in artificial photosynthesis 
 ( ap ) , a research area that falls within 
 the active and important realm of  renewable energy / solar fuels  , 
 the fuel- or o2-forming catalysts attached to a light absorber 
 ( e.g. , semiconductor ) can be regarded as electrocatalysts since they 
 catalyze the respective electrochemical half cell reactions . ideal 
 ap catalysts should exhibit high activity and not require a large , 
 wasteful overpotential to drive the reaction ; however , a minimal overpotential 
 requirement corresponds most closely to reversibility , whereas it 
 is beneficial to render the reaction as unidirectional as possible , 
 thus trapping the electron or hole and opposing recombination . under the condition of low overpotential that 
 is beneficial for efficiency , it is interesting and important to consider 
 how the relative rates of catalysis for forward and reverse directions 
 can be biased in favor of the desired direction . in principle , 
 such a bias can be provided if the carrier density 
 of the semiconductor material changes substantially within a potential 
 region close to the reduction potential of the reaction of interest . electrochemical kinetics at semiconductors depend on the density of 
 charge carriers in the space - charge region adjacent to the solution 
 interface . a useful physical characteristic of a semiconductor , 
 unrestricted by its chemical nature , but of general relevance , is 
 the flatband potential ( efb ) . for an n - type 
 semiconductor , as the applied potential becomes more negative than efb , the increasing electron density at the surface 
 gives rise to an accumulation layer ; the conduction and valence bands 
 bend downward and the semiconductor approaches metallic - like behavior . in contrast , when the applied potential is increased relative to efb , a depletion layer forms and the bands bend 
 upward , resulting in a barrier for electron transfer from the catalyst 
 to the semiconductor . at efb the space - charge region may extend well into the 
 material ( values in the range 10 nm to 1 m being reported ) . for h2 production , platinum 
 is well established as a 
 reversible catalyst , but it could never be scaled up for large - area 
 ap , and there is much interest in finding alternatives . for co2 reduction , there are no examples of reversible catalysts based on materials or small molecules . although a variety 
 of catalysts are being explored to drive h / co2 reduction to fuels , the high overpotential requirements and/or low turnover rates command 
 much room for improvement . in contrast , many enzymes are now known 
 to behave as reversible electrocatalysts , particularly 
 those catalyzing h and co2 reduction , and they are valuable models for understanding electrocatalysis 
 at a more fundamental level . the buried active site of an enzyme is 
 much less likely to be perturbed by the nature of the electrode material 
 than a small molecule catalyst . since an enzyme has specialized parts 
 ( long - range electron transfer and storage , substrate / product transport , 
 catalysis by inner - sphere proton - coupled electron transfers ) the component 
 steps of electrocatalysis can be resolved , often exploiting genetic 
 engineering of the enzyme structure . two particular classes of enzyme 
 have been extensively studied by protein film electrochemistry ( pfe ) : 
 hydrogenases ( h2ase ) and carbon monoxide dehydrogenases 
 ( codh ) each possess highly active fe- and ni - containing active sites 
 and exhibit turnover frequencies often measured in thousands ( 1010 ) per second at minimal overpotential , 
 achieving h and co2 reduction at 0.45 
 to 0.55 v vs she under neutral aqueous conditions . durrant et al . reported a series of studies 
 dealing with the electrochemistry of cytochrome c and hemoglobin immobilized on tio2 film electrodes . they found irreversible reduction of the proteins active 
 sites at potentials substantially more negative than the solution - based 
 redox potentials , similar to observations with simple redox couples 
 such as potassium ferricyanide k3[fe(cn)6 ] . the tio2 electrode thus exerts a 
 rectifying effect : however , these electrochemical reductions are not 
 catalytic . we now explore the directionality that can be imposed 
 on electrocatalytic 
 behavior when certain enzymes and , by extension , any reversible electrocatalysts are attached to a semiconductor ( figure 1 ) . although materials will differ considerably in 
 their chemical properties , the concept of efb provides a useful reference point . since a reversible electrocatalytic 
 reaction changes direction across a small potential region , the effect 
 of a change in carrier density across this region should be particularly 
 influential . we have therefore compared the reversibility of h / h2 and co2/co interconversion by h2ase and codh at a metallic - like graphite electrode and at 
 tio2 and cds electrodes ( n - type semiconductors ) by analyzing 
 the electrocatalytic voltammograms recorded under h2 or 
 co2/co mixtures . schematic representation of the formation of 
 an accumulation layer 
 at the surface of cds or tio2 due to the increased electron 
 density at the surface when eappl is lowered 
 relative to efb . the increased electron 
 density and subsequent downward band bending facilitate efficient 
 electron transfer to the enzyme active site via fes clusters to catalyze 
 h2 production or co2 reduction . we studied the [ nifese]-h2ase ( henceforth , simply h2ase ) from desulfomicrobium baculatum ( the  se refers to the fact that one of 
 the cysteine ligands normally coordinating the ni is replaced by selenocysteine ) 
 and codh i ( henceforth , simply codh ) from carboxydothermus 
 hydrogenoformans , which has a [ ni4fe-4s ] 
 active site . these enzymes contain electron - transfer relays comprised 
 of fes clusters , and when attached to a metallic 
 electrode ( including pyrolytic graphite , a semimetal ) , they are excellent 
 examples of reversible electrocatalysts . voltammograms of a solution containing both oxidized and reduced 
 forms of the substrate ( the redox - active molecules in solution ) intersect 
 the zero current axis at the equilibrium potential with little or 
 no inflection . the electrocatalytic behavior of each enzyme 
 was compared on three 
 different materials : graphite , and cds and tio2 ( both n - type 
 semiconductors ) . 0.52 
 v vs she , ph 6 , respectively ) are more 
 negative than the equilibrium potentials for h / h2 ( 0.36 v vs she ) and co2/co ( 0.46 v ) , 
 and nanoparticles of cds and dye - sensitized tio2 ( anatase 
 or p25 , a mixed rutile - anatase phase ) modified by attaching h2ase or codh have been shown to be excellent systems for photoreducing 
 h2o and co2 . synthesized porous thin films of hexagonal phase 
 cds and tio2 ( degussa p25 ) on indium tin oxide ( ito ; supporting information ) and fluorine doped tin 
 oxide ( fto ) 
 were converted into electrodes by making an ohmic contact with cu 
 wire on the uncoated area of the conducting glass and sealed with 
 nonconducting epoxy ( figure 2 and supporting information , figure s2 ) . sem images 
 of cds ( a ) and tio2 ( b ) film electrodes . the scale bars are ( a ) 2 and 1 m ( inset ) and ( b ) 1 m 
 and 200 nm ( inset ) . the cds thin film consists 
 of a highly porous 3-dimensional network 
 of cds nanoflakes / sheets . the pore size of cds varies from 700 nm to approximately 
 1 m . the tio2 thin film exhibits porous structures 
 consisting of nanoparticles ( of the order of 20 nm ) , which have formed 
 aggregates , as expected upon calcination at 450 c . pyrolytic 
 graphite  edge  ( pge , figure s1 , 
 supporting information ) electrodes of geometric surface area 
 0.03 cm were constructed in house as described previously . to study the electrocatalytic behavior 
 of the enzymes on cds , tio2 , and pge , aliquots of enzyme 
 samples ( either codh or h2ase , < 10 pmol ) were drop cast 
 onto the electrode and left 
 for a few minutes until partially dry . in the case of pge , the electrode 
 was abraded using p400 tufbak durite sandpaper and sonicated to remove 
 carbon debris before attachment of enzyme . the electrode was then 
 placed in an electrochemical cell and used as the working electrode 
 with ag / agcl ( 1 m kcl ) as reference and pt wire as counter . all potentials 
 are quoted vs the standard hydrogen electrode ( she ) using the conversion eshe = eag / agcl + 
 234 mv at 20 c . the electrolyte was 0.2 m mes at ph 6.0 , and 
 all solutions were prepared using purified water ( millipore , 18 m 
 cm ) . electrochemical measurements were made with an autolab potentiostat 
 ( pgstat30 ) controlled by nova software ( ecochemie ) . impedance measurements 
 were conducted using an ivium technologies compactstat.e portable 
 electrochemical interface and impedance analyzer , controlled by iviumsoft 
 software . cyclic voltammograms were recorded prior to and after impedance 
 and chronoamperometric experiments with enzyme - modified electrodes , 
 in order to ensure that the catalytic activity of the enzyme had remained 
 steady throughout the measurements . precise gas mixtures ( boc gases ) 
 were created using mass flow controllers ( sierra instruments ) , and 
 the electrochemical cell was constantly purged with the gas mixture 
 throughout the experiments . because of the oxygen sensitivity of the 
 enzymes , all experiments were performed inside an anaerobic glovebox 
 ( belle technologies , o2 < 3 ppm ) and in the dark , unless 
 otherwise stated . isolation , purification and activity measurements 
 of codh i from carboxydothermus hydrogenoformans were 
 carried out according 
 to previously described procedures . the [ nifese]-hydrogenase sample 
 from desulfomicrobium baculatum was obtained according 
 to previously published methods . the specific 
 h2 oxidation activity of the h2ase was determined 
 as 1700 mol min mg , 
 using conventional room - temperature assays after a few minutes under 
 h2 to fully activate the enzyme . chronoamperometry experiments 
 depicting the stability of codh and h2ase on the three 
 different electrode materials used in this report are shown in figure s3 ( supporting information ) . row a of figure 3 shows cyclic voltammograms 
 ( cvs ) of codh attached to pge ( codh - pge ) , cds ( codh - cds ) , and tio2 ( codh - tio2 ) in separate experiments with 100% 
 co2 or a mixture of 50% co2/50% co gently bubbling 
 through the cell solution . the catalytic current is directly related 
 to the rate of catalysis in either direction . a positive current corresponds 
 to substrate oxidation , and a negative current corresponds to reduction . 
 rather than reporting current densities , we display current vs voltage 
 curves in this report : in pfe it is rarely possible to ascertain the 
 electroactive coverage of enzyme as this requires the detection and 
 integration of reversible signals ( for example , due to fes redox couples ) , 
 which are usually vanishingly small . instead , pfe provides precise and continuous 
 measurement of the relative catalytic rates in either 
 direction . importantly , the shapes of the cvs on pge in the presence 
 of both the oxidized and reduced substrates reveal the inherent catalytic 
 bias of the enzymes to function more effectively in one particular 
 direction . a dominant factor in determining the bias of an enzyme 
 is the reduction potential ( eox / r ) of the relay center of the enzyme that serves as the electron entry / exit 
 point for transfer to the buried catalytic center . in contrast to codh at pge , the voltammograms for 
 the codh - cds 
 and codh - tio2 electrodes are dominated by co2 reduction ( vide infra ) ; i.e. , the bias appears reversed ( with respect to the bias observed at pge ) to favor instead the 
 formation of co ; in fact co oxidation is negligible at cds and extremely 
 sluggish at tio2 . the effect is all the more pronounced 
 if we consider that co is a natural inhibitor of co2 reduction 
 by codh ( product inhibition ) ; i.e. , the 
 exponentially increasing reductive current is suppressed when both 
 oxidized and reduced substrate is present . voltammograms recorded 
 under 100% co2 reveal the further increase in current that 
 is obtained when co is not introduced . cyclic voltammograms 
 ( 10 mv s ) of unmodified 
 ( black ) and enzyme - modified electrodes ( red , blue ) , 0.2 m mes , ph 
 6.0 , 20 c . voltammograms recorded under 100% co2 ( codh - pge , 
 codh - cds and codh - tio2 ) or 100% n2 ( h2ase - pge , h2ase - cds and h2ase - tio2 ) are depicted in blue . experiments in a 50% co2/50% co 
 gas mixture ( codh - pge , codh - cds and codh - tio2 ) bubbling 
 through the cell or with 100% h2 bubbling through the cell 
 ( h2ase - pge , h2ase - cds , and h2ase - tio2 ) are shown in red . row b shows analogous experiments for h2ase on 
 pge , 
 cds and tio2 while h2 is gently bubbled through 
 the cell solution . once again , the effect of product inhibition is 
 clearly visible in the case of h2ase - pge . electrocatalysis in the reduction direction is strongly 
 inhibited by h2 , whereas cvs recorded in the absence of 
 h2 reveal the higher rate of h reduction by 
 uninhibited enzyme . as with codh , comparison with pge shows that the 
 h2 oxidation currents at cds or tio2 are greatly 
 suppressed relative to the rates of h reduction . the potential dependencies of the low - level co and h2 oxidation 
 currents at cds and tio2 varied slightly between 
 different experiments : this scatter is most likely due to nonidealities 
 such as dispersion in interactions between enzyme and semiconductor 
 surface and direct contact between some enzyme molecules and the conducting 
 oxide support . typical ratios of currents for reduction vs oxidation 
 are shown graphically in figure s4 ( supporting 
 information ) . comparing background - subtracted oxidation and 
 reduction currents at 0.15 v either side of the thermodynamic 
 potential for co2/co and h / h2 interconversion ( figure 3 ) clearly reveals the changes in catalytic 
 bias for each enzyme . for h2ase the current ratio ( h2 oxidation / h reduction ) drops from 8.1 on pge 
 to 0.81 ( cds ) and 0.11 ( tio2 ) , i.e. , attenuation factors 
 of approximately 10- and 80-fold , respectively . for codh the current 
 ratio ( co oxidation / co2 reduction ) drops from 2.2 ( pge ) 
 to 0.04 ( cds ) and 0.05 ( tio2 ) , i.e. , an attenuation factor 
 of approximately 50-fold in each case . these results demonstrate that 
 although the residual oxidizing currents for both enzymes differ at 
 tio2 and cds , the oxidation reactions are essentially rendered 
 irreversible . previously , we reported photoinduced co2 reduction using 
 codh - tio2 and codh - cds nanoassemblies comprising the respective 
 nanoparticles and enzymes we have now used in this work . using gas chromatography , we were able to monitor the catalytic 
 reduction of co2 to co. in this work we used instead a 
 selective inhibitor of codh to prove that the reduction currents at 
 each electrode correspond to the catalytic conversion of co2 into co. we recently established that cyanate ( nco ) , an isoelectronic co2 analogue , targets the active site 
 of codh by intercepting the reduced cred2 level and blocking 
 co2 reduction . cyanate is 
 released as the potential is raised and does not significantly inhibit 
 co oxidation . figure 4 depicts the selective 
 and quantitative 
 inhibition of co2 reduction on codh - modified tio2 and cds electrodes after adding kocn to a final concentration of 
 20 mm to codh - tio2 ( figure 4a ) and 
 60 mm to codh - cds ( figure 4b ) . the portions 
 of the cathodic currents due to co2 reduction ( before introducing 
 the inhibitor , blue traces ) are quenched . instead , for codh - tio2 , the voltammogram resembles the characteristic shape of a 
 tio2 electrode at low potential . in the codh - cds system , 
 the voltammogram also closely resembles the shape of the bare electrode 
 after introduction of the inhibitor . for tio2 , the increase in reduction current for both 
 h and co2 coincides closely with the trumpet - shaped 
 increase in electrode charging current that is observed in the blank 
 experiments . in chemical terms , the background 
 waveshape can be ascribed to the reversible reduction of ti surface centers , the effect of which 
 is to increase the carrier density as the potential is lowered . an 
 analogous observation is not apparent for cds , so to obtain more insight 
 we carried out impedance experiments in order to measure how the capacitance 
 varies with potential and thus gain an estimate of efb under different conditions . the flatband potential 
 ( efb ) values for cds and tio2 were determined to be ca . 0.50 v vs 
 she , respectively , from mott  schottky plots of capacitance 
 data at 1 khz ( figure 5a and b ) . the value measured for tio2 at ph 6.0/20 c agrees 
 well with the value of 0.52 v vs she predicted by a model 
 for nanostructured tio2 electrodes in aqueous solution , 
 given by eq 1.1 cyclic voltammograms showing selective 
 inhibition of co2 reduction on codh - modified tio2 ( a ) and cds ( b ) in the 
 presence of 20 and 60 mm kocn , respectively , under a mixture of 50% 
 co2 , 50% co bubbling through the cell . solutions contained 
 0.2 m mes ( ph 6.0 ) ; temperature 20 c ; scan rate 10 mv s. mott schottky 
 plots recorded on tio2 electrodes 
 in a different protic electrolyte solution confirmed that the flatband 
 potential is independent of the electrolyte ; i.e. , the capacitance of the helmholtz layer at the semiconductor electrolyte 
 interface is negligible compared to the capacitance of the space - charge 
 region ( figure s5 , supporting information ) . two further experiments were carried out with tio2 in 
 the presence of codh , first under  nonturnover  conditions , 
 i.e. , under 100% n2 without the presence of substrate ( figure 5c ; an equivalent experiment on codh - cds is shown 
 in the supporting information , figure s5 ) 
 and then under 100% co2 ( figure 5d ) . analogous substrate free  impedance experiments 
 could not be conducted using h2ase , as protons of the buffer 
 solution constitute the substrate . the experiments showed that the 
 presence of enzyme alone has a considerable effect on the shape of 
 the mott schottky plot ( figure 5c ) and 
 revealed an interesting effect when substrate is added ( figure 5d ) . the experiment carried out in the presence of 
 enzyme alone shows an unfeatured shape at mid - to - low frequencies , 
 whereas the much sharper plot obtained in the presence of co2 ( yielding efb = 0.59 v ) becomes 
 flattened as the frequency is raised . although the sem image in figure 2a shows the pores in the tio2 film to 
 be of approximately the same size as the dimensions of codh ( approximately 
 88  63  60  ) , our observations suggest the enzyme 
 adsorbs on the outer surface and acts mainly as a resistive film . however , when co2 is introduced , a charge - transport pathway 
 becomes available and a flatband potential can be measured at low 
 frequencies that allow electron transfer and catalysis to occur . these 
 experiments are at a preliminary stage and suggest important possibilities 
 for using impedance techniques to study catalytic electron transfer 
 in enzymes . schottky plots ( 1/c vs potential ) recorded 
 at 20 c , 1 khz for ( a ) tio2 , ( b ) cds , ( c ) codh - tio2 , and ( d ) codh - tio2 in the presence of co2 . each plot was measured at 1 khz under 100% n2 apart 
 from ( d ) , which was measured at 78 hz under 100% co2 . the 
 cell buffer solution was 0.2 m mes ( ph 6.0 ) in all experiments . semiconductor nanoparticles and 
 highly porous nanocrystalline electrodes 
 are considered to be too small to support an effective space - charge 
 region due to the very small crystallite sizes . in contrast , the tio2 particles in our nanocrystalline 
 films are interconnected and partly fused together ( figure 2 ) . studies on similarly structured tio2 electrodes suggest that the electronic properties of such aggregates 
 are not completely dominated by the individual particles but instead 
 exhibit bulk properties to some extent , rendering the formation of 
 a space - charge layer possible . the results depicted in figure 3 clearly 
 display a pronounced rectification of the otherwise reversible electrocatalysis 
 of h / h2 and 
 co2/co interconversion by enzymes , when measured at tio2 and cds electrodes instead of pge . according to established 
 models , the surface concentration of electrons ( the majority carrier 
 for an n - type semiconductor ) increases exponentially as the potential 
 is lowered : it is controlled , through a boltzmann - like relationship , 
 by the difference between efb and the 
 applied potential . as the electrode potential is lowered through efb , there is a transformation 
 to metallic - like character as an accumulation layer forms ( figure 1 ) : the increase in electron density at the semiconductor - catalyst 
 interface favors efficient electron transfer from the semiconductor 
 to the enzyme to drive reduction . in contrast , raising the applied 
 potential above efb results in a depletion 
 layer that poses a barrier for electron transfer from the enzyme to 
 the semiconductor . this reasoning explains the resistive effect observed 
 for the oxidation reactions when enzymes are coupled on cds and tio2 surfaces . the efb values broadly 
 lie within 0.25 v of the reduction potentials for co2/co and h / h2 under 
 the experimental conditions used : this is important because the catalysts 
 are operating in a nearly reversible manner and the respective currents 
 change direction ( oxidation  reduction ) across the same limited 
 potential region . the apparent catalytic bias of each enzyme is thus 
 shifted to favor fuel formation when coupled to cds and tio2 electrode surfaces . finally , we studied visible - light - driven 
 h2 production 
 at the h2ase - cds electrode , as cds has a band gap of around 
 2.3 ev ( 540 nm ) . cyclic voltammograms were first recorded on an unmodified 
 ( bare cds ) electrode , both in the dark and under illumination (  
 > 420 nm , intensity 16 mw cm ) . to eliminate 
 heating 
 effects , the bath temperature was monitored during experiments , and 
 no light - dependent variation was noted . upon irradiation , the unmodified 
 cds electrode exhibits an anodic photocurrent ( as expected for an 
 n - type semiconductor at a potential positive of efb ) at potentials above 0.4 v , figure 6a . in experiments performed on h2ase - cds 
 under illumination , direct contact of enzyme and the transparent conducting 
 oxide layer does not contribute to the observed photo - oxidation current 
 under light exposure , because ito is transparent under visible - light 
 irradiation . ( a ) cyclic voltammograms recorded under an inert atmosphere , 
 both 
 under dark conditions and with visible light irradiation (  
 > 420 nm ) , for an unmodified cds electrode , and following modification 
 with h2ase . scan rate 10 mv s. ( b ) 
 chronoamperometry experiments comparing the catalytic h2 evolution current of h2ase - cds in the dark and under 
 irradiation at 0.65 v vs she . the cell solution was 0.2 m 
 mes ( ph 6.0 ) at 20 c in both experiments . interestingly , under irradiation , the h reduction 
 current 
 is further enhanced by photoexcitation of electrons from the valence 
 band into the conduction band , from where they transfer to h2ase , and there is a small but noticeable shift in onset potential . 
 for an n - type semiconductor , at potentials far more negative than efb , illumination should have no effect on the 
 reductive current because the electrode behaves like a metal . in contrast , 
 at potentials closer to efb , illumination 
 can enhance the concentration of electrons available for transfer 
 to the enzyme ; the result thus provides further evidence that this 
 factor is important . the observation highlights the importance of 
 the enzyme as a reversible catalyst as the availability ( concentration ) 
 of electrons can be at least as important as the overpotential driving 
 the reaction . our results demonstrate 
 that semiconducting electrodes can be used 
 to impose directionality on reversible catalysts that operate in the 
 region of the flatband potential . the catalytic direction shifts in 
 response to the ( ideally ) exponential increase in surface electron 
 concentration that occurs as the potential is changed , thereby , in 
 the case of an n - type semiconductor , favoring reduction over oxidation . this principle of rectifying catalysis provides further design criteria 
 for artificial systems that convert light into storable chemical energy . the enzymes are so proficient that the current is controlled more 
 by the charge carrier availability in the semiconductor than by properties 
 of the catalyst . although the enzymes used in this work are wholly 
 unsuited for long - term , large - scale systems , our results provide valuable 
 insight for developing integrated artificial systems ( based ultimately 
 on abundant chemical catalysts ) where getting as close as possible 
 to reversible catalysis is important for efficiency .
the most efficient catalysts for 
 solar fuel production should operate 
 close to reversible potentials , yet possess a bias for the fuel - forming 
 direction . 
 protein film electrochemical studies of ni - containing carbon 
 monoxide dehydrogenase and [ nifese]-hydrogenase , each a reversible 
 electrocatalyst , show that the electronic state of the electrode strongly 
 biases the direction of electrocatalysis of co2/co and 
 h+/h2 interconversions . attached to graphite 
 electrodes , 
 these enzymes show high activities for both oxidation 
 and reduction , but there is a marked shift in bias , in favor of co2 or h+ reduction , when the respective enzymes are 
 attached instead to n - type semiconductor electrodes constructed from 
 cds and tio2 nanoparticles . 
 this catalytic rectification 
 effect can arise for a reversible electrocatalyst attached to a semiconductor 
 electrode if the electrode transforms between semiconductor- and metallic - like 
 behavior across the same narrow potential range ( < 0.25 v ) that 
 the electrocatalytic current switches between oxidation and reduction .