Interfacing Formate Dehydrogenase with Metal Oxides for the Reversible Electrocatalysis and Solar‐Driven Reduction of Carbon Dioxide

Abstract The integration of enzymes with synthetic materials allows efficient electrocatalysis and production of solar fuels. Here, we couple formate dehydrogenase (FDH) from Desulfovibrio vulgaris Hildenborough (DvH) to metal oxides for catalytic CO2 reduction and report an in‐depth study of the resulting enzyme–material interface. Protein film voltammetry (PFV) demonstrates the stable binding of FDH on metal‐oxide electrodes and reveals the reversible and selective reduction of CO2 to formate. Quartz crystal microbalance (QCM) and attenuated total reflection infrared (ATR‐IR) spectroscopy confirm a high binding affinity for FDH to the TiO2 surface. Adsorption of FDH on dye‐sensitized TiO2 allows for visible‐light‐driven CO2 reduction to formate in the absence of a soluble redox mediator with a turnover frequency (TOF) of 11±1 s−1. The strong coupling of the enzyme to the semiconductor gives rise to a new benchmark in the selective photoreduction of aqueous CO2 to formate.

Abstract: The integration of enzymes with synthetic materials allows efficient electrocatalysis and production of solar fuels. Here,w ec ouple formate dehydrogenase (FDH)f rom Desulfovibrio vulgaris Hildenborough (DvH) to metal oxides for catalytic CO 2 reduction and report an in-depth study of the resulting enzyme-material interface.Protein film voltammetry (PFV) demonstrates the stable binding of FDH on metal-oxide electrodes and reveals the reversible and selective reduction of CO 2 to formate.Q uartz crystal microbalance (QCM) and attenuated total reflection infrared (ATR-IR) spectroscopy confirm ah igh binding affinity for FDH to the TiO 2 surface. Adsorption of FDH on dye-sensitized TiO 2 allows for visiblelight-driven CO 2 reduction to formate in the absence of as oluble redox mediator with aturnover frequency (TOF) of 11 AE 1s À1 .T he strong coupling of the enzyme to the semiconductor gives rise to an ew benchmark in the selective photoreduction of aqueous CO 2 to formate.
Electrocatalyticand solar-driven fuel synthesis from the greenhouse gas CO 2 is adesirable approach to simultaneously produce sustainable energy carriers and combat increasing atmospheric CO 2 levels. [1] Formate is astable intermediate in the reduction of CO 2 and can be used as liquid energy carrier in fuel cells,asahydrogen storage material, or feedstock for the synthesis of fine chemicals. [2] Metals and synthetic molecular systems have been widely studied as electrocatalysts for CO 2 reduction to formate,b ut largely lack the required efficiency,s electivity or affordability to enable carbon capture and utilization technologies. [3,4] There is avid research into both biological and artificial CO 2 fixation. Semi-artificial photosynthesis provides ac ommon stage for these contrasting approaches as components from synthetic and biological origin can be combined in hybrid model systems. [5] To date,e nzyme-based visible-lightdriven CO 2 reduction to formate relies on diffusional mediators,such as methyl viologen (MV 2+ )and nicotinamide adenine dinucleotide (NAD + ). [6,7] Mediated processes are inefficient as they consume energy,a re kinetically slow,a nd cause short-circuit reactions.M V 2+ is toxic to microorganisms, [8] and NAD + is prohibitively expensive for fuel production. [6] In this work, we selected wild-type formate dehydrogenase (FDH)f rom Desulfovibrio vulgaris Hildenborough (DvH) as it has previously displayed robustness and high activity for the oxidation of formate in solution assays, [10,11] and the electrochemical reduction of CO 2 . [12] Initially,protein film voltammetry (PFV) was employed to study the interfacial electron transfer between FDH and porous indiumdoped tin oxide (ITO) and TiO 2 electrodes in the absence of amediator.Immobilization and loading of FDH on TiO 2 were then investigated using aquartz crystal microbalance (QCM) and attenuated total reflection infrared (ATR-IR) spectros- relays to the W-active site of FDH for the reduction of CO 2 to formate. The oxidized dye is regenerated by triethanolamine (TEOA). Ap rotein structure homologoust oDvH FDH is shown. [9] copy. FDH was finally coupled directly to dye-sensitized TiO 2 nanoparticles for the selective photocatalytic reduction of CO 2 to formate in adiffusional mediator-free colloidal system ( Figure 1).
Thee lectrocatalytic activity of FDH on metal-oxide electrodes was studied by PFV on mesoporous ITO (meso-ITO) and TiO 2 (mesoTiO 2 )electrodes with afilm thickness of approximately 2.5 mm(Supporting Information, Figure S1). [13] FDH (21.5 mm)was activated by incubation with the reducing agent dl-dithiothreitol (DTT,5 0mm) [9] and the resulting solution (2 mL) was drop-cast on the electrode surface.T he FDH-modified electrode was placed in an electrolyte solution containing CO 2 /NaHCO 3 and KCl at pH 6.5 under aC O 2 atmosphere. Figure 2A shows the electrochemically reversible interconversion of CO 2 and formate by FDH immobilized on ac onductive mesoITOe lectrode (mesoITO j FDH). The onset potential for both CO 2 reduction and formate oxidation was observed close to the thermodynamic potential (E 0' = À0.36 Vv s. standard hydrogen electrode (SHE), pH 6.5), [14] demonstrating that interfacial electron transfer by the [4Fe-4S] relays and catalysis at the W-active site are highly efficient. [15] Similar electrochemically reversible characteristics have previously only been reported for FDHsf rom Escherichia coli and Syntrophobacter fumaroxidans on graphite electrodes. [14,16,17] When FDH was immobilized on as emiconducting mesoTiO 2 electrode (mesoTiO 2 j FDH), asimilar onset potential for CO 2 reduction (À0.4 Vvs. SHE) was observed and the current density reached À100 mAcm À2 at À0.6 Vv s. SHE ( Figure 2B). Formate oxidation could not be observed for mesoTiO 2 j FDH electrodes as TiO 2 behaves as an insulator at the required potentials.C ontrolled-potential electrolysis (CPE) at À0.6 Vv s. SHE for 2h produced formate with aF aradaic efficiencyo f( 92 AE 5)% ( Figure 2B,i nset). Comparison of PFV scans before and after CPE showed that approximately 90 %ofthe initial FDH activity remains after 2h,d emonstrating the excellent stability of the immobilized enzyme.

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Communications 4602 www.angewandte.org probed by exposing the planarTiO 2 j FDH electrode to buffer solutions with different ionic strengths.Rinsing the QCM cell with an enzyme-free solution for 1hdesorbed only 6% of the preloaded FDH.U pon increasing the KCl concentration to 0.5-3.0 m KCl, 70-60 %o fFDH remained adsorbed on the TiO 2 surface.T he finding that 60 % FDH remained adsorbed on TiO 2 after multiple rinsing steps with high KCl concentrations suggests ac ontribution from chemisorption to the attachment of the enzyme. [20,21] Amino-acid residues exposed on the FDH surface are likely involved in binding.F or example,a spartic and glutamic acid have previously been suggested to form astrong interaction with TiO 2 . [22,23] Thea dsorption of FDH was also probed by surfaceselective ATR-IR spectroscopy using aS ip rism coated with a planar or a mesoTiO 2 layer (100 or 400 nm thickness, respectively). After the addition of FDH to the buffer solution covering the planarTiO 2 ( Figure 3B)o rmesoTiO 2 (Supporting Information, Figure S2) coated prism, the two characteristic amide Ia nd amide II bands of the protein backbone structure were detected at 1650 cm À1 and 1545 cm À1 ,r espectively. [24] Thep rotein adsorption was monitored in situ over 2hof incubation time and no (in the case of planarTiO 2 )orslight (in the case of mesoTiO 2 )changes to the band features in the amide-band region were observed, suggesting am ainly retained backbone structure of FDH on the surface of TiO 2 .D uring the adsorption process,a mide I and amide II band intensities showed an increase over time ( Figure 3B). Themajority of FDH remained adsorbed on the surface of planarTiO 2 (Supporting Information, Figure S3) upon increasing the ionic strength of the buffer, which agrees with the QCM experiments ( Figure 3A,i nset) and supports astronger than purely electrostatic interaction between FDH and TiO 2 .
After establishing the strong interface between FDH and TiO 2 ,v isible-light-driven CO 2 reduction to formate was investigated with FDH immobilized on dye-sensitized TiO 2 nanoparticles (dye j TiO 2 j FDH,F igures 1a nd 4). Thec olloidal system was self-assembled by adding FDH (pre-activated with DTT) to as uspension of TiO 2 nanoparticles containing TEOAand aphosphonate group-bearing dye,either aruthenium tris-2,2'-bipyridine complex (RuP)o radiketopyrrolopyrrole (DPP)a tp H6.5 and 25 8 8Cu nder N 2 atmosphere (to protect the enzyme from aerobic damage). Both dyes are known to adsorb onto TiO 2 via their phosphonate-anchoring groups and DPP provides aprecious-metal-free alternative to RuP. [25] CO 2 was introduced to the solution via the addition of NaHCO 3 .Upon UV-filtered irradiation, the photoexcited dye injects electrons into the conduction band (CB) of TiO 2 (E CB (TiO 2 ) = À0.67 Vv s. SHE at pH 6.5), [25] whereupon the electrons are conveyed to the catalytic W-center of FDH to drive CO 2 reduction. Theo xidized dye is regenerated by the sacrificial electron donor (Figure 1). [26] Thed ye j TiO 2 j FDH systems showed stable formate production for approximately 6h (Figure 4). Thef ormation of gaseous or dissolved side-products was not detected by gas chromatography,i on chromatography,a nd 1 HNMR spectroscopy.The activity of RuP j TiO 2 j FDH was not limited by the amount of dye or the light intensity (Supporting Information, Figures S4 and S5). Asolution assay monitoring the activity of FDH by UV/Vis spectroscopy (via formate oxidation in presence of 2mm MV 2+ )s howed that approximately 36 AE 7% FDH remained active after 24 ho fp hotocatalysis (Supporting Information, Figure S6), suggesting that inactivation of FDH is likely the main reason for activity loss. Thea ddition of MV 2+ as as oluble redox mediator to RuP j TiO 2 j FDH showed that not all FDH present in the system is accessible to direct electron transfer across the enzymematerial interface (Supporting Information, Figure S7). Control experiments demonstrated that all components are essential for formate production (Supporting Information, Figures S8 and S9) and support oxidative quenching and "through-particle" electron transfer as depicted in Figure 1 (Supporting Information, Figures S10 and S11). [26] Isotopiclabeling studies confirmed that formate was produced from CO 2 (Supporting Information, Figure S12).
Forp hotocatalytic experiments,a ne nzyme loading of approximately 0.03 pmol cm À2 was calculated assuming that all FDH is adsorbed on TiO 2 with asurface area of 50 m 2 g À1 . Saturation of the TiO 2 surface with FDH in the QCM experiment was only observed when two orders of magnitude higher amounts of FDH were adsorbed ( Figure 3A). As QCM and ATR-IR spectroscopy indicate stronger than purely electrostatic interactions,close-to-quantitative adsorption of FDH on the TiO 2 nanoparticle in the colloidal system is likely.Aturnover frequency (TOF) of 11 AE 1.0 and 5 AE 0.6 s À1 (based on CO 2 conversion after 6h)and approximately 4.9 AE 0.2 and 2.0 AE 0.2 mmol formate (after 24 h) were observed from CO 2 using RuP and DPP-sensitized TiO 2 , respectively ( Figure 4). Ther esults of all photocatalysis experiments are presented in Tables S1 and S2 in the Supporting Information. Table 1s hows ac omparison of state-of-the-art catalysts (enzymatic and synthetic) in combination with dye-sensitized TiO 2 nanoparticles without diffusional mediators for CO 2 reduction and H 2 evolution. Previous studies showed that enzymes outperform the synthetic systems in terms of TOF. [30]  Among the compared systems,t he presented RuP j TiO 2 j FDH system exhibits the highest TOFf or CO 2 reduction. The DPP j TiO 2 j FDH system shows that comparable activities can also be achieved in an entirely precious-metal-free system. In semi-artificial systems,rapid electron transfer from TiO 2 to the enzyme was previously found to be essential for efficient catalysis, [22,31] suggesting that the strong interfacial interaction plays an important role for the high activity and stability of dye j TiO 2 j FDH.P reviously reported photocatalyst systems employing NAD + -dependent FDHsf or CO 2 reduction to formate rely on soluble redox mediators and only produced TOFs in the range of 10-20 h À1 . [32] In summary, FDH immobilized on metal-oxide electrodes is established as ar eversible electrocatalyst for the selective conversion of CO 2 to formate.T he porous metal-oxide scaffolds allow for high FDH loading and consequently high current densities,w hich makes the protein-modified electrodes not only arelevant model system for CO 2 utilization, but also for formate oxidation in formate fuel cells.Anexcellent interface between TiO 2 and FDH is confirmed by QCM analysis and ATR-IR spectroscopy.T he direct (diffusional mediator-free) electron transfer across the enzyme-metaloxide interface is exploited for visible-light-driven CO 2 reduction to formate.T hese results underline the importance of characterizing the interactions at the enzyme-material interface and future improvements in performance may arise from more controlled immobilization and more efficient electron transfer with the directly wired FDH.