Orientated binding of photosynthetic reaction centers on gold using NiNTA self-assembled monolayers

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Abstract

Coupling of photosynthetic reaction centers (RCs) with inorganic surfaces is attractive for the identification of the mechanisms of interprotein electron transfer (ET) and for possible applications in construction of photo- and chemosensors. Here we show that RCs from Rhodobacter sphaeroides can be immobilized on gold surfaces with the RC primary donor looking towards the substrate by using a genetically engineered poly-histidine tag (His7) at the C-terminal end of the M-subunit and a NiNTA terminated self-assembled monolayer (SAM). In the presence of an electron acceptor, ubiquinone-10, illumination of this RC electrode generates a cathodic photocurrent. The action spectrum of the photocurrent coincides with the absorption spectrum of RC and the photocurrent decreases in response to the herbicide, atrazine, confirming that the RC is the primary source of the photoresponse. Disruption of the NiNTARC bond by imidazole leads to about 80% reduction of the photocurrent indicating that most of the photoactive protein is specifically bound to the electrode through the linker.

Introduction

The construction of functionalized substrates and electrodes using photosynthetic reaction centers (RCs) has attracted growing interest for their possible application in various chemo- and biosensor devices, (Brewster et al., 1995, Jockers et al., 1993a, Koblizek et al., 2002, Nakamura et al., 2003, Peters et al., 1997, Tamura et al., 1994) as well as in different types of photochemical cells (Hara et al., 1997, Kalabina et al., 1996, Katz et al., 1989, Nakamura et al., 2000, Tamura et al., 1991, Tamura et al., 1992, Yasuda et al., 1997). RCs are very efficient in light energy conversion with a quantum yield near 100% (Feher et al., 1989, Kirmaier et al., 1991, Sundsrom, 2000) making them potentially useful for photovoltaic devices and power sources. Additionally, RCs have both an optical and electrical response for herbicide detection with an internal herbicide sensor domain at the QB binding site for N′-(3,4-dichlorophenyl)-N,N-dimethylurea (DCMU), atrazine, and other chemicals widely used in agriculture (Jockers et al., 1993a, Jockers et al., 1993b; Koblizek et al., 2002, Nakamura et al., 2003) and their sensitivity can be considerably improved by RC genetic modifications (Lancaster and Michel, 1999). Furthermore, studies of the interaction of RC with electrodes are a good model for the analysis of mechanisms of interprotein electron transfer (ET) (Haas et al., 2001, Jeuken, 2003, Khoshtariya et al., 2003, Willner et al., 1999).

The advantages of using RCs from purple non-sulfur bacteria for electrode functionalization are in their simplicity since their molecular structure is well determined by X-ray crystallography (Allen et al., 1987, Deisenhofer et al., 1985, Lancaster and Michel, 1999) and they have a broad spectral sensitivity (from UV to near IR). These RCs are composed of three polypeptides H, L, and M. The polypeptides L and M, each forming five transmembrane α-helices, are arranged in two-fold symmetry, whereas the H polypeptide is located at the cytoplasmic surface and binds to both L and M. A dimer of bacteriochlorophylls, so called the primary donor (P), two monomer bacteriochlorophylls (BA and BB), two bacteriopheophytins (HA and HB), two quinones (QA and QB) and one iron are all non-covalently bound to the polypeptides and arranged into symmetrical branches.

Upon absorption of light, P is raised to its excited singlet state (P*), followed by electron transfer from the primary donor to the primary quinone (QA) along the L branch. This process occurs in ∼200 ps at room temperature and forms the charge separated state P+QA(Fig. 1). In about 100 μs, the electron is transferred from QA to QB. The oxidized end of RC can accept electrons from cytochrome complexes and reduced quinone (QB) becomes an electron donor (Feher et al., 1989, Gunner, 1991, Hoff and Deisenhofer, 1997, Kirmaier et al., 1991, Sundsrom, 2000).

A prerequisite for utilizing RC for device applications is the requirement to immobilize the protein on a surface while retaining its natural function. Moreover, to be effective most or all protein molecules must have the same desired orientation. Several strategies have been utilized for oriented immobilization of RC on electrodes, including electrostatic interactions of the polar H-subunit with the substrate surface; (Yasuda et al., 1994) sulfhydryl reactive self-assembled monolayers (SAMs) for immobilization through a single protein surface exposed thiol (Cys) group in the H-subunit of Rhodobacter sphaeroides (Katz, 1994) or SAMs containing nitrilotriacetic acid charged with Ni2+ (NiNTA) for immobilization through genetically engineered poly His tag in the H-subunit of the same bacterium (Nakamura et al., 2000). In all these cases the constructed surfaces have the RC primary donor looking to the solution and primary acceptor side looking to the electrode. In some cases to get a reasonable photocurrent in this configuration, the authors have to introduce an insoluble mediator, unbiquinone-50, between RC and the electrode (Katz, 1994).

In the present work, we immobilize RC on an electrode in the opposite configuration with the acceptor side looking to the solution. To this end we bound RC from R. sphaeroides to NiNTA terminated self-assembled monolayers on gold substrates through a poly-histidine tag (His7) constructed at the C-terminal end of the RC M-subunit (Goldsmith and Boxer, 1996). Though gold is known to quench pigment photoexcited states, we chose it as a substrate for the advantage of well known and defined alkanethiols/gold surface chemistry. Contrary to the previous studies, our orientation provides the primary donor looking to the electrode surface and mimics the RC configuration in its native complex with cytochrome (Axelrod et al., 2002) and thus potentially could improve the efficiency of electron transfer between RC and the electrode. Our results demonstrate that despite the location of the primary donor in close proximity to gold this configuration retains RC’s photochemical activity and photo-induced current at the electrode.

Section snippets

Materials

All reagents were used as received. Atrazine was purchase from Supelco. Nα,Nα-bis(Carboxymethyl)-l-lysine hydrate (lysine-NTA) was purchased from Fluka. 2,3-Dimethoxy-5-methyl-6-geranyl-1,4-benzoquinone (ubiquione-10) was obtained from Sigma. Dithiobis[succinimidylpropionate] (DSP) was purchased from Pierce. Gold on silica substrates (500 Å, Continuous Coating) was supplied by Asylum Research.

Protein purification

The poly-histidine tagged RCs were expressed and isolated from R. sphaeroides strain SMpHis constructed

Results and discussion

The scheme of RC immobilization is illustrated in Fig. 1A. As an electron acceptor (A) we used water-soluble ubiquinone-10 which has a redox potential closed to the internal ubiquinone (QB) (Fig. 1B) and thus can effectively accept electrons from RC (Katz, 1994). The concentration of the quinone was chosen to be high enough to exclude limitations of electron transfer at the RC acceptor side and sufficient enough for competition with the back reaction from QB to the oxidized primary donor (P+)

Conclusions

In conclusion, our results demonstrate that photosynthetic RCs can be immobilized on gold with the primary donor looking to the substrate. In this configuration the protein retains its photochemical and ET activity and the close location of the primary donor to the gold does not quench the RC photoexcited state. Moreover, this configuration generates a significant photocurrent. Thus the NiNTA terminated SAM on gold provides a useful platform for the immobilization of RC for the creation of

Acknowledgements

We thank J. Allen (ASU) for providing us with the R. sphaeroides clone having a His7 tag at the C-terminal end of the M-subunit. We appreciate E. Gantt and F. Cunningham (UMd) for the opportunity to perform some steps of protein purification in their lab. We appreciate F. Ligler, D. Lowy, and W. Dressick (NRL) for reading the manuscript and fruitful comments. This work was supported by ONR and DARPA. J. Zullo summer internship was supported by NRC.

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