Metal-Containing Formate Dehydrogenases, a Personal View.

Mo/W-containing formate dehydrogenases (FDH) catalyzes the reversible oxidation of formate to carbon dioxide at their molybdenum or tungsten active sites. The metal-containing FDHs are members of the dimethylsulfoxide reductase family of mononuclear molybdenum cofactor (Moco)- or tungsten cofactor (Wco)-containing enzymes. In these enzymes, the active site in the oxidized state comprises a Mo or W atom present in the bis-Moco, which is coordinated by the two dithiolene groups from the two MGD moieties, a protein-derived SeCys or Cys, and a sixth ligand that is now accepted as being a sulfido group. SeCys-containing enzymes have a generally higher turnover number than Cys-containing enzymes. The analogous chemical properties of W and Mo, the similar active sites of W- and Mo-containing enzymes, and the fact that W can replace Mo in some enzymes have led to the conclusion that Mo- and W-containing FDHs have the same reaction mechanism. Details of the catalytic mechanism of metal-containing formate dehydrogenases are still not completely understood and have been discussed here.

Infrared Spectroscopy Elucidates the Inhibitor Binding Sites in a Metal-Dependent Formate Dehydrogenase.

Biological carbon dioxide (CO2 ) reduction is an important step by which organisms form valuable energy-richer molecules required for further metabolic processes. The Mo-dependent formate dehydrogenase (FDH) from Rhodobacter capsulatus catalyzes reversible formate oxidation to CO2 at a bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. To elucidate potential substrate binding sites relevant for the mechanism, we studied herein the interaction with the inhibitory molecules azide and cyanate, which are isoelectronic to CO2 and charged as formate. We employed infrared (IR) spectroscopy in combination with density functional theory (DFT) and inhibition kinetics. One distinct inhibitory molecule was found to bind to either a non-competitive or a competitive binding site in the secondary coordination sphere of the active site. Site-directed mutagenesis of key amino acid residues in the vicinity of the bis-MGD cofactor revealed changes in both non-competitive and competitive binding, whereby the inhibitor is in case of the latter interaction presumably bound between the cofactor and the adjacent Arg587.

An Integrated CO2 Electrolyzer and Formate Fuel Cell Enabled by a Reversibly Restructuring Pb-Pd Bimetallic Catalyst.

A single device combining the functions of a CO2 electrolyzer and a formate fuel cell is a new option for carbon-neutral energy storage but entails rapid, reversible and stable interconversion between CO2 and formate over a single catalyst electrode. We report a new catalyst with such functionalities based on a Pb-Pd alloy system that reversibly restructures its phase, composition, and morphology and thus alters its catalytic properties under controlled electrochemical conditions. Under cathodic conditions, the catalyst is relatively Pb-rich and is active for CO2 -to-formate conversion over a wide potential range; under anodic conditions, it becomes relatively Pd-rich and gains stable catalytic activity for formate-to-CO2 conversion. The bifunctional activity and superior durability of our Pb-Pd catalyst leads to the first proof-of-concept demonstration of an electrochemical cell that can switch between the CO2 electrolyzer/formate fuel cell modes and can stably operate for 12 days.

Mechanistic studies of formate oxidase from Aspergillus oryzae: A novel member of the glucose-Methanol-choline oxidoreductase enzyme superfamily that oxidizes carbon acids.

Formate oxidase (FOX) from Aspergillus oryzae is the only GMC member that oxidizes a carbon acid rather than alcohols; thus, its catalytic mechanism may be different from that of other GMC members. We have used pH, solvent viscosity, and deuterium kinetic isotope effects, to investigate the catalytic mechanism of FOX. The enzyme followed a Bi-Bi sequential steady-state kinetic mechanism. The kcat value was pH-independent between pH 2.8 and 6.8, suggesting a lack of ionizable groups in kinetic step(s) that limit the overall turnover of the enzyme. The kcat/Kformate value decreased from a value of 10,000 M-1s-1 at low pH with a pKa value of 4.4, consistent with the requirement of a protonated group for substrate binding. An inverse viscosity dependence on the kcat/Kformate value indicated an isomerization of the Michaelis complex. The kcat/Koxygen value was 340,000 M-1s-1 and pH independent up to pH 6.0. The Dkcat and D(kcat/Kformate) values were 2.5 and 1.9, respectively, indicating that substrate CH bond cleavage is rate-limiting for FOX catalysis. Analytical ultracentrifugation indicated a concentration dependence of the oligomeric state of FOX. The appkred,H value was ∼75% that of kcat,H, indicating that the anaerobic reduction of FOX was dependent on the oligomeric state of FOX.

Fabric-based alkaline direct formate microfluidic fuel cells.

Fabric-based microfluidic fuel cells (MFCs) serve as a novel, cost-efficient alternative to traditional FCs and batteries, since fluids naturally travel across fabric via capillary action, eliminating the need for an external pump and lowering production and operation costs. Building on previous research with Y-shaped paper-based MFCs, fabric-based MFCs mitigate fragility and durability issues caused by long periods of fuel immersion. In this study, we describe a microfluidic fabric-based direct formate fuel cell, with 5 M potassium formate and 30% hydrogen peroxide as the anode fuel and cathode oxidant, respectively. Using a two-strip, stacked design, the optimized parameters include the type of encasement, the barrier, and the fabric type. Surface contact of the fabric and laminate sheet expedited flow and respective chemical reactions. The maximum current (22.83 mA/cm2 ) and power (4.40 mW/cm2 ) densities achieved with a 65% cotton/35% polyester blend material are a respective 8.7% and 32% higher than previous studies with Y-shaped paper-based MFCs. In series configuration, the MFCs generate sufficient energy to power a handheld calculator, a thermometer, and a spectrum of light-emitting diodes.

An improved alkaline direct formate paper microfluidic fuel cell.

Paper-based microfluidic fuel cells (MFCs) are a potential replacement for traditional FCs and batteries due to their low cost, portability, and simplicity to operate. In MFCs, separate solutions of fuel and oxidant migrate through paper due to capillary action and laminar flow and, upon contact with each other and catalyst, produce electricity. In the present work, we describe an improved microfluidic paper-based direct formate FC (DFFC) employing formate and hydrogen peroxide as the anode fuel and cathode oxidant, respectively. The dimensions of the lateral column, current collectors, and cathode were optimized. A maximum power density of 2.53 mW/cm(2) was achieved with a DFFC of surface area 3.0 cm(2) , steel mesh as current collector, 5% carbon to paint mass ratio for cathode electrode and, 30% hydrogen peroxide. The longevity of the MFC's detailed herein is greater than eight hours with continuous flow of streams. In a series configuration, the MFCs generate sufficient energy to power light-emitting diodes and a handheld calculator.

A microfluidic direct formate fuel cell on paper.

We describe the first direct formate fuel cell on a paper microfluidic platform. In traditional membrane-less microfluidic fuel cells (MFCs), external pumping consumes power produced by the fuel cell in order to maintain co-laminar flow of the anode stream and oxidant stream to prevent mixing. However, in paper microfluidics, capillary action drives flow while minimizing stream mixing. In this work, we demonstrate a paper MFC that uses formate and hydrogen peroxide as the anode fuel and cathode oxidant, respectively. Using these materials we achieve a maximum power density of nearly 2.5 mW/mg Pd. In a series configuration, our MFC achieves an open circuit voltage just over 1 V, and in a parallel configuration, short circuit of 20 mA absolute current. We also demonstrate that the MFC does not require continuous flow of fuel and oxidant to produce power. We found that we can pre-saturate the materials on the paper, stop the electrolyte flow, and still produce approximately 0.5 V for 15 min. This type of paper MFC has potential applications in point-of-care diagnostic devices and other electrochemical sensors.

Cu-Modified Palladium Catalysts: Boosting Formate Electrooxidation via Interfacially OHad-Driven Had Removal.

Direct formate fuel cells have gained traction due to their eco-friendly credentials and inherent safety. However, their potential is hampered by the kinetic challenges of the formate oxidation reaction (FOR) on Pd-based catalysts, chiefly due to the unfavorable adsorption of hydrogen species (Had). These species clog the active sites, hindering efficient catalysis. Here, we introduce a straightforward strategy to remedy this bottleneck by incorporating Pd with Cu to expedite the removal of Pd-Had in alkaline media. Notably, Cu plays a pivotal role in bolstering the concentration of hydroxyl adsorbates (OHad) on the surface of catalyst. These OHad species can react with Had, effectively unblocking the active sites for FOR. The as-synthesized catalyst of PdCu/C exhibits a superior FOR performance, boasting a remarkable mass activity of 3.62 A mg-1. Through CO-stripping voltammetry, we discern that the presence of Cu in Pd markedly speeds up the formation of adsorbed hydroxyl species (OHad) at diminished potentials. This, in turn, aids the oxidative removal of Pd-Had, leveraging a synergistic mechanism during FOR. Density functional theory computations further reveal intensified interactions between adsorbed oxygen species and intermediates, underscoring that the Cu-Pd interface exhibits greater oxyphilicity compared to pristine Pd. In this study, we present both experimental and theoretical corroborations, unequivocally highlighting that the integrated copper species markedly amplify the generation of OHad, ensuring efficient removal of Had. This work paves the way, shedding light on the strategic design of high-performing FOR catalysts.

A remarkable peroxidase-like behavior of the catalase KatA from the pathogenic bacteria Helicobacter pylori: The oxidation reaction with formate as substrate and the stabilization of an [Fe(IV) = O Trp•] intermediate assessed by multifrequency EPR spectroscopy.

We have characterized the catalytic cycle of the Helicobacter pylori KatA catalase (HPC). H. pylori is a human and animal pathogen responsible for gastrointestinal infections. Multifrequency (9-285 GHz) EPR spectroscopy was applied to identify the high-valent intermediates (5 ≤ pH ≤ 8.5). The broad (2000 G) 9-GHz EPR spectrum consistent with the [Fe(IV) = O Por•+] intermediate was detected, and showed a clear pH dependence on the exchange-coupling of the radical (delocalized over the porphyrin moiety) due to the magnetic interaction with the ferryl iron. In addition, Trp• (for pH ≤ 6) and Tyr• (for 5 ≤ pH ≤ 8.5) species were distinguished by the advantageous resolution of their g-values in the 285-GHz EPR spectrum. The unequivocal identification of the high-valent intermediates in HPC by their distinct EPR spectra allowed us to address their reactivity towards substrates. The stabilization of an [Fe(IV) = O Trp•] species in HPC, unprecedented in monofunctional catalases and possibly involved in the oxidation of formate to the formyloxyl radical at pH ≤ 6, is reminiscent of intermediates previously identified in the catalytic cycle of bifunctional catalase-peroxidases. The 2e- oxidation of formate by the [Fe(IV) = O Por•+] species, both at basic and acidic pH conditions, involving a 1H+/2e- oxidation in a cytochrome P450 peroxygenase-like reaction is proposed. Our findings demonstrate that moonlighting by the H. pylori catalase includes formate oxidation, an enzymatic reaction possibly related to the unique strategy of the neutrophile bacterium for gastric colonization, that is the release of CO2 to regulate the pH in the acidic environment.

Modulating the Interfacial Water Network of Dual-Site Pd/FeOx/C Catalyst for Efficient Formate Electrooxidation.

The rational design of electrocatalysts for formate oxidation reaction (FOR) in alkaline media is crucial to promote the practical applications of direct formate fuel cells (DFFCs). The FOR kinetic on palladium (Pd) based electrocatalysts is strongly hindered by unfavorably adsorbed hydrogen (Had) as the major intermediate species blocking the active sites. Herein, we report a strategy of modulating the interfacial water network of dual-site Pd/FeOx/C catalyst to significantly enhance the desorption kinetics of Had during FOR. Aberration-corrected electron microscopy and synchrotron characterizations revealed the successful construction of Pd/FeOx interfaces on carbon support as a dual-site electrocatalyst for FOR. Electrochemical tests and in situ Raman spectroscopy results showed that Had could be effectively removed from the active sites of the as-designed Pd/FeOx/C catalyst. CO-stripping voltammetry and density functional theory calculations (DFT) demonstrated that the introduced FeOx could effectively accelerate the dissociative adsorption of water molecules on active sites, which accordingly generates adsorbed hydroxyl species (OHad) to facilitate the removal of Had during FOR. This work provides a novel route to develop advanced FOR catalysts for fuel cell applications.

Ultrathin PdCu Nanosheet as Bifunctional Electrocatalysts for Formate Oxidation Reaction and Oxygen Reduction Reaction.

The development of robust nonplatinum electrocatalysts to enhance the performance of formate oxidation reaction (FOR) and oxygen reduction reaction (ORR) is one of the key issues for the commercialization of direct formate fuel cells (DFFCs), but still challenging. Herein, a structurally controlled 3D flower-like PdCu nanosheet (NS) catalyst is synthesized by a method of oil bath reduction under mild conditions as a bifunctional electrocatalyst for DFFCs. Under the dual tuning on the composition and intermetallic phase, the PdCu nanosheet catalyst exhibits 8.67 times higher mass activity for anodic formate oxidation reaction than the commercial Pd/C. For the cathodic ORR, a positive shift half-wave potential is obtained for PdCu nanosheets exceeding Pt/C. Moreover, after a long-term stability test, the current density of the PdCu nanosheet catalyst for FOR and ORR can be well maintained with the least activity decay. When the PdCu NSs are used as optimized anode and cathode electrodes for DFFCs enable a peak power density as high as 53 mW cm-2 at room temperature, which is about 1.3 times higher than that of the commercial Pd/C and Pt/C as anode and cathode electrodes. This work provides a potential strategy to improve the catalytic performance of non-Pt-based nanocatalysts.

A Novel NADP-Dependent Formate Dehydrogenase From the Hyperthermophilic Archaeon Thermococcus onnurineus NA1.

The genome of the hyperthermophilic archaeon Thermococcus onnurineus NA1 contains three copies of the formate dehydrogenase (FDH) gene, fdh1, fdh2, and fdh3. Previously, we reported that fdh2, clustered with genes encoding the multimeric membrane-bound hydrogenase and cation/proton antiporter, was essential for formate-dependent growth with H2 production. However, the functionality of the other two FDH-coding genes has not yet been elucidated. Herein, we purified and characterized cytoplasmic Fdh3 to understand its functionality. The purified Fdh3 was identified to be composed of a tungsten-containing catalytic subunit (Fdh3A), an NAD(P)-binding protein (Fdh3B), and two Fe-S proteins (Fdh3G1 and Fdh3G2). Fdh3 oxidized formate with specific activities of 241.7 U/mg and 77.4 U/mg using methyl viologen and NADP+ as electron acceptors, respectively. While most FDHs exhibited NAD+-dependent formate oxidation activity, the Fdh3 of T. onnurineus NA1 showed a strong preference for NADP+ over NAD+ as a cofactor. The catalytic efficiency (k cat /K m) of Fdh3 for NADP+ was measured to be 5,281 mM-1 s-1, which is the highest among NADP-dependent FDHs known to date. Structural modeling suggested that Arg204 and Arg205 of Fdh3B may contribute to the stabilization of the 2'-phosphate of NADP(H). Fdh3 could also use ferredoxin as an electron acceptor to oxidize formate with a specific activity of 0.83 U/mg. Furthermore, Fdh3 showed CO2 reduction activity using reduced ferredoxin or NADPH as an electron donor with a specific activity of 0.73 U/mg and 1.0 U/mg, respectively. These results suggest a functional role of Fdh3 in disposing of reducing equivalents by mediating electron transfer between formate and NAD(P)H or ferredoxin.

Enhanced Electrocatalytic Oxidation of Formate via Introducing Surface Reactive Oxygen Species to a CeO2 Substrate.

Direct formate fuel cells (DFFCs) as promising energy technologies have been applied for portable and wearable devices. However, for the formate oxidation reaction (FOR), the deficiency of catalysts has prevented DFFCs from practical applications. Herein, we prepared a Pd-loaded CeO2 catalyst through a simple steam treatment at 400 °C to enhance the catalytic FOR performance. In comparison with the counterpart of Pd/CeO2 without stream treatment, the as-prepared Pd/CeO2-ST catalyst has a lower onset potential of 381 mV and a lower peak potential of 0.64 V with a higher peak current of 10.62 mA cm-2. The experimental results show that the enhanced FOR properties of Pd/CeO2-ST are ascribed to the introduction of surface reactive oxygen species to the CeO2 substrate, which substantially promotes the desorption of adsorbed hydrogen (H*) intermediates. Density functional theory (DFT) calculations reveal that on the surface of CeO2, the abundant oxygen vacancies boost the OH* adsorption ability and accelerate the kinetics of the potential-limiting step. This work not only proposes a new strategy for enhancing the activity of FOR catalysts but also highlights the understanding of the FOR mechanism in alkaline media for DFFC applications.

Bifunctional Palladium Hydride Nanodendrite Electrocatalysts for Hydrogen Evolution Integrated with Formate Oxidation.

The rational design of advanced electrocatalysts and energy-saving electrolysis strategies is highly desirable for achieving high-efficiency electrochemical H2 generation yet challenging. In this work, we report highly branched Pd hydride nanodendrites (PdH-NDs) formed by a very facial solvothermal method and a succedent chemical H intercalation method in N,N-dimethylformamide. The electrocatalytic performance of PdH-NDs is experimentally and theoretically correlated with the morphology and composition, which has demonstrated substantially enhanced electrochemical activity and stability for formate oxidation reaction and hydrogen evolution reaction in alkaline electrolyte compared with Pd nanodendrites. Density functional theory calculations suggest a downshift of the Pd d-band center of PdH-NDs due to the dominant Pd-H ligand effects that weaken the binding energies of the intermediate catalytic species and toxic carbon monoxide. The asymmetric formate electrolyzer based on bifunctional PdH-ND electrocatalysts is first constructed, which only requires a low voltage of 0.54 V at 10 mA cm-2 for continuous H2 generation. This study reveals significant insights about the morphology/composition-performance relationship for palladium hydrides with bifunctional electroactivity.

Engineering Two-Dimensional PdAgRh Nanoalloys by Surface Reconstruction for Highly Active and Stable Formate Oxidation Electrocatalysis.

Promoting the formate oxidation reaction (FOR) is central to develop promising direct formate fuel cells, but current electrocatalysts are suffering from low activity and ultrapoor stability. Herein, the ternary PdAgRh nanoalloys with ultrathin two-dimensional architecture are for the first time synthesized and employed as a novel class of electrocatalysts for the FOR. Benefitting from unique nanostructure as well as oxophilic Rh surface oxides, the Pd55Ag30Rh15/C electrocatalyst demonstrates an exceptional FOR activity of 1.85 A mgPd-1, showing a 4.74-fold improvement compared to the commercial Pd/C, and retains the current density of 150 mA mgPd-1 after a long-term test, representing the greatest durability among all available FOR electrocatalysts. More strikingly, extending the upper limit potential (ULP) of cyclic voltammetry is revealed to facilitate the surface reconstruction of the Pd55Ag30Rh15/C electrocatalyst to in situ form Ag surface oxides (Ag-O), resulting in a highly active and stable Pd/Ag-O interface at the atomic scale, which considerably boost the FOR performance. In particular, the reconstructed Pd55Ag30Rh15/C electrocatalyst exhibits a mass activity of 3.26 A mgPd-1 with 74.2% of initial activity retained after 1000 cycles. This work showcases an effective strategy to tune surface reconstruction on multimetallic nanoalloys for robust FOR electrocatalysts and beyond.

Carbon-nanotube-caged microbial electrodes for bioelectrocatalysis.

A method to stably immobilize microbes on electrodes was developed. Resting cells of Methylobacterium extorquens AM1(MeAM1) were caged within multiwalled carbon nanotubes (MWNTs)by adding the cells to a water dispersion of MWNTs then allowing the resulting mixture to dry on electrodes. The MeAM1-MWCNTs electrode thus obtained displayed excellent activities in the bidirectional bioelectrocatalysis due to formate dehydrogenase(s) in the resting cells; formate oxidation and carbon dioxide reduction proceeded at steady-state catalytic current densities of 0.6 ±â€¯0.1 and -0.8 ±â€¯0.1 mA cm-2, respectively, using methyl viologen as mediator under very mild conditions (pH 7.0, atmospheric pressure, and 37 °C). In addition, the catalytic signal was stable for more than one week under continuous operation.

Formate: an Energy Storage and Transport Bridge between Carbon Dioxide and a Formate Fuel Cell in a Single Device.

We demonstrate the first device to our knowledge that uses a solar panel to power the electrochemical reduction of dissolved carbon dioxide (carbonate) into formate that is then used in the same device to operate a direct formate fuel cell (DFFC). The electrochemical reduction of carbonate is carried out on a Sn electrode in a reservoir that maintains a constant carbon balance between carbonate and formate. The electron-rich formate species is converted by the DFFC into electrical energy through electron release. The product of DFFC operation is the electron-deficient carbonate species that diffuses back to the reservoir bulk. It is possible to continuously charge the device using alternative energy (e.g., solar) to convert carbonate to formate for on-demand use in the DFFC; the intermittent nature of alternative energy makes this an attractive design. In this work, we demonstrate a proof-of-concept device that performs reduction of carbonate, storage of formate, and operation of a DFFC.

Biogenic concrete protection driven by the formate oxidation by Methylocystis parvus OBBP.

The effectiveness of Microbiologically Induced Carbonate Precipitation (MICP) from the formate oxidation by Methylocystis parvus OBBP as an alternative process for concrete protection was investigated. MICP was induced on Autoclaved Aerated Concrete (AAC), the model material, by immersing the material in 10(9) M. parvus cells mL(-1) containing 5 g L(-1) of calcium formate. A 2 days immersion of the material gave the maximum weight increase of the specimens (38 ± 19 mg) and this was likely due to the deposition of calcium carbonate, biomass, and unconverted calcium formate. The solid deposition mainly occurred in the micropores of the specimen, close to the outer surface. A significantly lower water absorption was observed in the bacterially treated specimens compared to the non-treated ones (up to 2.92 ± 0.91 kg m(-2)) and this could be attributed to the solid deposition. However, the sonication test demonstrated that the bacterial treatment did not give a consolidating effect to the material. Overall, compared to the currently employed urea hydrolysis process, the formate-based MICP by M. parvus offers a more environmentally friendly approach for the biotechnological application to protect concrete.

Molybdenum-containing membrane-bound formate dehydrogenase isolated from Citrobacter sp. S-77 having high stability against oxygen, pH, and temperature.

Membrane-bound formate dehydrogenase (FDH) was purified to homogeneity from a facultative anaerobic bacterium Citrobacter sp. S-77. The FDH from Citrobacter sp. S-77 (FDHS77) was a monomer with molecular mass of approximately 150 kDa. On SDS-PAGE, the purified FDHS77 showed as three different protein bands with molecular mass of approximately 95, 87, and 32 kDa, respectively. Based on the N-terminal amino acid sequence analysis, the sequence alignments observed for the 87 kDa protein band were identical to that of the large subunit of 95 kDa, indicating that the purified FDHS77 consisted of two subunits; a 95 kDa large subunit and a 32 kDa small subunit. The purified FDHS77 in this purification did not contain a heme b subunit, but the FDHS77 showed significant activity for formate oxidation, determined by the Vmax of 30.4 U/mg using benzyl viologen as an electron acceptor. The EPR and ICP-MS spectra indicate that the FDHS77 is a molybdenum-containing enzyme, displaying a remarkable O2-stability along with thermostability and pH resistance. This is the first report of the purification and characterization of a FDH from Citrobacter species.