Shuichi Ajiki

Oriented immobilization of bacterial photosynthetic membrane.

SummaryWe have examined a method for oriented immobilization of photosynthetic membrane fragments on a solid surface by specific avidin-biotin interaction. Photosynthetic membrane fragments from the purple non-sulphur photosynthetic bacterium Rhodopseudomonas viridis, of which the H-subunit of the photosynthetic reaction centre was biotinylated, was immobilized on an avidin-adsorbed plate. Orientation of the immobilized membrane on the plastic plate was checked by an antisera binding assay that could react to the respective sides of the membrane: the H-subunit side was selectively adsorbed on the plate. Light-induced potential and current responses could be measured when the membrane immobilized on the SnO2-coated glass plate was dried and sandwiched with a counter electrode of Hg. The electrical response in the immobilized membrane was much improved in comparison with the control (membranes were simply adsorbed on the plate), supporting the idea that the membranes have an orientation on solid surfaces.

Thermal stability of dried photosynthetic membrane film for photoelectrodes

Abstract Thermal stability was examined on a photoelectrode made from dried film of a photosynthetic membrane (chromatophore) isolated from a photosynthetic bacterium Rhodopseudomonas viridis . Below 60°C, neither the optical absorbance of the film nor its photoelectric response were affected when heated for 1 h. When the temperature was raised over 120 °C, both the absorbance and the response were lost. The temperature of half-denaturation of the chromatophore was 90 °C. X-ray diffraction of the film showed that heating did not cause the complete denaturation of the proteins of chromatophores. Heating did not destroy the secondary structure ( α -helix) of the proteins but the bundles of α -helices were distorted.

Light-induced electrical responses of dried chromatophore film: effect of the addition of cytochrome c

Chromatophores, photosynthetic membranes of photosynthetic bacteria, were isolated and cast on a transparent electrode to form films. The absorption spectrum of the film was similar to that of the native chromatophore. This indicated that the chromophore-protein structures were maintained through drying. Electrical transients of the film on the light pulsed excitation were examined. The amplitude and decay of the electrical transient of the film were altered by the addition of horse heart cytochrome c to the chromatophore of Rhodobacter sphaeroides R-26, which lacks the reaction centre-bound cytochrome c. The addition of cytochrome c into the film resulted in an increase in the light-induced current and a decrease in current decay.

Light-induced electrical response of chromatophore film in a semi-wet photocell with an agar layer containing an electron mediator

Abstract Light-induced electrical response (open-circuit voltage and short-circuit current) was measured for chromatophore films in a semi-wet photocell. Chromatophores, which are membrane fragments of photosynthetic organelle, were isolated from a photosynthetic bacterium, Rhodopseudomonas viridis, and were immobilized on a substrate electrode by an electrodeposition method. The semi-wet photocell was chromatophore film layer and an agar layer containing an electron mediator, K4[Fe(CN)6], sandwiched between two ITO electrodes. The photoresponses were enhanced by a factor of 1000 when an electron mediator added to the chromatophore layer: the open-circuit voltage and the short-circuit current were 120 mV and 450 μA cm−2, respectively.

Reconstitution and immobilization of photo-reaction units from photosynthetic bacterium Rhodopseudomonas viridis

Abstract Photo-reaction units (PRUs) were solubilized from chromatophores of the photosynthetic bacterium Rhodopseudomonas viridis under high salt concentration using Triton X-100 as a detergent. The absorption spectrum of isolated PRUs was similar to that of native chromatophores. Isolated PRUs were composed of reaction centers (RCs) and light-harvesting (LH) proteins. Purified PRUs were reconstituted into liposomes. The Q Y absorption bands derived from bacteriochlorophyll (BChl) b in LH subunits of PRUs were red-shifted from 1000 nm in the isolated PRUs solution to 1006 nm in the reconstituted liposomes, contrasting with the 1015 nm for native PRUs in chromatophores. To make a photo-electrochemical conversion layer for photocell or biosensor, we deposited PRU liposomes on a solid substrate by electrodeposition. The orientation angle between the molecular axis of PRUs and the disk normal was 2.4° for the deposited PRU liposomes, compared with 20.5° for purified chromatophores deposited. This demonstrates that the vertical alignment of protein molecules with the disk normal was superior for electrodeposited protein molecules immobilized using PRU liposomes compared with those using purified chromatophores.

Topological characterization and immobilization of a chromatophore membrane from Rhodopseudomonas viridis for application as a photoelectrical device

Abstract We characterized topological orientation of bacterial photosynthetic membranes (chromatophores) from Rhodopseudomonas viridis using antibodies against H- and C-subunit of photosynthetic reaction center protein (RC). About 40% of the membranes were unsealed vesicles in the inside-out orientation exposing the cytoplasmic side whereas 20% of those were unsealed vesicles in the right side-out orientation expo-sing the periplasmic side. The residual 20% was in the form of a flat sheet structure. We immobilized the chromatophore membranes onto an indium–tin-oxide (ITO) electrode by the electro-deposition method. The electro-deposited film (ED film) showed photocurrent and photovoltage between different electrodes such as ITO and gold (Au). Addition of buffer salts such as sodium phosphate enhanced very much the photoelectrical response in the ED film.