Photocurrent Generation by Photosynthetic Purple Bacterial Reaction

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Photocurrent generation by photosynthetic purple bacterial reaction centers interfaced with a porous antimony-doped tin oxide (ATO) electrode Anne-Marie Carey, Haojie Zhang, Daniel G Mieritz, Alex M. Volosin, Alastair T. Gardiner, Richard J. Cogdell, Hao Yan, Dong-Kyun Seo, Su Lin, and Neal W. Woodbury ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07940 • Publication Date (Web): 30 Aug 2016 Downloaded from http://pubs.acs.org on September 3, 2016

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ACS Applied Materials & Interfaces

Photocurrent generation by photosynthetic purple bacterial reaction centers interfaced with a porous antimony-doped tin oxide (ATO) electrode Anne-Marie Carey,* §,‡ HaoJie Zhang, Ϩ,‡ Daniel Mieritz, Ϩ Alex Volosin, Ϩ Alastair T. Gardiner, ϸ Richard J. Cogdell, ϸ Hao Yan, Ϩ,Ϯ Dong-Kyun Seo, Ϩ Su Lin, §, Ϩ and Neal W. Woodbury §, Ϩ

§

Biodesign Center for Innovation in Medicine at Biodesign Institute, Arizona State University,

Tempe, AZ 85287 USA; Ϩ

School of Molecular Sciences, Arizona State University, Tempe, AZ 85287-1604 USA;

ϸ

Institute of Molecular Cell and Systems Biology, University of Glasgow, Glasgow G12 8QQ,

Scotland, UK Ϯ

Biodesign Center for Molecular Design and Biomimetics at Biodesign Institute, Arizona State

University, Tempe, AZ 85287 USA

‡

These authors contributed equally to this work.

ACS Paragon Plus Environment

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KEYWORDS Reaction center; Electron Transfer; Cytochrome c; Photocurrent; Porous electrode; Antimonydoped tin oxide (ATO)

ABSTRACT

The ability to exchange energy and information between biological and electronic materials is critical in the development of hybrid electronic systems in biomedicine, environmental sensing and energy applications. While sensor technology has been extensively developed to collect detailed molecular information, less work has been done on systems that can specifically modulate the chemistry of the environment with temporal and spatial control.

The bacterial

photosynthetic reaction center represents an ideal photonic component of such a system in that it is capable of modifying local chemistry via light driven redox reactions with quantitative control over reaction rates and has inherent spectroscopic probes for monitoring function. Here a wellcharacterized model system is presented, consisting of a transparent, porous electrode (antimonydoped tin oxide) which is electrochemically coupled to the reaction center via a cytochrome c molecule. Upon illumination, the reaction center performs the 2-step, 2-electron reduction of a ubiquinone derivative which exchanges with oxidized quinone in solution. Electrons from the electrode then move through the cytochrome to reoxidize the reaction center electron donor. The result is a facile platform for performing redox chemistry that can be optically and electronically controlled in time and space.


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INTRODUCTION Generating a facile interface between redox-active biochemical complexes and electronic materials is challenging for a number of reasons. The attachment of biological complexes to surfaces is often nonspecific and unstable, with the current exchange limited due to a poor connection to the electrode, and such systems are frequently difficult to assemble, control and monitor. Historically, the majority of work interfacing redox proteins with conductive materials has focused on pyrolitic graphite and alkanethiol/alkylthiol-modified gold

1-2

.

Limitations

arising from monolayer deposition, denaturation of the protein(s) of interest and the lack of optical transparency of the substrate (which precludes both spectroscopic analysis and limits optoelectronic applications) prompted the development of transparent (semi)conductive oxides (TCOs)

3

.

TCOs were a significant step forward allowing spectroelectrochemistry of

immobilized redox proteins and, from an applied perspective, electrochromic and optoelectronic applications of the biohybrid system. becoming increasingly available

3-5

Novel nano- and mesostructured porous TCOs are

. The 3D architecture of porous TCO materials greatly

increases the effective surface area (hundreds of fold) and provides protective cavities for association with macromolecules such as proteins or DNA. This serves to both concentrate the redox protein of interest (and thus increase the optical density for spectroscopic investigations and applications) and to promote functional stability by providing protective encapsulation

5-9

.

Although proteins have been incorporated into other porous transparent conductive oxides, such as WO3TiO2 and SnO2 6-9, the excellent transparency and optical properties of porous indium tin oxide (ITO) have made it the favored TCO

4, 10-11

. Incorporation of high concentrations of the

small redox protein cytochrome c into ITO has been reported (on the order of hundreds of


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pmol/cm2 rather than the tens of pmol/cm2 reported for monolayer studies), with the redox properties intact. Porous antimony-doped tin oxide (ATO) is now emerging as a preferred alternative to porous ITO due to its lower cost and very high optical transparency and conductivity 12-14. The incorporation of functionally intact DNA nanostructures into ATO pores has been demonstrated

12-13

. More recently the redox proteins cytochrome c and azurin were

successfully interfaced with the porous ATO surfaces and shown to retain their redox activity 1415

. In Kwan et al. (2011), the authors reported extremely high loading of cytochrome c into

porous ATO (over 1000 pmol/cm2 for cytochrome c, an order of magnitude greater than that reported previously for cytochrome c in porous in ITO) and used spectroelectrochemistry techniques to demonstrate that cytochrome c had retained its redox activity upon adsorption on ATO pore surfaces 14. Photosynthetic reaction centers (RCs) perform light-driven reduction of a terminal acceptor molecule that is then available for subsequent chemistry. This makes them an ideal photonic element for interfacing with electrodes and controlling local redox chemistry in time and space via optical and electronic signals. Because the redox reactions are light driven, as opposed to requiring a substrate, the control of turnover rate is directly and instantaneously connected to light level at a particular time and place. The reaction centers from the bacterium Rhodobacter (Rb.) sphaeroides are well suited for this purpose. The structure of these RCs is known, their electron transfer reactions have been well characterized and they are very stable. Furthermore, Rb. sphaeroides RCs are amenable to protein engineering and easily expressed in large quantities when grown in the dark chemoautotrophically. The conversion of absorbed solar energy into chemical redox energy is initiated in Rb. sphaeroides RCs by excitation of the primary electron donor, P, which is an excitonically


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coupled pair of bacteriochlorophylls

16-18

. A series of electron transfer reactions takes place

down a redox gradient from the excited state of P, through a monomeric bacteriochlorophyll (BA) and a bacteriopheophytin (HA) resulting in reduction of a molecule of ubiquinone, ‘QA’ with near unity quantum yield on a time scale of about 200 ps 16-18. The electron then passes from QA to the second ubiquinone, QB, forming the semiquinone anion of this cofactor. A second photoinduced electron transfer to QB completes the two electron reduction forming a dihydroquinone which leaves the binding pocket and diffuses through the membrane to the cytochrome b/c1 complex where it drives proton pumping and ultimately ATP production. The oxidized primary donor (P+) is then re-reduced via electron transfer from a soluble cytochrome c; cytochrome c docks with the RC and an electron transfer reaction takes place with a rate constant on the order of 10 microseconds

16-18

. The interaction between the RC and cytochrome c is electrostatic, with the

positively charged lysine residues surrounding the heme in cytochrome c interacting with the negatively charged carboxylate groups in the P binding pocket of the RC 19. There have been a number of previous reports of bacterial RCs interfaced with electrodes for use in integrated optical electronic devices, photovoltaics and biosensors (e.g. 20-30). In the field of molecular electronics, for example, RCs isolated from Rb. sphaeroides have been inserted into nanogaps between gold electrodes to act as a switch, which can be controlled by the voltage applied across the electrodes 20. In addition, the sensitivity of RCs to certain herbicides, such as triazines (which block electron transfer by displacement of the secondary quinone) has been used to develop herbicide sensors based on inhibition of the measured photocurrent 21-22. A major issue in many of the previous studies has been the connection between the reaction center and the electrode

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. Lebedev et al. (2006) demonstrated that cytochrome c could be

employed as a redox wire, reducing the electron tunneling distance to P and thus improving the


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connection between P and the electrode surface. Photocurrents generated by HIS-tagged RCs interfaced with an NTA-terminated SAM on a gold electrode were improved by an order of magnitude when cytochrome c was incorporated into the system

31

. Another key limitation is

monolayer deposition of RCs on the electrode surface. Typically, a monolayer of reaction centers can only produce nanoamps of current per cm2 (in terms of a reduced product formed by the RC, 1 nanoamp/cm2 = ~10 femtomoles reduced product per cm2). Lu et al. (2005) and Lukashev et al. (2008) achieved photocurrents on the µA/cm2 scale (picomoles/cm2 of reduced product) by interfacing RCs with a porous titanium oxide electrode (doped with tungsten trioxide in the case of Lu et al. 2005) to achieve multiple layers of RCs 32-33. However, the later studies involved the RC donating an electron to the doped tin oxide electrode (rather than receiving an electron as described below). In the study presented, Rb. sphaeroides RCs are electrochemically coupled to transparent, porous ATO electrode films via cytochrome c to produce a biohybrid platform in which 2electron reduction and release of a soluble quinone can be controlled both optically and electronically.

EXPERIMENTAL SECTION Preparation of ATO film on FTO glass A previously established sol-gel route for producing highly mesoporous ATO coatings was adapted for the fabrication of macroporous ATO coatings

34

. Briefly, 1.00 g of SnCl4·5H2O

(Alfa Aesar, 98%) and 1.00 g of deionized water were added to a solution of 0.05 g SbCl3 (Alfa Aesar, ≥99.9%) in 6.50 g of n-BuOH (J.T. Baker, 99.9%) in an Ultra-Turrax tube drive with a rotor-stator element. A separate mixture containing 12.4 wt.% carbon black, acetylene (Alfa


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Aesar, 50% compressed ≥99.9%, surface area = 75 m2/g, bulk density of 0.095 – 0.103 g/cm3), 25.8 wt.% polyethylene glycol bisphenol A, epichlorohydrin copolymer (PEG) (Sigma-Aldrich, 15-20 kDa) and 61.8 wt.% ±-epichlorohydrin (Fluka, ≥99.9%) was homogeneously blended in a separate bottle. From this mixture, 6.15 g was transferred to the metal salt solution in the tube drive. The tube drive was run on speed 3 for 10 seconds, followed by vigorous shaking. Subsequently, the mixing was carried out with a setting of six for one minute followed by vigorous shaking, and finally the tube drive run on the maximum setting for one minute to produce the reaction mixture. For producing coatings, fluorine-doped tin oxide (FTO, Hartford Glass, sheet resistance 8 Ω/sq.) was washed with soapy water and sonicated in an acetone bath. Two opposing edges were masked by applying double-thick tape (Scotch Transparent Tape) to cover 1 mm of the FTO face, and a single layer of tape was applied to a third edge. About 0.25 mL of the reaction mixture was pipetted on the single tape layer, and was drawn smoothly and quickly across the entire slide length with a Pasteur pipette. After drying for one hour under ambient conditions, the tubes were placed in a tube furnace that was flushed with oxygen for one hour before increasing the temperature to 500 °C. The initially black coatings were calcined at 500 °C for four hours under an oxygen flow to yield translucent, pale blue coatings. Scanning electron micrographs of ATO films are shown in the Supporting Information (Fig. S1).

Incorporation of bacterial reaction centers into ATO films RCs were isolated from anaerobically grown cultures of Rb. sphaeroides 2.4.1 and purified. Harvested cells were re-suspended in 20mM Tris.HCl (pH 8.0) and broken by three passes through a French Press (15,000 kpsi) in the presence of a small amount of DNase and MgCl2.


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Unbroken material was removed by low speed centrifugation (2700 g, 10min) and membranes were pelleted with high speed ultracentrifugation (250,000 g, 90min). Membranes were adjusted to OD 50 and solubilized in 20mM Tris.HCl (pH 8.0) containing 0.25% LDAO, in the presence of 150 mM NaCl to preferentially solubilize the RC from the chromatophores, for 60 min at 20ºC. The solubilized RCs were centrifuged at 250,000 g for 60 min to pellet unsolubilized material and the supernatant was then dialyzed overnight to remove NaCl. Solubilized RCs were purified by ammonium sulphate precipitation followed by anion exchange and size exclusion chromatography. ATO slides were pre-soaked in cytochrome c to facilitate orientated binding of the RC at the electron-accepting P end. In the intracytoplasmic membrane of Rb. sphaeroides cytochrome c docks at the P end of the reaction center and donates an electron to the specialized pair of Bchls to re-reduce the reaction center. It can thus act both as an oriented docking component and as a redox wire, connecting the RC to the ATO electrode as illustrated in Fig. 2 A. Cytochrome c has previously been shown to retain its conformation and redox properties within ATO pores 14, and has been used to orientate RCs on a SAM-coated gold electrode

31

. Lebedev and coworkers

reported a significant improvement in photocurrent when equine cytochrome c was included in their system, which was independent of the initial redox state of the cytochrome c

31

.

Cytochrome c (equine heart, Sigma) was adsorbed to ATO by incubating ATO slides in a solution of 150 µM cytochrome c in nanopure water (18.2 MΩ cm-1) for 30 minutes as described in Kwan et al. (2011). Excess solution was withdrawn by pipette and ATO surfaces were then washed repeatedly with nanopure water and left to air dry under cover. A solution of Rb. sphaeroides RCs was then applied to the ATO films using a pipette and the slides were again left


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to air dry under cover. Steady-state UV–vis absorption spectra (Varian Cary® 50 UV-Vis Spectrophotometer, Agilent) were collected before and after RC application (Fig. 1).

Photocurrent measurements and cyclic voltammetry Photoelectrochemical measurements and cyclic voltammetry (CV) were performed on a CHI 650D electrochemical station electrochemistry workstation (CHI Instrument Co., USA), with a three-electrode system containing a Ag/AgCl (CHI 111) reference electrode and a platinum wire counter electrode. For each experiment, an ATO slide was set as the working electrode. The electrochemical cell was covered with black tape with a 1 cm2 window to allow illumination and the working electrode was inserted such that the ATO side of the slide faced the cell window. ATO slides were illuminated using a Titanium:Sapphire laser (5W Millennia pumped Tsunami, Spectra Physics).

The light intensity immediately outside the cell was recorded using a

FieldMate Laser Power Meter (Coherent) and adjusted using a neutral density filter.

The

electrolyte buffer was 0.1 M Tris buffer (+/- ubiquinone-0). A potential equal to the dark open circuit potential was applied to the ATO slide during each photocurrent measurement and the effect of illumination was recorded.


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RESULTS AND DISCUSSION Cytochrome c is the natural redox partner of the RC from Rb. sphaeroides and exposing the ATO/cytochrome c electrode system (See Kwan et al. 14) to RCs results in the specific and stable assembly of RCs onto the surface. The highly porous architecture of the transparent ATO films allows multiple layers of cytochrome c/RCs, analogous to the invaginated membranes of natural systems. The incorporation of cytochrome c and Rb. sphaeroides RCs was confirmed with UVvis spectroscopy (Fig. 1). The positions and relative intensities of the Qy absorption maxima demonstrated that the RCs retain their conformation and function when assembled in the ATO pores (Fig. 1). Based on the extinction coefficients for cytochrome c (106 mM-1 cm-1 at 410 nm) and the RC (288 mM-1 cm-1 at 802 nm), the spectroscopic surface coverage was calculated to be 2.3 and 0.45 nmol/cm2, respectively, equating to a ratio of approximately 5 molecules of cytochrome c for every one RC. This high incorporation of cytochrome c is on the same order of magnitude as that reported for cytochrome c in porous ATO by Kwan et al. (2011)

14

and an

order of magnitude higher than that reported for porous ITO 5.


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Fig. 1 Steady-state for an ATO slide following soaking in cytochrome c (red) and a cytochrome c-soaked ATO slide following application of RC solution (blue). Data are normalized at the 407 nm Soret peak for cytochrome c. Slides have been left to air dry after each step. Inset shows a photo of an ATO film on FTO glass, following soaking in 150 µM cytochrome c and washing to remove the unbound molecules. Film dimension is 1 cm2.

To characterize the function of the ATO-cyt c-RC system, photocurrents were measured using the electrochemical set-up illustrated in Fig. 2A. Various sample conditions were tested to understand the origin of the photocurrent generated from the ATO-cyt c-RC system and the results are summarized in Figs. 2B and S2. Only slides containing both cytochrome c and RCs


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showed detectable photocurrent (Fig. 2B).

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ATO slides which had been pre-soaked in

cytochrome c but not treated with RCs showed no detectable photocurrent (Fig. S2A). ATO slides which were not first pre-soaked in cytochrome c but treated with RCs also showed no detectable photocurrent (Fig. S2B).

Slides not pretreated with cytochrome c also did not

appreciably bind RCs; the RC absorbance after incubation was tenfold lower than slides that were first pre-soaked in cytochrome c. The ATO-cyt c-RC complexes proved to be extremely stable, with slides still capable of producing photocurrents weeks after initial measurements when stored under darkness at 4 °C.

Fig. 2 A Photosynthetic RCs interfaced with an ATO electrode via a cytochrome c bridge. The cytochrome c serves to both orientate the RC such that the electron-accepting P end is facing the


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electrode and as redox wire to facilitate electron transfer from the electrode to P. In this system, the RC performs one electron photochemistry but generates a two electron carrier (the reduced ubiquinone, which leaves the quinone binding pocket and must be replaced by another ubiquinone molecule from the electrolyte solution). B Photocurrent detected from ATO films containing cytochrome c and RCs, with either 100 µM Ubiquinone-10 (top) or 100 µM Ubiquinone-0 (bottom) in the 0.1 M Tris electrolyte buffer. For the bottom panel, RCs have also been pre-soaked overnight in a 10-fold Molar excess of Ubiquinone-0. Slides were illuminated with a continuous wave Titanium:Sapphire laser set to 800 nm, with an illumination intensity of 5 mW/cm2.

When fully assembled ATO-cyt c-RC slides were illuminated continuously at 800nm and 5 mW/cm2 (light intensities were intentionally kept well below saturation, see below), an initial photocurrent density of 2 µA/cm2 was observed (Fig. 2B and S2C). However, the initial high photocurrent rapidly decreased to a sustained level of ~ 0.5 µA/cm2. The RCs naturally contain ubiquinone-10, which contains a long, hydrophobic isoprenoid tail, and is thus nearly insoluble in aqueous solution. Upon including 100 µM of ubiquinone-0 (no isoprenoid tail and thus considerably more soluble than ubiquinone-10) in the electrolyte buffer, a sustained photocurrent between 1.5 and 2 µA/cm2 was obtained (Figs. 2B and S2D). Apparently, after the initial reduction of bound quinone, the current was limited by the exchange of the reduced quinone with oxidized quinone in the solvent and subsequent migration to the counter electrode. The presence of the more soluble quinone alleviated this limitation, at least in part. The light intensity dependence of the system was determined by varying the power of the continuous, 800 nm Titanium:Sapphire laser illumination incrementally from 1.3 to 53


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mW/cm2), and recording the resulting photocurrent (Fig. 3).

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The photocurrent is half saturated

at about 5 mW/cm2 and largely saturated by 50 mW/cm2 giving a maximum photocurrent of nearly 3 µA/cm2. The light intensities used for the studies reported below (99% of the doubly reduced ubiquinone molecules released by the RC, which have a potential near 0V, SHE, are simply donating their electrons back to the working electrode (which for the experiments described has a potential ≥ 0.1V, SHE). Thus the amount of reduced quinone produced in the vicinity of the working electrode, and thus potentially available for further chemistry, is likely in the many nanomole per cm2 per second range.

CONCLUSION Layering cytochrome c on porous ATO electrodes allows the assembly of RCs onto the surface and provides a specific binding configuration and electrical connection between the electrode and the RC initial electron donor. The high effective surface area of the transparent macroporous electrode greatly increases the number of RCs that can absorb light and generate reduced quinones. The resulting photocurrent (~ 2 µA/cm2 with no applied potential and >10 µA/cm2 when a small negative potential is applied) represents a convenient measure that is proportional to the amount of redox activity taking place. Perhaps most importantly from a systems perspective, each component can be individually and quantitatively controlled and monitored. The level of cytochrome c reduction can be controlled by potential. The rate of reaction center photoexcitation can be controlled by light intensity. The rate of exchange between the final electron acceptor in the reaction center and the soluble quinones which carry electrons to the counter electrode is sensitive to both the concentration of free quinone and to specific inhibitors of the secondary quinone pocket. This represents a prototype for a type of integrated biohybrid system in which 2-electron redox chemistry can be controlled in time and space both optically and electronically.


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ASSOCIATED CONTENT Supporting Information. Scanning electron micrographs of the porous ATO films, Photocurrents detected for control samples (in addition to reaction center samples), Action spectra for the initial phase of photocurrent detected for ATO-cyt c-RC samples (in addition to the stable phase presented in the main text), Potential dependence data (photocurrent detected under different applied potentials) that was used to generate the Nernst curves presented in the main text, Absorption spectra for cytochrome c soaked ATO slides collected under a range of applied potentials (showing the relative differences in the absorption maxima that reflects their varying degrees of cytochrome c reduction). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Anne-Marie Carey; [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS This work was funded by the DOD MURI award W911NF-12-1-0420 and NSF grants MCB1157788; Alastair Gardiner and Richard Cogdell were supported by Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Basic Energy Sciences under Award # DE-SC0001035 and by the Royal Society UK/USA International Exchanges Scheme. We thank Douglas Daniel for his help in constructing the electrochemical cell and for designing and building bespoke housing to allow accurate spectroscopy of our ATO slides.

ABBREVIATIONS RC, reaction center; ATO, antimony-doped tin oxide; ITO, indium tin oxide; TCO, Transparent conducing oxide.


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Table of Contents Graphic


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Photocurrent Generation by Photosynthetic Purple Bacterial Reaction

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