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

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 (His(7)) 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.

[1]  H. Frank,et al.  Electrochemical reactions of redox cofactors in Rhodobacter sphaeroides reaction center proteins in lipid films. , 2001, Bioelectrochemistry.

[2]  Alan S. Haas,et al.  Cytochrome c and Cytochrome c Oxidase: Monolayer Assemblies and Catalysis , 2001 .

[3]  Rolf D. Schmid,et al.  Specific binding of photosynthetic reaction centres to herbicide-modified grating couplers , 1993 .

[4]  L. Jeuken.  Conformational reorganisation in interfacial protein electron transfer. , 2003, Biochimica et biophysica acta.

[5]  J. Deisenhofer,et al.  Photophysics of photosynthesis. Structure and spectroscopy of reaction centers of purple bacteria , 1997 .

[6]  P. Dutton,et al.  Langmuir-Blodgett monolayer films of bacterial photosynthetic membranes and isolated reaction centers: preparation, spectrophotometric and electrochemical characterization. , 1991, Biochimica et biophysica acta.

[7]  Itamar Willner,et al.  Integration of a Reconstituted de Novo Synthesized Hemoprotein and Native Metalloproteins with Electrode Supports for Bioelectronic and Bioelectrocatalytic Applications , 1999 .

[8]  C. Kirmaier,et al.  Charge separation in a reaction center incorporating bacteriochlorophyll for photoactive bacteriopheophytin , 1991, Science.

[9]  T. Mattioli,et al.  Structure, spectroscopic, and redox properties of Rhodobacter sphaeroides reaction centers bearing point mutations near the primary electron donor. , 1993, Biochemistry.

[10]  T O Yeates,et al.  Structure of the reaction center from Rhodobacter sphaeroides R-26: membrane-protein interactions. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Rolf D. Schmid,et al.  The photoreaction center of Rhodobacter sphaeroides: a ‘biosensor protein’ for the determination of photosystem-II herbicides? , 1997 .

[12]  M. Gunner,et al.  Trapping conformational intermediate states in the reaction center protein from photosynthetic bacteria. , 2001, Biochemistry.

[13]  G. Feher,et al.  X-ray structure determination of the cytochrome c2: reaction center electron transfer complex from Rhodobacter sphaeroides. , 2002, Journal of molecular biology.

[14]  J. Miyake,et al.  Self-assembling photosynthetic reaction centers on electrodes for current generation. , 2000, Applied biochemistry and biotechnology.

[15]  W. Mäntele,et al.  Electrochemical redox titration of cofactors in the reaction center from Rhodobacter sphaeroides , 1991, FEBS letters.

[16]  C. Nakamura,et al.  Rapid and specific detection of herbicides using a self-assembled photosynthetic reaction center from purple bacterium on an SPR chip. , 2003, Biosensors & bioelectronics.

[17]  H. Michel,et al.  Refined crystal structures of reaction centres from Rhodopseudomonas viridis in complexes with the herbicide atrazine and two chiral atrazine derivatives also lead to a new model of the bound carotenoid. , 1999, Journal of molecular biology.

[18]  V. Shuvalov,et al.  COUPLING OF PHOTOINDUCED CHARGE SEPARATION IN REACTION CENTERS OF PHOTOSYNTHETIC BACTERIA WITH ELECTRON-TRANSFER TO A CHEMICALLY MODIFIED ELECTRODE , 1989 .

[19]  Y. Yasuda,et al.  Displacement current measurements of the dynamic charge transfer of photosynthetic reaction centers in monolayer LB films , 1997 .

[20]  M. Gunner.  The Reaction Center Protein from Purple Bacteria: Structure and Function , 1991 .

[21]  J. Kong,et al.  Differentiating the orientations of photosynthetic reaction centers on Au electrodes linked by different bifunctional reagents. , 2002, Biosensors & bioelectronics.

[22]  S. Boxer,et al.  Rapid isolation of bacterial photosynthetic reaction centers with an engineered poly-histidine tag , 1996 .

[23]  V. Sundström,et al.  Light in elementary biological reactions , 2000 .

[24]  R. Strongin,et al.  Vectorially oriented membrane protein monolayers: profile structures via x-ray interferometry/holography. , 1994, Biophysical journal.

[25]  Kazuhiro Nomura,et al.  Immobilization of Photosynthetic Reaction Center Complexes onto a Hydroquinonethiol-Modified Gold Electrode , 1999 .

[26]  Electron transfer in gel-immobilized photosynthetic reaction centers , 1997 .

[27]  J. Miyake,et al.  Control of protein orientation in molecular photoelectric devices using Langmuir—Blodgett films of photosynthetic reaction centers from Rhodopseudomonas viridis , 1994 .

[28]  D. Waldeck,et al.  Charge-transfer mechanism for cytochrome c adsorbed on nanometer thick films. Distinguishing frictional control from conformational gating. , 2003, Journal of the American Chemical Society.

[29]  J. Deisenhofer,et al.  Structure of the protein subunits in the photosynthetic reaction centre of Rhodopseudomonas viridis at 3Å resolution , 1985, Nature.

[30]  E. Katz,et al.  Photobioelectrodes on the basis of photosynthetic reaction centres. Study of exogenous quinones as possible electron transfer mediators , 1992 .

[31]  G. Feher,et al.  Structure and function of bacterial photosynthetic reaction centres , 1989, Nature.

[32]  S. Zaitsev,et al.  Polymer ultrathin films with immobilized photosynthetic reaction center proteins , 1996 .

[33]  Eugenii Katz,et al.  Application of bifunctional reagents for immobilization of proteins on a carbon electrode surface: Oriented immobilization of photosynthetic reaction centers , 1994 .

[34]  Roberto Pilloton,et al.  A biosensor for the detection of triazine and phenylurea herbicides designed using Photosystem II coupled to a screen-printed electrode. , 2002, Biotechnology and bioengineering.

[35]  Rolf D. Schmid,et al.  Herbicide biosensor based on photobleacing of the reaction centre of Rhodobacter sphaeroides , 1993 .

[36]  James F. Allen,et al.  Specific alteration of the oxidation potential of the electron donor in reaction centers from Rhodobacter sphaeroides. , 1994, Proceedings of the National Academy of Sciences of the United States of America.