Photocurrent Generation from Surface Assembled Photosystem I on

Feb 4, 2013 - Weapons and Materials Research Directorate, United States Army Research Laboratory, Aberdeen Proving Ground, Maryland. 21005, United ...
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Photocurrent Generation from Surface Assembled Photosystem I on Alkanethiol Modified Electrodes Amy K. Manocchi,† David R. Baker,† Scott S. Pendley,‡ Khoa Nguyen,§ Margaret M. Hurley,‡ Barry D. Bruce,§,∥ James J. Sumner,† and Cynthia A. Lundgren*,† †

Sensors and Electron Devices Directorate, United States Army Research Laboratory, Adelphi, Maryland 20783, United States Weapons and Materials Research Directorate, United States Army Research Laboratory, Aberdeen Proving Ground, Maryland 21005, United States § Biochemistry Cellular and Molecular Biology Department, University of Tennessee, Knoxville, Tennessee 37996, United States ∥ Chemical and Biomolecular Engineering Department, University of Tennessee, Knoxville, Tennessee 37996, United States ‡

S Supporting Information *

ABSTRACT: Photosystem I (PSI) is a key component of oxygenic photosynthetic electron transport because of its light-induced electron transfer to the soluble electron acceptor ferredoxin. This work demonstrates the incorporation of surface assembled cyanobacterial trimeric PSI complexes into a biohybrid system for light-driven current generation. Specifically, this work demonstrates the improved assembly of PSI via electrophoretic deposition, with controllable surface assembled PSI density, on different self-assembled alkanethiol monolayers. Using artificial electron donors and acceptors (Os(bpy)2Cl2 and methyl viologen) we demonstrate photocurrent generation from a single PSI layer, which remains photoactive for at least three hours of intermittent illumination. Photoelectrochemical comparison of the biohybrid systems assembled from different alkanethiols (hexanethiol, aminohexanethiol, mercaptohexanol, and mercaptohexanoic acid) reveals that the PSI generated photocurrent is enhanced by almost 5 times on negatively charged SAM surfaces as compared to positively charged surfaces. These results are discussed in light of how PSI is oriented upon electrodeposition on a SAM.

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INTRODUCTION Photosynthesis has evolved into a highly efficient and advanced mechanism of converting solar energy to chemical energy in plants, algae, and some bacteria. Photosystem I (PSI) is one of two light harvesting pigment proteins in the oxygenic photosynthetic pathway, and functions as a biological photodiode for the unidirectional transport of electrons across the thylakoid membrane.1 Cyanobacterial PSI is a supramolecular trimeric protein complex, with each monomer possessing 12 different protein subunits, 96 light harvesting chlorophyll a molecules, and 1 electron transfer chain.2 Upon illumination, excitons are rapidly transferred from the antennae chlorophyll to the special pair, P700 (a chlorophyll a dimer), where charge separation takes place. This phenomenon drives the transfer of an electron through PSI’s internal electron transfer chain, from P700 (located on the luminal face) to the terminal iron−sulfur center, FB (located on the stromal face of PSI), with an internal quantum efficiency near unity.3,4 This high efficiency is unmatched by any man-made photoelectronic device,5 making PSI an ideal candidate for the improvement of solar devices using biomaterials. The integration of PSI and other reaction center proteins6−10 into chemical or electrical energy generating devices has become a key area of investigation. PSI has been isolated on a range of surfaces11−15 with the help of self-assembled monolayers (SAMs)16−22 or polymer matrices23 with most of © 2013 American Chemical Society

these reports showing that PSI retains its activity on surfaces, depending on the underlying composition. However, in nature, electron transport to PSI is assisted by charge carrier proteins, either plastocyanin or cytochrome c6.24 Without the dynamic docking of this charge carrier protein in surface assembled PSI systems, the P700 site remains generally inaccessible to the surrounding environment due to outer protein chains on PSI. Thus, photoactivity in these surface assembled systems is severely limited by the impaired transfer of electrons from the electrode to PSI. As a result, the photoactivity of PSI on these surfaces is quite low, and researchers have resorted to applying large amounts of PSI in order to acquire higher photocurrent magnitudes.25 In this work, a technique is presented for the successful integration of cyanobacterial PSI monolayer films and SAM modified electrode surfaces using an osmium based redox mediator, Os(bpy)2Cl2.27 The influence of various electrode surface modifications on PSI assembly and photoelectrochemistry is also demonstrated. As shown in Scheme 1a, alkanethiol SAMs were first formed on the surfaces of freshly evaporated Au electrode surfaces. Four alkanethiol molecules, hexanethiol (HT), aminohexanethiol (AHT), mercaptohexanol (MHO), Received: November 9, 2012 Revised: January 16, 2013 Published: February 4, 2013 2412

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Scheme 1. Schematic of PSI Assembly on Electrodes Modified with Alkanethiolsa

(a) Gold electrodes are first modified with alkanethiols to form SAMs, followed by (b) PSI assembly. (c) Light induced electron transfer associated with surface assembled PSI on SAM modified electrodes. Os(bpy)2Cl2 and methyl viologen (MV2+) are used in solution as redox mediators. (d) Os(bpy)2Cl2 and (e) MV2+chemical structures. (f) Energy level diagram relating redox potentials of mediators and PSI.26 The Os(bpy)2Cl2 and MV2+ energy levels were measured experimentally via cyclic voltammetry. a

excessive amounts of surface assembled PSI by using non-native redox mediators.

and mercaptohexanoic acid (MHA), were utilized in order to provide a range of surface functionalities. PSI was then assembled on freshly prepared SAMs (PSI/SAM) via electrophoretic deposition, as shown in Scheme 1b. Finally, as shown in Scheme 1c, photoelectrochemistry was used to test the photoactivity of PSI/SAM electrodes. The experiment was conducted in a three electrode cell with the Os(bpy)2Cl2 redox mediator (Scheme 1d) and the redox mediator methyl viologen (MV2+, Scheme 1e) present in solution. In this test, Os(bpy)2Cl2 transfers the electron between the electrode and the P700 site in PSI. The methyl viologen (MV2+) mediator is a redox couple which is reduced when it scavenges electrons from the FB site of PSI and oxidized at the Pt counter electrode (Figure 1f). This study demonstrates the enhanced assembly of PSI on Au electrode surfaces via electrophoretic deposition, where the amount of surface assembled PSI was easily tailored, as compared to adsorption. Also, this work clearly shows the effect of surface modification on PSI assembly, where PSI bound in higher densities to hydrophilic uncharged or negatively charged SAMs (MHO, MHA respectively). The photoactivity of surface assembled PSI was investigated, where photocurrent magnitude was related to the amount of surface assembled PSI and the type of surface modification. Finally, the longevity of the PSI/ SAM electrode system was analyzed, where PSI/SAM electrodes were stable and improved over time, for at least three hours of illumination. Combined, these results demonstrate that significant photocurrent can be achieved without the need for

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MATERIALS AND METHODS Materials. Silicon wafers were acquired from WRS Materials (San Jose, CA), and indium tin oxide (ITO) coated glass slides (15−25 Ω resistivity) were acquired from Cytodiagnostics (Burlington, ON, Canada). The 200 proof ethanol, methyl viologen (MV2+), hexanethiol (HT), mercaptohexanol (MHO), mercaptohexanoic acid (MHA), and 2-(N-morpholino)ethanesulfonic acid (MES) were purchased from Sigma Aldrich and used as received. Aminohexane thiol (AHT) was purchased from Dojindo Molecular Technologies, Inc. (Rockville, MD). Preparation of Gold Substrates and SAMs. Gold substrates were prepared by evaporating Ti (50 Å) and Au (3000 Å) in sequence on either silicon wafers (for AFM imaging purposes only) or ITO coated glass slides (for electrochemical characterization). Flattened Au surfaces (silicon only) were prepared by intermittent flaming. Au/ITO substrates were not flattened prior to use. Self-assembled monolayers (SAMs) were formed by immersing the Au substrate in 1 mM thiol in 200 proof ethanol for 36 h. The substrates were then rinsed with ethanol and dried with N2 gas. PSI Isolation from Thermosynechoccus elongatus. PSI was isolated from the thylakoid membranes of the thermophilic cyanobacteria Thermosynechoccus elongatus (T.e.) as described previously.28 Briefly, T.e. cells were exposed to lysozyme for 2 h at 37 °C, and then lysed via French press. Unlysed cells were removed via centrifugation and the thylakoid membranes 2413

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reproducibility. Electrophoretic deposition was performed by applying a potential of −2 V for 5 min (unless otherwise specified), where the SAM/Au electrode functioned as the anode. PSI/SAM electrodes were thoroughly washed with deionized water and dried with N2 gas. PSI/SAM electrodes were used immediately or stored at 4 °C in a dark box until use. Atomic Force Microscopy. PSI assembly on flattened Au or flattened SAM/Au electrodes was measured via tapping mode atomic force microscopy (AFM) (Veeco) using TAP150-Al tips (Budget Sensors). Osmium Mediator Synthesis. Os(bpy)2Cl2 was synthesized using a method previously described by Habermüller et al.30 Briefly, a 20 mL solution of 100 mM 2,2′-bipyridyl and 50 mM K2OsCl6 in dimethylformamide (DMF) was stirred and refluxed in a round-bottom flask for 1 h. The KCl precipitate was filtered out of solution. A 250 mL portion of diethyl ether was added dropwise to the remaining liquid phase while stirring, and then stirred for an additional hour. The resulting precipitate was filtered and dried. The filtrate was resuspended in a 15 mL solution of 2:1 by volume DMF/methanol. A 100 mL solution of 57 mM Na2S2O4 was slowly added to the resuspended solution in an ice bath while stirring. After filtering, the filter cake was washed with deionized water, ethanol, and methanol, and then dried in vacuum overnight. The dried Os(bpy2)Cl2 complex was then diluted with water to a concentration of 27 μM, as the complex was sparingly soluble in water. The solution was diluted to 13.5 μM for use in photoelectrochemical tests. Photoelectrochemical Analysis. Photoelectrochemical measurements were conducted using a Gamry Reference 600 potentiostat (Warminster, PA) in a three electrode configuration with an Ag/AgCl (3 M KCl) reference electrode, and Pt wire counter electrode in a quartz cell. The electrolyte was an aqueous solution of 0.1 M MgCl2, containing 250 μM methyl viologen (MV2+) and 13.5 μM Os(bpy)2Cl2. The applied potential in the cell was −0.1 V versus Ag/AgCl. The applied potential was selected on the basis of a series of photoelectrochemical tests of a SAM/PSI electrode measured at a range of applied potentials (data not shown.) The −0.1 V applied potential resulted in the highest photocurrent magnitude; thus, this applied potential was selected for further experiments. The sample was illuminated by 1.4 mW/cm2 incident light produced by a Newport lamp and filtered by a 676 nm band-pass filter (to target chlorophyll’s long wavelength absorbance maximum, Figure S5a). The illuminated electrode area was 1 cm2. All photocurrent measurements were aerobic, based on observations by Badura et al.23

Figure 1. PSI assembly on unfunctionalized flattened gold electrodes via (a) 5 min adsorption, and (b) 5 min, (c) 30 min, and (d) 60 min electrophoretic deposition.

collected. The membranes were washed with 3 M NaBr, and the surfactant N-dodecyl-β-D-maltoside (DDM) (Glycon Biochemicals, Luckenwalde, Germany) was added to stabilize the protein. The supernatant was then purified via a 10−30% linear sucrose gradient (10 mM CaCl2, 10 mM MgCl2, 20 mM MES pH 6.4, and 0.03% w/v DDM). The lower green band was collected and purified using anion exchange chromatography via HPLC. PSI was used at a final concentration of 0.1 mg/mL in a supporting solution of 200 mM MES, 0.03% DDM, at pH 7.4. It is important to note that all PSI handling occurred in the dark in order to prevent photodegradation of the protein. PSI Deposition. PSI was deposited onto SAM/Au electrodes via electrophoretic deposition in a two electrode cell using a Gamry Reference 600 potentiostat (Warminster, PA).29 The SAM/Au electrode (working electrode) was immersed in the 0.1 mg/mL PSI solution in a 1 cm cuvette with a Pt wire electrode (counter and reference electrodes). The working electrode and Pt wire were spaced 1 cm apart for deposition

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RESULTS AND DISCUSSION PSI Surface Assembly. The surface assembly of cyanobacterial PSI on unfunctionalized Au electrodes is simple and tunable through adsorption or electrophoretic deposition, as shown in the AFM images of Figure 1. First, in the case of PSI adsorption, freshly evaporated Au electrodes were flattened via intermittent flaming, incubated at room temperature in PSI solution for 5 min, and then thoroughly rinsed with deionized water and dried with nitrogen gas. As shown in Figure 1a, PSI adsorbed to the bare Au surface in low densities and assembled either stromal or luminal side down (or a mixture of conformations) as confirmed by the cross-sectional size and shape of the PSI complex. It is important to note that cyanobacterial PSI, used here, naturally exists as a trimer measuring approximately 7 nm tall and 22 nm in diameter.2,31 2414

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This size measurement was confirmed via AFM as shown in Figure S1. In order to increase PSI binding to the unfunctionalized Au electrodes, electrophoretic deposition was employed by applying a −2 V potential (vs Pt wire) in a two-electrode cell.29 Figure 1b,c,d) show 5, 30, and 60 min electrophoretic depositions, respectively, of PSI on flattened Au electrodes. The 5 min electrophoretic deposition, Figure 1b, showed an increased amount of PSI on the surface (∼315 PSI/μm2), as compared to the 5 min adsorption (∼115 PSI/μm2). The orientation of the PSI trimer on the electrode surface is of high importance, as the electron transfer within the protein is unidirectional from luminal side to stromal side. Therefore, for electron transfer from the electrode to PSI, it would be ideal for all of the PSI trimers to be oriented luminal side down. While the exact orientation of the PSI complex on the Au surface remains open to future investigation, it is noted that results are consistent with the proposed assembly of the PSI trimers, either stromal or luminal side down (see Figure S1). This is in contrast to previous studies of plant PSI on derivatized Au surfaces21 where a mixture of conformations was obtained. This is likely due to the structural differences between the plant form of PSI, which exists as an ellipsoid shaped monomer,32 and the disc shaped cyanobacterial PSI trimer. Analysis of the crystal structure of the PSI complex shows a very strong dipole, running from luminal surface to stromal surface, and oriented at a moderate angle to the complex central axis (and the surface normal) (Figure S2).2,33,34 It is therefore likely that PSI orients itself in the electric field applied during electrophoretic deposition. This observation, combined with the AFM measurements of PSI trimer size (Figure S1), gives further credence to the hypothesis of stromal or luminal face orientation on the bare Au surface. Next, longer electrophoretic deposition times resulted in increased PSI surface assembly, with the 30 min electrophoretic deposition (Figure 1c) reaching near monolayer coverage. The 60 min electrophoretic deposition formed multilayers of PSI, as evidenced by the tall plateaus of PSI found in the AFM images in Figure 1d. Combined, the results in Figure 1 show that PSI assembly is enhanced (by almost 3 times) using electrophoretic deposition, as compared to adsorption, with longer deposition times resulting in higher amounts of PSI on the surface Despite the clear advantage of increased PSI assembly using electrophoretic deposition, the use of bare Au surfaces is not optimal for protein assembly, as specific surface functionalities could enhance PSI assembly and stability. Thus, PSI assembly was next examined on alkanethiol self-assembled monolayer (SAM) modified Au electrodes, as shown in the AFM images in Figure 2. In general, as the alkyl chain length of an alkanethiol SAM increases, the SAM packing becomes more dense and organized.35 These highly organized SAMs block the electrode surface more significantly and limit or even prevent electron transfer through the SAM under a low voltage bias. In this work, the SAM blocks the electron donor Os(bpy)2Cl2 from directly interacting with the electrode, and Os(bpy)2Cl2 is reduced at the SAM surface instead. Thus, a long chain SAM could hinder electron transfer from the electrode to the surface of the SAM, and in turn could hinder electron transfer to Os(bpy)2Cl2 and then to PSI. For this reason, six-carbon alkyl chain length SAMs were utilized because the length was short enough to allow for sufficient electron transfer through the SAM, yet long enough to form organized layers. SAMs were

Figure 2. PSI assembly on SAM modified gold electrodes via 5 min electrophoretic deposition on (a) hexanethiol (HT), (b) aminohexanethiol (AHT), (c) mercaptohexanol (MHO), and (d) mercaptohexanoic acid (MHA).

formed on flattened Au electrodes by 36 h immersion in 1 mM thiol ethanolic solution. PSI was then assembled via 5 min electrophoretic deposition, as above (see Figure 1b). First, Figure 2a shows the results of 5 min electrophoretic deposition of PSI on hexanethiol (HT) SAMs. The AFM image clearly shows that negligible amounts of PSI (1−5 PSI/μm2) assembled on the highly hydrophobic surface, as was previously reported.20 Next, Figure 2b shows 5 min PSI electrophoretic deposition on aminohexanethiol (AHT) SAMs, where PSI binding was enhanced (∼830 PSI/μm2) as compared to the 5 min depositions on HT (Figure 2a) and bare Au (∼315 PSI/ μm2 Figure 1b). Figure 2c,d shows the electrophoretic deposition of PSI onto mercaptohexanol (MHO) (∼875 PSI/ μm2) and mercaptohexanoic acid (MHA) (∼925 PSI/ μm2), respectively. Analysis of the PSI crystal structure localizes the hydrophobic regions largely to the sides of the protein complex, in keeping 2415

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with its function as an integral membrane protein complex (Figure S3).24 However, the PSI trimer complexes with surfactant in solution, minimizing the hydrophobic characteristics of the protein surface.36 Thus, it is expected that the PSI complex binds in low density to the hydrophobic HT electrode surface, and in higher density to the hydrophilic electrode surfaces (AHT, MHO, MHA), again with a preferred stromal or luminal surface orientation. This phenomenon has also been observed in prior work by other groups.18,20,22 Combined, the results presented here show that PSI bound in highest density to the negatively charged surface (MHA) or the polar surface (MHO).20 This likely occurred because the stromal and luminal faces of PSI are positively charged in order to act as binding sites for cytochrome c6 and ferredoxin.3 Additionally, the luminal face of the PSI complex is composed of many polar residues. This is visualized in Figure S4. Once again, PSI assembly on these surfaces are likely to be predominantly oriented either stromal or luminal side down. PSI Photoelectrochemistry. Upon illumination of PSI, photons induce electron−hole pair separation, resulting in the oxidation of the P700 special pair (electron acceptor site) and the reduction of FB (the electron transfer chain terminus). This phenomenon induces the transfer of a single electron from P700 to FB through the electron transfer chain.3 However, it is well-known that re-reduction of the photogenerated hole at the P700 site is rate limiting without the specific docking of the native electron donor cytochrome c6, and that removal of the electron from the terminal FB site requires the docking of ferredoxin in the native system. Alternatively, the use of the redox mediators Os(bpy)2Cl2 and methyl viologen (MV2+) are employed in this work to facilitate in electron transfer to P700+ site and away from the FB site, respectively (Figure 1f). The photoactivity of surface assembled PSI was analyzed electrochemically by examining the PSI induced photocurrent, as shown in Figure 3. For this, the PSI/SAM electrodes were illuminated with 676 nm light for 60 s on/off cycles for a total of 5 min, at a fixed potential of −0.1 V (vs Ag/AgCl) in the presence of the redox mediators Os(bpy)2Cl2 and MV2+. Figure 3a shows the photocurrent measurements in the presence of both Os(bpy)2Cl2 and MV2+ for a SAM with no PSI (black dashed line), and 5 min electrophoretic depositions of PSI on HT (red line), AHT (orange line), MHO (green line), and MHA (blue line). The control sample is a blank MHA SAM, which had undergone a 5 min “electrodeposition” treatment with MES buffer and no PSI. The blank sample shows no current increase when the electrode was exposed to red light. Although a blank MHA SAM is shown here, HT, AHT and MHO SAMs were also tested similarly in the absence of PSI, and none of them showed a current increase upon exposure to light (data not shown). The PSI/HT sample shows a small light driven current jump of ∼2 nA, and the PSI/AHT sample shows a higher photocurrent magnitude of ∼17 nA. The highest PSI density samples, PSI/MHO and PSI/MHA, gave much higher photocurrent magnitudes of ∼60 and ∼75 nA, respectively. It was expected that the PSI/HT sample did not generate significant photocurrent, as very few PSI trimers were bound to this surface (Figure 2a). It was also expected that the PSI/ MHO and PSI/MHA samples generated the most photocurrent, as these samples had the highest amount of surface assembled PSI. The PSI/MHO and PSI/MHA electrodes generated approximately 4.5 and 5 electrons/s per PSI, respectively. Interestingly, the PSI/AHT electrode only gave about 1.2 electrons/s per PSI, which was much lower than the

Figure 3. PSI generated photocurrent on HT, AHT, MHO, and MHA SAMs in the presence of (a) Os(bpy)2Cl2 and MV2+, (b) Os(bpy)2Cl2 only, and (c) MV2+ only. Note: current/time plots in this figure are offset for comparison purposes.

PSI/MHO and PSI/MHA samples. Since the amount of PSI on the AHT surface (Figure 3b) was comparable to that of MHO and MHA, this implies that the amount of PSI on the surface is not the only factor affecting photocurrent magnitude, and that the characteristics of the surface may also play a role. A possible factor could be that the Os(bpy)2Cl2 possesses an overall neutral charge when reduced, and a positive charge when oxidized. Since oxidation occurs at the P700 site, the Os 2416

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complex possesses a positive charge which may influence its rate of reduction by the SAM modified electrode. In other words, the negatively charged SAM surface of MHA could attract the Os complex, whereas the positively charged AHT surface would repel the Os(bpy)2Cl2 complex following its oxididation during the re-reduction of P700+. Additionally, the negative charge of the MHA surface likely attracts the luminal or stromal faces of PSI, resulting in improved association of PSI to the surface. Finally, the luminal face of PSI is composed of many polar residues, resulting in improved and possibly oriented binding of PSI to MHO. In Figure 3b, the photocurrent of the PSI/HT, PSI/AHT, PSI/MHO, and PSI/MHA samples were measured in the presence of Os(bpy)2Cl2 only (no MV2+). Figure 3b clearly shows that, despite the SAM type or assembled PSI amount, there is little measurable photocurrent. This is likely due to the inability of PSI to rid the FB site of photogenerated electrons in the absence of ferredoxin or MV2+, therefore prohibiting the continued movement of electrons through the electron transfer chain. Similarly, the photocurrent of these samples in the presence of MV2+ only (no Os(bpy)2Cl2) is shown in Figure 3c. Low photocurrent magnitudes are observed for all samples, indicating the poor transfer of electrons from the electrode surface to the P700 site in the absence of the Os(bpy)2Cl2 mediator. Importantly, the control samples (no PSI) in Figure 3 show no significant photocurrent in the absence of PSI. This indicates that the photocurrents observed in this work were not generated by photoinduced electron transfer between the two redox mediators. Additionally, the absorbance spectra of Os(bpy)2Cl2, shown in Figure S5b, indicates that Os(bpy)2Cl2 does not absorb significantly at 676 nm, and thus does not contribute directly to photocurrent. Combined, the results shown in Figure 3 demonstrate the importance of the two redox mediators incorporated in this study; Os(bpy)2Cl2 and MV2+. In the absence of MV2+, electrons were not effectively removed from the terminal FB site on PSI. Similarly, in the absence of Os(bpy)2Cl2 electrons were not effectively transferred from the electrode to the P700 site. However, in the presence of both redox mediators, significant photocurrent magnitudes were measured for monolayers of PSI on various SAMs, where photocurrent magnitude corresponds well to PSI density and SAM characteristics. The exact nature of the interaction between the PSI complex and the Os(bpy)2Cl2 complex is poorly understood. A computational study is currently underway to probe this phenomenon in atomistic detail. Preliminary results demonstrate considerable flexibility of the ligands around the central Os to accommodate docking to the PSI surface, as well as a sufficiently varied electrostatic surface to ensure attachment on some portion of the PSI luminal (or stromal) face. The longevity of photocurrent derived from surface assembled PSI was examined next, as shown in Figure 4. In this experiment, the photocurrent test was performed for the PSI/MHO and PSI/MHA samples (as described above in Figure 3) with longer on/off light cycles of one hour each, for a total of six hours. The PSI/MHO and PSI/MHA samples both showed approximate photocurrent magnitudes of 35 nA during the first hour of illumination, which fluctuated slightly over the full hour. Current fluctuations and noise over the course of the entire six hour test are likely due to environmental variances, such as heat fluctuations.

Figure 4. Photocurrent longevity test of PSI assembled on MHO and MHA modified electrodes via 5 min electrophoretic deposition. Note: current/time plots in this figure are offset for comparison purposes.

Interestingly, the magnitude of photocurrent increased with each illumination. The PSI/MHO sample showed increased photocurrents of approximately 50 and 62 nA, during the second and third hours of illumination, respectively. Similarly, the PSI/MHA sample showed photocurrent magnitudes of 70 and 80 nA during the second and third hours, respectively. This phenomenon could possibly be related to the electron transfer between the SAM surface and the P700 site of PSI, which relies on the diffusion of the Os(bpy)2Cl2 molecule (in the case of luminal surface orientation) to the region between the SAM surface and PSI. The binding between PSI and the MHA or MHO surface is strong, due to hydrogen bonding, leaving PSI tightly bound to the SAM surface. Thus, the transport of Os(bpy)2Cl2 to the SAM/PSI interface may be diffusion limited, possibly resulting in increased photocurrent magnitude over time as more Os(bpy)2Cl2 is able to diffuse into the PSI/ SAM interface. The electrochemical cell temperature did not fluctuate throughout this experiment, and remained approximately constant at room temperature (22 °C), therefore negating the temperature effects that could have affected the electron transfer kinetics over time. Importantly, the PSI photocurrent did not decrease during the time scale tested, clearly indicating that the PSI films are stable for long periods of illumination.

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CONCLUSIONS The information summarized in this report demonstrates the effective and controllable assembly of active single layers of PSI on electrode surfaces. First, the improved deposition of PSI via electrophoretic deposition was shown, where PSI binding was increased by almost 3-fold on unfunctionalized Au surfaces, as compared to assembly via adsorption. Next, the assembly of PSI on four SAMs (HT, AHT, MHO, and MHA) was demonstrated. PSI assembled in high density to the uncharged or negatively charged hydrophilic surfaces (MHO, MHA) and in slightly lower amount to the positively charged surface (AHT). PSI binding was negligible on the hydrophobic HT surface. 2417

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The generation of photocurrent in PSI single layer films was also demonstrated in this report, where the use of the redox molecule Os(bpy)2Cl2 was essential to the transfer of electrons from the electrode to the P700 site in PSI. Similarly, the redox mediator MV2+ was vital in the removal of photogenerated electrons from the terminal FB site. Photocurrent magnitude corresponded with PSI quantity in the case of the MHO and MHA surfaces, but showed significantly reduced turnover rates in the AHT system (4.5 and 5 electrons/s per PSI in MHO and MHA, versus 1.2 electron/s per PSI in AHT). This implies that photocurrent magnitude was not driven solely by the amount of PSI on the surface, but also by the underlying surface composition. Finally, the longevity of this active biofilm was demonstrated, where PSI monolayers were stable and active for at least three hours of illumination. Photocurrent magnitude increased over time for both the MHO and MHA electrodes, likely due to the increased amount of Os(bpy)2Cl2 molecules that were able to diffuse to PSI over time. Combined, these results demonstrate the effect of surface composition on PSI assembly and photoactivity.

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ASSOCIATED CONTENT

S Supporting Information *

PSI size measurement via AFM, chlorophyll a and Os(bpy)2Cl2 absorbance spectra, PSI dipole vector, and PSI surface characteristics. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the U.S. Department of the Army and U.S. Army Materiel Command are gratefully acknowledged. Research was supported, in part, by a contractual appointment to the U.S. Army Research Laboratory Postdoctoral Fellowship Program administered by the Oak Ridge Associated Universities (A.K.M., D.R.B., S.S.P.). B.D.B. was partially supported by a NSF grant, EPS-1004083.

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ABBREVIATIONS PSI, photosystem I; Os, Os(bpy)2Cl2; MV2+, methyl viologen; SAM, self-assembled monolayer; HT, hexanethiol; AHT, aminohexanethiol; MHO, mercaptohexanol; MHA, mercaptohexanoic acid

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