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J . Phys. Chem. 1990, 94, 6151-6153
Vectorial Photocurrents and Photoconductivity in Metalized Chloroplastst E. Greenbaum Oak Ridge National Laboratory.$ P.O. Box 2008, Bldg. 4500N, Oak Ridge, Tennessee 37831 -61 94 (Received: April 19, 1990)
A novel photobiophysical phenomenon was observed in isolated spinach chloroplasts that were metalized by precipitating colloidal platinum onto the surface of the thylakoid membranes. A two-point irradiation and detection system was constructed in which a continuous-beam helium-neon laser (A = 632.8 nm) was used to irradiate the platinized chloroplasts at varying perpendicular distances (Figure 1) from a single linear platinum electrode in pressure contact with the platinized chloroplasts. No external voltage bias was applied to the system. The key objective of the experiments reported in this report was to measure the relative photoconductivity of the chloroplast-metal composite matrix. Unlike conventional photosynthetic electrochemical cells, in which irradiated chloroplasts are in close proximity to an electrode or linked to the electrode by an electrode-active mediator, the flow of photocurrent was through the biocomposite material. A sustained steady-state vectorial flow of current in the plane of the entrapped composite from the point of laser irradiation to the wire electrode was measured. The absolute value of the measured photocurrent and the maximum perpendicular distance between the point of irradiation and the linear electrode for which sustained current could be measured depended on the experimental procedure used to prepare the platinized chloroplasts. Figure 2 and Table 1 summarize the data reported in this report.
Photosynthesis is vectorial photochemistry. Light quanta that are captured in photosynthetic reaction centers initiate a primary electron-transfer reaction, resulting in spatial separation of electrostatic charge across the photosynthetic membrane. The vectorial nature of photosynthesis lies in the intrinsic orientation of the reaction centers embedded in the membranes. Electron flow is from the inner membrane surface of the flattened saclike vesicles to the outer surface.’ In normal photosynthesis, electrons from the reducing end of photosystem I are used for the enzymatic reduction of NADP+ to NADPH. This reduction is mediated by ferredoxin and ferredoxin NADP-reductase. NADPH serves as the electron carrier to the Calvin cycle for the enzymatic reduction of atmospheric carbon dioxide to plant matter. It has been shown that colloidal platinum can be precipitated onto the surface of photosynthetic membranes so that water is photobiocatalytically split into molecular hydrogen and oxygen upon illumination with light of any wavelength in the chlorophyll absorption spectrum.* Since no electron mediator such as ferredoxin or methyl viologen was present, the colloidal platinum must have been precipitated sufficiently close to the photosystem I reduction site to allow interfacial electron transfer from the membrane to the platinum. The presence of a reticulated network of platinum particles embedded in the chloroplast matrix suggests that metallike properties can, at least partially, be imparted to the chloroplasts. In this work, the relative photoconductivity of the material was measured by the flow of photocurrent in the plane of the entrapped platinized chloroplasts of uniform thickness from the point of laser irradiation to the linear platinum wire electrode. Figure I is a schematic illustration of the experimental arrangement. The helium-neon laser and the front surface reflecting mirror are mounted on a linear translation stage whose position is controlled by a stepper motor. Figure 1 is a cross section of the entrapped platinized chloroplasts and linear electrode, viewed parallel to the axis of the electrode. D is the perpendicular distance from the electrode to the point of irradiation of the laser beam, whose diameter was approximately 0.4 mm. Light scatter was reduced to negligible proportions with neutral density filters. This was verified both by visual inspection with the dark-adapted eye and with a calibrated silicon photodiode. Type-C chloroplasts were prepared according to the procedure of Reeves and in which the chloroplast envelope is osmotically ruptured, exposing the photosynthetic membranes to the external aqueous medium. Solutions of chloroplatinic acid neutralized to pH 7 with NaOH ‘Research sponsored by the Division of Chemical Sciences, Office of Basic Energy Sciences, US.Department of Energy, and the Materials Laboratory, Wright-Patterson Air Force Base. *Operated by Martin Marietta Energy Systems, Inc., for the U S . Department of Energy under Contract DE-AC05-840R21400.
TABLE I: Vectorial Photocurrents and Electron-Transfer Distances in Platinized ChloroDlasts as a Function of Platinum Loading line no. I,,? nA D,,., mm [Pt]) mg/mL 1 0 0 0.24 2 3 4
0 3.6
7.8
,
0.49 0.97
1.94
“ I 0 is defined as the measured photocurrent when D = 0. This corresponded to the laser beam position when it was directly over the linear electrode and was the maximum current measured in each run. bAqueous-phaseplatinum concentration from which the platinum precipitation step was performed. Lines 1 and 2 are the average of two runs each. Line 3 is the average of three runs. Line 4 is the average of six runs.
were prepared. The amount of precipitated platinum was varied by adjusting the aqueous-phase concentration of hexachloroplatinate. The temperature of the platinized chloroplast film assembly was held constant with a water-jacketed cell connected to a constant temperature bath. The current was measured with a Keithley Model 485 picoammeter. Table I presents data for four platinum concentrations. A 5-mL suspension of spinach chloroplasts (containing 3 mg of chlorophyll) was used. Platinization of chloroplasts is feasible because hexachloroplatinate can be converted to metallic platinum at pH 7 and room t e m p e r a t ~ r e . ~These are experimental conditions that preserve photoactivity of the isolated chloroplasts. The platinized chloroplasts were entrapped on a thin, fiberglass filter pad (Millipore, AP40) and were moistened with Walker’s assay medium,s in which the chloroplasts were suspended. The fiberglass filter pad was 0.3 mm thick, had an active filtration area of 10.4 cm2, and contained no binder resins. The thickness of the chloroplast film was estimated to be between 0.01 and 0.1 mm. A silver-silver chloride reference electrode was placed in pressure contact with the filter; a straight, single, platinum wire of 0.2-mm diameter was placed in pressure contact with the entrapped platinized chloroplasts. The electrodes, platinized chloroplasts, and filter paper were held together with Lucite plates and compression screws. To prevent the electrochernistry of atmospheric oxygen from interfering with cathode reactions by providing an alternative electron pathway, the entire assembly was placed in a small glass chamber sealed with an O-ring. The O-ring was pierced to allow Amesz, J., Ed. Photosynthesis; Elsevier: Amsterdam, 1987. Greenbaum, E. Science 1985, 230, 1373-1375. Reeves, S. G.; Hall, D. 0. Methods Enzymol. 1980, 69, 85-94. Anderson, J. R. Structure of Metallic Catalysts; Academic Press: New 1975. (5) Walker, D. A. Methods Enzymol. 1980, 69, 94-104.
(1) (2) (3) (4) York,
0022-3654/90/2094-615 1%02.50/0 , 0 1990 American Chemical Society I
0 2.3 3.4
6'152 The Journal of Physical Chemistry, Vol. 94, No. 16, 1990
Long-Distance Photoelectron
Letters Photoconductivity of Platinized Chloroplasts
Transfer
In Platinized Chloroplasts t
Percolatlon Model
*
SURFACE MIRROR
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Wire Electrode 1
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FILTER PAPER
__REFERENCE ELECTRODE
a 0- v o
-D-
Figure 1. Schematic illustration of the experimental apparatus used to measure the relative photoconductivity of platinized chloroplasts. The figure is a cross-sectional view of the apparatus viewed parallel to the linear platinum wire electrode. D is the perpendicular distance from the point of laser irradiation to the electrode. The helium-neon laser and
front surface mirror are rigidly mounted to a linear translation stage that was used to vary the distance. The filter paper was impregnated with KCI-containing electrolyte. The reference electrode was a silver-silver chloride electrode in pressure contact with the filter paper. The output of the helium-neon laser was attenuated with a neutral density filter so that the photocurrent response was in the linear region of the light saturation curve. This figure is not drawn to scale. In particular, the thickness of the filter paper in relation to the platinized chloroplasts and silver-silver chloride reference electrode is exaggerated. 12,
,
I
,
I
'
I
'
'
'
I
J
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0
-i
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1.0
1.5
2.0
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3.0
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4.0
4.5
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DISTANCE (mm)
Figure 2. Photocurrent vs distance in platinized chloroplasts. The general shape of the photocurrent vs distance curve of Figure 1 was similar to that for most of the entries in Table I; however, the numerical values for Io and D varied from run to run as indicated in Table I. 1, was the value of the photocurrent when the laser beam was positioned directly over the linear platinum electrode. The maximum value of D corresponded to the distance a t which the photocurrent fell to approximately 3% of I,.
the passage of two narrow-diameter wires for establishing electrical contact with the electrodes. Premoistened helium gas flowed through the chamber to flush out atmospheric oxygen. After about 45 min, the oxygen concentration of the chamber was below 3 ppm as measured by a calibrated Hersch electrogalvanic cell. Calibration was achieved with an in-line electrolysis cell and Faraday's law of electrochemical equivalence. The platinum precipitation step was performed in a waterjacketed reactor cell containing 8.0 mL of suspension maintained at 20 OC. Molecular hydrogen was passed over the head space of the reactor while a Teflon-coated magnetic stirrer was used to gently stir the chloroplast suspension in a neutral hexachloroplatinate solution. Purge times of 30-60 min were used. After incubation and precipitation, the reactor chamber was opened to air, the contents were filtered onto the filter pad, and the cell was assembled as described previously. This coprecipitation step was essential. The novel observation in this research is the effect of the precipitated platinum on the photoconductivity of the chloroplast matrix. Figure 2 illustrates the current vs distance profile for a sample that contained an initial platinum concentration of 1.94 mg of Pt/cm3. Each data point represents a steady-state flow of current. Although steady state was-achieved within-a few minutes of each change in the laser beam position, sustained photocurrent could be observed for hours. Each data point represents a dwell time of 15-20 min.
"
c> ' (ti-
Reaction Center
Figure 3. Schematic illustration of the flow of photocurrent in the platinized chloroplast film. A model in which the platinum colloid particles form a reticulated pathway from the point of irradiation to the linear wire electrode is shown.
Figure 2 shows that the photocurrent had a maximum value when the laser beam was directly over the platinum wire. Although the laser beam was partially blocked by the wire electrode when the beam was positioned directly over it, the loss of light was not sufficient to cause a drop in photocurrent. This observation is reasonable since the diameter of the laser beam is larger than the diameter of the platinum wire electrode. Also, the close physical proximity of the laser light and wire on the umbral periphery of the wire provided for efficient charge collection. The extent to which the laser beam could be moved from the wire and still generate measurable photocurrent depended on the concentration of the solution from which the platinum was precipitated. Figure 3 is a schematic illustration of the flow of photocurrent in the platinized chloroplast film. Table I is a summary of the data of initial currents and maximum distances that were observed for varying distances. A systematic study of photocurrents vs platinum loading is beyond the scope of this report; it is being pursued with additional experiments. The origin of the photocurrent can be understood as follows: It has previously been shown that colloidal platinum can make electrical contact with the reducing end of photosystem I of photosynthesis. This contact was demonstrated by (1) the photocatalytic evolution of molecular hydrogen2 and (2) the observation of photocurrent in a sandwichlike photobioelectrochemical Upon illumination, the platinum electrode in pressure contact with the platinized chloroplasts swung negative with respect to the silver-silver chloride electrode that was in pressure contact with the electrolyte-impregnated filter pad. No external bias was placed on the electrodes to force the direction of photocurrent flow. A reasonable model for these results, based on the generally accepted structure of photosynthetic membranes,' is that colloidal platinum precipitated onto the external surface of the thylakoid membranes forms an isopotential surface whose distance is determined by the connectivity of the reticulated colloidal particles that are in the metallic component of the chloroplast-metal composite matrix. This distance is a statistical parameter whose average value is determined by the nature of the platinum precipitation process. When platinum is precipitated, it does so in a nonspecific manner on the external surface of the thylakoid membranes. There is, however, an electrostatic interaction between the negative charge of the hexachloroplatinate ions and the local positive charge of the lysine residues constituting part of the polypeptides of the photosystem I proteins.s The experimental system described in this report differs qualitatively from prior research performed with photosynthesis-based bioelectrochemical cells that utilized various organelles and components to generate photocurrents. Examples of prior ( 6 ) Greenbaum, E. Bioelecrrochem. Bioenerget. 1989, 21, 171-177. (7) Marder, J . B.; Barber, J. Plant, Cell Enuiron. 1989, 12, 595-614. (8) Colvert. K . K.; Davis, D. J. Arch. Biochem. Biophys. 1983, 225, 936-943.
J . Phys. Chem. 1990, 94, 6153-6156 research include chlorophyll liquid crystals:*1° pigmented bilayer membranes,” chloroplasts,I2 chloroplast membra ne^,'^-'^ algae,16 and photosynthetic bacterial reaction centers.I7J8 For example, a photosystem 11 enriched submembrane fraction in a photoelectrochemical cell operated in potentiostatic mode was used by Lemieux and Carpentier to generate photocurrent^.'^ The cell included artificial electron acceptors acting as charge-transfer mediators between the photosynthetic membrane and the working electrode. Trissl and Kunze took another approach to generating and measuring photoelectric signals; they studied primary electrogenic reactions in chloroplasts probed by picosecond flash-induced dielectric polarization.20 Seibert and Kendall-Tobias measured photoelectrochemical properties of electrodes coated with photoactive-membrane vesicles isolated from photosynthetic bacteriaS2’ In their work chromatophores, isolated from the (9) Aizawa, M.; Hirano, M.; Suzuki, S.J. Membr. Sci 1978,4,251-259. (IO) Aizawa, S.; Hirano, M.; Suzuki, S. Electrochim. Acta 1979, 24, 89-94. ( I I ) Tien, H. T. Photochem. Photobiol. 1976, 24, 97-1 16. (12) Haehnel, W.; Hochheimer, H . J. Eioelectrochem. Eioenerget. 1979, 6, 563-574. (13) Allen, M. J.; Curtis, J . A,; Kerr, M. W . Eioelectrochem. Eioenerget. 1974, 1 , 408-417. (14) Allen, M. J.; Crane, A. E. Bioelectrochem. Bioenerget. 1976, 3, 85-91, ( 1 5) Allen, M. J. In Liuing Sysfems us Energy Converfers; Buret, R., Allen, M. J., Massue, J . P., Eds.: North Holland: New York, 1977; pp 271-274. (16) Ochiai, H.; Shibata, H.; Sawa, Y.; Katoh, T. Proc. Natl. Acad. Sci. U . S . A . 1980. 77. 2442-2444. (17) Drachev; L. A,; Kondrashin, A. A,; Samuilov, V. D.; Skulachev, V. P. FEES Left. 50, 219-222. ( I 8) Janzen, A. F.; Seibert, M. Nature 1980, 286, 584-585. (19) Lemieux, S.;Carpentier, R. J. Photochem. Photobiol. B: Biology 1988. 2. 221-231. (20) Trissl, H. W . ;Kunze, U. Eiochim. Eiophys. Acta 1985,806, 136-144. (21) Seibert, M.; Kendall-Tobias, M. W . Eiochim. Eiophys. Acta 1982, 681, 504-511.
6153
photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, were prepared as a film or tin oxide electrodes, and the response to red light was examined in a liquid junction photoelectrochemical cell. Alexandrowicz and Berm measured photovoltages in chloroplast extract bilayer membranes stimulated by micromolar amounts of oxidants and reductants.22 The distinguishing feature of the work presented in this report is that the planar composite matrix of precipitated platinum and chloroplast membranes is the conductive medium. That is to say, unlike chloroplast suspensions coupled to electrodes by redox-active mediators (there are no mediators in the preparation) or chloroplast preparations or films in close physical proximity to the electrodes, the composite photobioelectronic material itself is the photoconductive pathway. It was demonstrated that the concentration of the solution from which the platinum was precipitated directly affects the relative photoconductivity of the sample. In conclusion, it has been demonstrated that electrical contact with the reducing end of photosystem I was achieved by precipitating colloidal platinum in the presence of isolated chloroplasts. The presence of the platinum had a significant effect on the photoconductivity of the metal-biological composite material. This work is technologically significant because the photosynthetic reaction centers are nanometer structures with picosecond switching times. This work demonstrates that the electrontransport chain of photosynthesis can be electrically contacted and that the larger structural matrix of the platinized chloroplasts demonstrates enhanced photoconductivity.
Acknowledgment. I thank B. Z. Egan for comments and criticism of the manuscript. I also thank J. P. Eubanks for technical support and D. J. Weaver and S. A. Hoglund for secretarial assistance. (22) Alexandrowicz, G.; Berns, D. S . Photobiochem. Phorobiophys. 1980, I , 353-360.
Rotationally Mediated Intramolecular Vibrational Redistribution in Jet-Cooled trans-Ethanol at 2990 cm-‘ Jungsug Go, G. A. Bethardy, and David S . Perry* Department of Chemistry, The University of Akron, Akron, Ohio 44325-3601 (Received: April 24, 1990) Rotationally resolved infrared absorption spectra of jet-cooled ethanol reveal extensive mixing of the zero-order methyl C-H stretch vibration with bath rotational-vibrational states. The fragmentation of single rotational lines into a multiplet of molecular eigenstates is the spectroscpic signature of intramolecular vibrational redistribution (IVR). The number of resolved molecular eigenstates increases from about 2 at J ’ = 0 to about 20 at J’ = 4 but is independent of K,’. The density of spectral features suggests that the IVR explores both the gauche and the trans forms of ethanol and that K,’ is not a good quantum number.
Intramolecular vibrational redistribution (IVR) is evident in high-resolution spectra as the splitting of individual rotationalvibrational lines into a multiplet of lines.’+ Zero-order levels may be mixed with bath states by anharmonic, Coriolis, or centrifugal coupling.’ When such mixing occurs, the transition ( I ) Abramson, E.; Field, R. W.;Imre, D.; Innes, K. K.;Kinsey, J. L. J. Chem. Phys. 1984,80, 2298. (2) Dai, H. L.; Korpa, C. L.; Kinsey, J. L.: Field, R. W . J. Chem. Phys. 198582, 1688. Dai, H . L.; Field, R. W.;Kinsey, J. L. J. Chem. Phys. 1985, 82, 2161. (3) Guyer, D. R.; Polik, W. F.; Moore, C . B. J. Chem. Phys. 1986, 84, 65 19. (4) Lehmann, K. K.; Coy, S.L. J . Chem. Soc., Faraday Trans. 2 1988, 84. 1389. ( 5 ) de Souza. A. M.: Kaur, D.; Perry, D. S.J. Chem. Phys. 1988,88,4569; Eer. Bunsen-Ges. Phys. Chem. 1988, 92, 424. (6) Mcllroy, A.; Nesbitt, D. J . J. Chem. Phys. 1990, 90, 2229.
intensity to the zero-order state is distributed among a number of molecular eigenstates, resulting in a multiplet or “clump” of lines in the spectrum. Anharmonic coupling can be distinguished from the rotationally mediated mechanisms such as Coriolis and centrifugal coupling by the dependence of the coupling parameters on the rotational quantum numbers, J’and KL,of the vibrationally excited state.2 In the acetylenic C-H stretch region of 1-butyne, a multiplet of lines was observed at J‘ = 0 and similar multiplets were assigned to J’ = 1 and 2, which points to an anharmonic coupling mechanis” This conclusion has been recently confirmed by McIlroy and Nesbitt.6 Preliminary work in the methyl C-H stretching region of 1-butyne is also consistent with an anharmonic coupling mechanism.8 We present here a rotationally resolved (7) Knight, A. E. W. In Excited Stares; Lim, E. C., Innes, K. K., Eds.; Academic: New York, 1988; Vol. 7, pp 1-78.
0022-3654/90/2094-6153S02.50/00 1990 American Chemical Society
