Photoelectrochemical H2 Evolution with a Hydrogenase Immobilized on a TiO2‐Protected Silicon Electrode

Abstract The combination of enzymes with semiconductors enables the photoelectrochemical characterization of electron‐transfer processes at highly active and well‐defined catalytic sites on a light‐harvesting electrode surface. Herein, we report the integration of a hydrogenase on a TiO2‐coated p‐Si photocathode for the photo‐reduction of protons to H2. The immobilized hydrogenase exhibits activity on Si attributable to a bifunctional TiO2 layer, which protects the Si electrode from oxidation and acts as a biocompatible support layer for the productive adsorption of the enzyme. The p‐Si|TiO2|hydrogenase photocathode displays visible‐light driven production of H2 at an energy‐storing, positive electrochemical potential and an essentially quantitative faradaic efficiency. We have thus established a widely applicable platform to wire redox enzymes in an active configuration on a p‐type semiconductor photocathode through the engineering of the enzyme–materials interface.

Chong-Yong Lee,Hyun S. Park, Juan C. Fontecilla-Camps,and Erwin Reisner* Abstract: The combination of enzymes with semiconductors enables the photoelectrochemical characterization of electrontransfer processes at highly active and well-defined catalytic sites on al ight-harvesting electrode surface.Herein, we report the integration of ah ydrogenase on aT iO 2 -coated p-Si photocathode for the photo-reduction of protons to H 2 .T he immobilized hydrogenase exhibits activity on Si attributable to abifunctional TiO 2 layer,which protects the Si electrode from oxidation and acts as ab iocompatible support layer for the productive adsorption of the enzyme.T he p-Si j TiO 2 j hydrogenase photocathode displays visible-light driven production of H 2 at an energy-storing,p ositive electrochemical potential and an essentially quantitative faradaic efficiency.W eh ave thus established aw idely applicable platform to wire redox enzymes in an active configuration on ap-type semiconductor photocathode through the engineering of the enzyme-materials interface.
Hydrogenases are enzymes that catalyze the reversible reduction of protons to H 2 at record rates,and have therefore attracted much attention as an oble-metal-free benchmark catalyst in the fuel-forming reaction of water splitting. [1] Research into hydrogenases has resulted in an in-depth understanding of catalytic function and inspired the design of both structural [2] and functional [3] H 2 evolution catalysts.
Hydrogenases have also emerged recently as model electrocatalysts in photocatalytic H 2 generation schemes,i n which the enzyme is coupled to light-harvesters,s uch as carbon nitride,d ye-sensitized TiO 2 ,C d-based quantum dots, and organic dyes such as Eosin Y. [4] In these systems,t he hydrogenase can efficiently collect photo-excited electrons via its intraprotein FeScluster relays and deliver them to the embedded active site for H 2 generation at benchmark turnover rates.H owever,asacrificial electron donor is required in all of these systems,w hich limits the utility of the overall redox chemistry and prevents solar water splitting in these systems.P reviously,ahydrogenase has also been adsorbed onto asemiconductor electrode composed of n-type CdS-and TiO 2 -based materials. [5] In this case,light-driven H 2 evolution was only possible at apotential more negative than the thermodynamic equilibrium potential in the dark and consequently the storage of light energy in H 2 could not be demonstrated.
The[ NiFeSe]-hydrogenase isolated from Desulfomicrobium baculatum (Dmb)i saparticularly suitable H 2 evolution catalyst for water splitting as it displays as trong bias towards H 2 evolution in the presence of O 2 . [6][7][8] As such, we were able to demonstrate quantitative water splitting and the net storage of light energy as H 2 using this hydrogenase, wired to aphotosystem II-based photoanode,inaphotoelectrochemical cell. [9] However,t his system relies on the hydrogenase being adsorbed onto ah ierarchical indium-tin oxide electrode,w hich requires an applied bias to perform proton reduction. [9] Bias-free water splitting may be achieved by the incorporation of the hydrogenase on as uitable p-type semiconducting electrode and complementing this to as uitable photoanode.However,immobilization of functional enzymes onto ap -type semiconductor material remains am ajor challenge, [10] largely owing to the intrinsic instability of the available materials and their fragile interface with biological materials.P hotoelectrocatalytic H 2 production with ah ydrogenase on ap-type semiconductor electrode is unknown.
Thec hallenge is therefore to find as uitable p-type photocathode material that is stable,a llows af avorable interaction with the hydrogenase and has suitable band levels to enable visible-light-driven proton reduction. p-Type silicon has been widely regarded as one of the most promising photocathode materials as it has as mall band gap of 1.1 eV with ac onduction-band edge position of approximately À0.6 Vv ersus the standard hydrogen electrode (SHE). [11] However,enzyme integration with abare Si surface is limited by the electrode instability in aqueous solution owing to the formation of an insulating SiO 2 layer. Herein, we present ap-Si j TiO 2 j hydrogenase photocathode,w hich contains an amorphous TiO 2 protection layer between the semiconductor and the enzyme.Amorphous and thin TiO 2 is aconductor on Si and known to protect the Si surface. [12] In addition, we show that biocompatibility of the amorphous TiO 2 film enables productive adsorption of the hydrogenase.T he engineered interface in this semiconductor-enzymeelectrode allows us to assemble ah ydrogenase-based photocathode capable of storing light energy (i.e., showing light-induced cathodic response at apotential more positive than E o '(H + /H 2 )). Figure 1s ummarizes the key features of the proposed p-Si j TiO 2 j hydrogenase photocathode:photoexcitation of Si by visible light generates low-potential electrons in the semiconductor conduction band, which are transferred to the immobilized hydrogenase via the thin layer of TiO 2 .T he electrons enter the enzyme through the distal FeScluster and reach the [NiFeSe] active site,where the reduction of protons to H 2 occurs. [7] This mechanistic consideration is facilitated by the TiO 2 conduction-band potential at approximately À0.6 V versus SHE, which is located between the conduction band of Si and the H + /H 2 reduction potential. Thus,T iO 2 can be considered as ac onductor under the reducing conditions provided by the excited Si. [4b,c,e, 12b,e] First, we examined the interaction of Dmb [NiFeSe]hydrogenase with amorphous TiO 2 coated on af luoridedoped tin oxide (FTO) electrode.A na morphous TiO 2 layer was prepared by drop-casting TiCl 4 in toluene onto an FTOcoated glass substrate (3 mLo f2m m solution per cm 2 )a nd hydrolysis in air, and this step was repeated twice resulting in af ilm thickness of approximately 500 nm (Figure S1 ai nt he Supporting Information). Thef ormation of TiO 2 was confirmed by X-ray photoelectron spectroscopy (XPS) and by energy dispersive X-ray spectroscopy (EDX) elemental analysis ( Figures S1 ba nd S2). This method to generate an amorphous TiO 2 coating through solution processing at room temperature is simple and widely applicable, [13] which is in contrast to conventionally employed atomic-layer deposition or sputtering technologies to produce TiO 2 films. [12a,c] The FTO j TiO 2 surface was subsequently rinsed with water, the enzyme (3 mLo f8mm solution per cm 2 )w as drop-cast onto the TiO 2 surface and the enzyme-modified electrode rinsed with the electrolyte solution prior electrochemical measure-ments (50 mm MES (2-(N-morpholino)ethanesulfonica cid), at pH 6.0). Figure 2s hows the protein film voltammogram [14] of the FTO j TiO 2 j hydrogenase electrode in at hree-electrode configuration, with aA g/AgCl and aP tw ire as reference and counter electrodes,respectively (see Supporting Information for details). Thec atalytic current is associated to the activity of the immobilized hydrogenase, [1b,14] and the voltammograms display the characteristic features of ah ydrogenase wired onto the electrode.P roton reduction and H 2 oxidation currents can be observed with as mall overpotential. In the absence of an immobilized enzyme,n oc atalytic response is observed and only TiO 2 -characteristic charging currents can be seen in the cyclic voltammogram ( Figure S3).
Controlled potential electrolysis (CPE) was performed to determine the stability and Faradaic yield of H 2 evolution. Thehydrogenase was adsorbed onto amorphous TiO 2 ,and an applied potential (E appl )ofÀ0.35 Vversus SHE was applied, which corresponds to 0V versus the reverse hydrogen electrode (RHE) at pH 6.0. [15] After 1h CPE under aN 2 atmosphere,acharge of 18 mC had passed and 90 AE 5nmol of H 2 accumulated, which corresponds to aF aradaic yield of 96 AE 6%.T hese results demonstrate that amorphous TiO 2 acts as as uitable interfacial layer for the immobilization of electroactive Dmb [NiFeSe]-hydrogenase;t his interaction is believed to occur at the surface-exposed glutamate and aspartate residues in close proximity to the distal Fe-S cluster. [1f, 6,7] Protein film voltammetry with hydrogenases on crystalline TiO 2 had been reported previously, [4b, 16] but the preparation of these metal oxide films required high-temperature annealing, which is not compatible with the integration on as ilicon electrode (see below).
Theamorphous TiO 2 layer (3 mLof2mm TiCl 4 in toluene solution per cm 2 )was subsequently applied on apretreated p-Si with an atomically flat H-terminated surface.This step was repeated twice,followed by rinsing with water and adsorption of the enzyme (3 mLof8mm solution per cm 2 )using the same  The voltammograms were recorded for astirred sample, under an atmosphere of N 2 (blue trace) and 1bar H 2 (red trace) at ascan rate of 10 mVs À1 .Acontrol experiment(black trace) in the absence of enzyme is also shown. The inset shows the CPE trace at E appl = À0.35 Vversus SHE under N 2 for FTO j TiO 2 (black) and FTO j TiO 2 j hydrogenase(blue). All experiments were performed in MES (50 mm)electrolyte solution with aAg/AgCl reference and Pt counter electrode at pH 6.0 at 20 AE 2 8 8C.

Angewandte Chemie
Zuschriften method as on the FTO-coated glass electrodes (see Supporting Information for details). Thep rotein film photoelectrochemical response under chopped white-light illumination (10 mW cm À2 )r ecorded under an N 2 atmosphere is summarized in Figures 3a and Figure S4. Thep -Si j TiO 2 j hydrogenase photocathode showed an onset photocurrent of approximately À0.1 Vversus SHE (i.e., approximately 0.25 Vm ore positive than E 0 '(H + /H 2 )), thereby demonstrating the capability to operate at athermodynamic underpotential with this electrode.I nc ontrol experiments,t he linear sweep voltammograms show the expected low photoactivity of the TiO 2free p-Si electrodes (corresponding to bare p-Si and p-Si j hydrogenase,w here the enzyme was adsorbed on bare p-Si); aresult of the fast formation of an insulating SiO 2 layer on the semiconductor surface. [17] Thee nzyme-free p-Si j TiO 2 electrode displays ap hoto-response in the cathodic region (presumably owing to charging of the TiO 2 conduction band), but the current density is significantly lower at lessnegative potentials than that of the p-Si j TiO 2 j hydrogenase, indicating significantly faster H 2 evolution kinetics with the enzyme-semiconductor hybrid system. No photocurrent response was observed with an amorphous TiO 2 film on FTO-coated glass ( Figure S5).
Thus,t he p-Si j TiO 2 j hydrogenase electrode exhibits an enhanced photoresponse compared to the TiO 2 -a nd hydro-genase-free p-Si electrodes,i np articular at less-negative potentials,where energy from irradiation can be stored. These higher photocurrents are due to the efficient collection of conduction-band electrons by the electroactive enzyme.T his work demonstrates the integration of ahydrogenase on Si and optimizations to achieve higher photocurrent densities are currently in progress.T he photocurrent density is currently limited by the sub-optimal integration of the hydrogenase in the TiO 2 protection layer (i.e., the absence of ap orous morphology for high protein loading) and the low light intensity (10 mW cm À2 )e mployed in this study.
CPE was performed to confirm photoelectrochemical H 2 formation and to study the robustness of p-Si j TiO 2 j hydrogenase.T he electrolysis experiments were performed at E appl = À0.35 Vv ersus SHE (0 Vv s. RHE) under N 2 and in the dark for 60 s, followed by white-light illumination (10 mW cm À2 )f or 1h (Figure 3b). An initial decrease in the photocurrent was observed, followed by stabilization of the photoresponse with p-Si j TiO 2 j hydrogenase.A fter 1h, ac harge of 5.1 AE 0.2 mC had passed and 25 AE 2nmol of H 2 were detected, which corresponds to aF aradaic yield of 95 AE 6%.I nt he absence of hydrogenase or TiO 2 ,s ubstantially lower photocurrents were observed and we were unable to reliably detect H 2 in the control experiments with p-Si j TiO 2 and p-Si j hydrogenase.
Finally,f urther control experiments were performed to unambiguously demonstrate that the enzyme was indeed the active catalyst on the electrode.T he inset in Figure 3b shows the current-time profile at E appl = À0.35 Vversus SHE under an alternating N 2 and 10 %COinN 2 atmosphere,and during white-light illumination. CO was selected as it is ar eversible inhibitor of the Dmb [NiFeSe]-hydrogenase active site. [7] The photocurrent was substantially reduced upon addition of CO. Removal of CO by N 2 purging of the electrolyte solution shows the recovery of the photocurrent. This response is consistent with the characteristic of reversible inhibition of the hydrogenase and can be repeated for several cycles.Some photocurrent remained even in the presence of CO,w hich is attributed to the background photocurrent of the amorphous TiO 2 layer (see above).
In summary,w eh ave established an easily applicable methodology for interfacing redox enzymes with p-type semiconductor electrodes to promote light-driven reductive reactions.Indoing so,wehave also extended the use of TiO 2 protection layers from anchoring synthetic catalysts to biological catalysts.S pecifically,w ed emonstrate with this interfacial engineering approach that ah ydrogenase can be coupled to ap -type silicon electrode,w hich allows for photocatalytic H 2 generation with ah ydrogenase on as emiconductor in the absence of as acrificial agent and electrochemical overpotential. This work takes us closer to the construction of semi-biological photoelectrochemical systems and the assembly of an all-enzyme driven, bias-free photoelectrochemical water-splitting cell with the O 2 -tolerant Dmb [NiFeSe]-hydrogenase.W ork is currently in progress to enhance the performance of the photocathode through rational materials design for an optimized immobilization of the hydrogenase. . Protein film photoelectrochemistry with p-Si j TiO 2 j hydrogenase. a) Photoresponse under chopped irradiation (10 mWcm À2 ;g ray shading indicates response in the dark) performed at ascan rate of 10 mVs À1 under aN 2 atmosphere. b) CPE at E appl = À0.35 Vversus SHE (pH 6.0) during irradiation under aN 2 atmosphere;t he color labeling of the traces from (a) applies also to the CPE traces in (b). Inset shows the effect of 10 %COi njections (highlighted in gray) on the photocurrent response of p-Si j TiO 2 j hydrogenase at E appl = À0.35 Vv ersus SHE, followed by flushing with 100 %N 2 .A ll experiments were performed in MES (50 mm)electrolyte solution with aAg/AgCl reference and Pt counter electrode at pH 6.0 at 20 AE 2 8 8Cu nder white-light illumination with an intensity of 10 mWcm À2 .