. coli FDH-H shows that the essential residues (SeCys158, His159, and Arg407) at the active site are conserved. The active site is accessible via a positively charged tunnel, while product release may be facilitated, for H + by buried waters and protonable amino acids and for CO 2 through a hydrophobic channel.