171

Bioelectrochemistry and Bioenergetics, 21 (1989) 171-117 A section of J. Electroanai. Chem, and constituting Vol. 275 (1989) Elsevier Sequoia S.A., Lausarme - Printed in The Netherlands

Biomolecular electronics: observation of oriented photocurrents by entrapped platinized chloroplasts

E. Greenbaum

Chemical Technology Diuision, Oak Ridge National Laboratory Oak Ridge, TN 37831-6194 (U.S.A.)

(Received 24 August 1988; in revised form 10 December 1988)

ABSTILWT

Electrical contact of the electron transport chain of photosynthesis has been achieved by precipitating colloidal platinum onto the surface of thylakoid membranes. This composite metal-biological membrane material was immobilized on filter paper and sandwiched between metal gauze electrodes. Upon irradiation, oriented photocurrents were observed. The direction of the flow of photocurrent was consistent with the vectorial model of photosynthesis, which predicts that the electric potential of the external surface of photosynthetic membranes swings negative with respect to the internal surface. Control experiments indicated that the oriented nature of the observed photocurrents could not be attributed to structural asymmetries associated with electrodes or to light gradients associated with the direction of illumination.

INTRODUCTION

The conversion of light energy into chemical energy by the photosynthetic apparatus occurs in and across membranes that contain specialized reaction centers where the primary photo-induced electron transfer reactions take place. The prevail- ing molecular model of the structure of photosynthetic membranes indicates that the photochemistry associated with photosynthesis is of a vectorial nature. Oriented reaction centers are embedded in the photos~thetic membranes that are composed of grana stacks and stroma lamellae, structures that are topologically equivalent to spheres. Therefore, irrespective of the rotational orientation of the thylakoid mem- branes, photosynthetic electrons emerge from the membrane and enter the stroma region of the chloroplast. The ability to observe sustained photocurrents and photovoltages from chloroplast-based systems depends on the ability of the reduc- ing end of photosynthesis to communicate electrically with an electrode. The key result of the experiments described in this report is the observation of oriented

0302-4598/89/$03.50 0 1989 Elsevier Sequoia S.A.

172

photocurrents from platinized chloroplast membranes that have been entrapped on fiberglass filter paper. What distinguishes these experiments from previous research is the strict symmetric electrode structure used for the construction of the photobio- electrochemical cell and the control experiments, which exclude the effect of light gradients in the photosynthetic tissue.

Numerous ingenious and elegant electrochemical structures have been used previously to measure photovoltages and photocurrents derived from chloroplast- based systems. These structures include working electrodes covered with films of chloroplasts or chloroplast particles [l-7], bacterial membrane vesicles [8] and even living microorganism [9]. They also include more conventional multi-compartment electrochemical cells in which an aqueous suspension of components interacts with electrodes either in the presence or absence of electrode-active mediators [lo-141. However, as pointed out by Becker et al. [15], the photoelectrochemical properties of chloroplast-based devices depends on the details of the way in which the experiment is performed. They have shown that the photovoltage of suspensions of chloroplasts can be attributed to the Dember effect that arises when inhomogeneous light absorption gives rise to a gradient of positive and negative charges along the direction defined by the propagation vector of the light, which is also the direction joining the two electrodes.

The key objective of this communication is to provide a simple demonstration of the inherent vectorial properties of intact photosynthetic thylakoid membranes and to show that the directionality of the flow of photocurrent can only be explained by the orientation of the photosynthetic reaction centers within the membranes. For this purpose, platinized chloroplasts [16] were entrapped on fiberglass filter paper and contacted with a platinum gauze electrode. Simultaneous experiments were performed in which counter electrodes of either platinum or silver/silver chloride were used.

EXPERIMENTAL

Type C chloroplasts were prepared according to the procedure described by Reeves and Hall [17]. In this preparation, the chloroplast envelope is ruptured osmotically, exposing the thylakoid membranes to the external aqueous medium. A solution of chloroplatinic acid (5.34 mg/ml), neutralized to pH 7 with NaOH, was prepared separately. One milliliter of this solution was combined with 5 ml of chloroplast suspension (containing 3 mg of chlorophyll) in Walker’s assay medium [18]. The 6-ml volume was placed in a temperature-controlled, water-jacketed chamber fitted with O-ring connectors to provide a hermetic seal and with inlet and outlet ports for hydrogen flow. The mixture was gently stirred and purged with molecular hydrogen in the headspace above the liquid, and the sample temperature was held at 21 o C.

In the platinum precipitation step, it was determined empirically that a hydrogen incubation time of - 30 min was needed to obtain photoactive material. Times of 60 to 90 min were typically used. As indicated schematically in Fig. 1, after incubation,

173

/pt

Fig. 1. Schematic illustration of entrapped platinized chloroplasts. Colloidal platinum was precipitated

onto the external surface of photosynthetic membranes and entrapped on KCl-impregnated fiber glass

filter paper.

the contents were filtered onto fiberglass filter paper (Millipore, AP40). The platinum precipitation reaction had a marked effect on filtration properties. Whereas control experiments without hydrogen incubation produced a chloroplast mixture that filtered immediately, the platinized chloroplast required a considerably longer time, typically 5 to 30 min. Also, the platinized chloroplasts were dark green, as opposed to the normal bright green of higher plant chloroplasts. The presence of insoluble platinum on the platinized-chloroplast filter paper composition was de- termined by X-ray fluorescence analysis, after the filter paper was rinsed in 2 1 of continuously stirred distilled water for 1 h.

As indicated in Fig. 2, electrical contact with the platinized chloroplasts was achieved by contacting the surface of the filter-paper-entrapped material with a fine-mesh platinum gauze. Either fine-mesh silver/silver chloride gauze or platinum gauze placed on the opposite side of the filter paper was used as the counter electrode. All of the components were held in close proximity in a lucite sandwich-like assembly with nylon compression screws. Platinum wires were used to make electrical contact with the gauzes, and all electrical and gas evolution measurements were performed in a helium atmosphere.

The photoreactions took place in a specially constructed chamber, which allowed the simultaneous measurement of the electrical properties of two platinized chloro- plast disks. Each of these disks had a diameter of approximately 1 cm. In order to avoid electrochemical reactions with atmospheric oxygen, all of the experiments were performed in a helium atmosphere. Two platinized chloroplast disks were placed in the chamber side by side. One disk had a symmetric electrode structure consisting of two platinum gauze electrodes. One electrode, the top electrode in Fig. 2, made direct pressure contact with the platinized chloroplasts, and the second electrode made pressure contact with the bottom of the moist electrolyte-containing filter paper. The second disk assembly in the chamber was identical to the first with the exception that the bottom electrode was a silver/silver chloride gauze screen. The chamber containing the two disks was opaque except for two illumination ports. The ports consisted of glass tubes that extended into the chamber and reached to a depth just above the disks. Fiber optic bundles were inserted into the tubes, and a red cut-off filter was placed between the optical bundle light source and each platinized chloroplast disk. The use of the red cut-off filter served as a control and precautionary measure to ensure that the photocurrents observed were

PLATINUM CONTACT

LIGHT

Fig. 2. Schematic illustration of interface detail of outer membrane interface. Electrical contact with the

reducing end of Photosystem I was made by metal-to-metal pressure contact.

attributed to reactions sensitized by chlorophyll. This was, however, redundant since additional control experiments indicated that no photocurrents were observed in the absence of chloroplasts. Polychromatic illumination gave the same results as those obtained with the red cut-off filters.

The flow of photocurrent from each of the two disks was measured simulta- neously by two Keithley Model 617 electrometers. Four platinum lead wires pierced the O-ring of the main chamber. This allowed for electrical contact with the platinized-chloroplasts-electrode assembly through an external measuring system, while maintaining the assembly in a helium atmosphere. The outputs of the electrometers were used to drive a two-pen chart recorder, which documented the

175

data. Electrical contact between the platinized chloroplasts and the platinum gauze was achieved by pressure contact on the side of the filter paper disk that contained the chloroplasts. Either silver/silver chloride or platinum gauze reference electrodes made electrolytic contact with the electrolyte entrained in the filter paper on the side opposite the chloroplasts.

RESULTS AND DISCUSSION

Figure 3 demonstrates the flow of photocurrent as a function of incident light intensity. Figure 4 illustrates the measurement of simultaneous photocurrents from two disks. The upper trace is for the platinum/silver electrode structure; the lower trace is the plat~~/pla~um electrode structure. There are several noteworthy aspects to the data of Fig. 4. First, it can been seen in Fig. 4 that the direction of the flow of photocurrent from the platinized chloroplast assembly is same for both the plat~um/plat~um and platin~/s~ver electrodes. Second, the orientation of the photocurrent is independent of the direction of incident light. Control experiments were performed in which the sandwich-like cell assembly was irradiated from either direction. Of course, when the cell was irradiated from the “back” side, the light was somewhat attenuated. However, the el~~ol~e-impregnate filter paper is by no means opaque, and photocurrents were easily seen in this mode of irradiation. The key result of this control experiment was that the direction of the flow of photocurrent was the same, irrespective of the direction of incident light. This observation eliminated Dember-like effects associated with light gradients as the origin of the orientation of the photocurrents. Third, the orientation of the mea- sured photocurrent is consistent with the prevailing vectorial model of photosynthe-

12

10

0

0 2 4 6 a 10 12 14 16 18

INCIDENT LIGHT/W k2

Fig. 3. Photocurrent vs. incident light intensity (Pt/Ag electrode assembly). The direction of flow photocurrent was consistent with the generally accepted vectorial model of photosynthesis.

of

H 20 min

Fig. 4. Simultaneous photocurrents as a function of counter-electrode. The direction of flow of

photocurrent was preserved with a symmetric electrode structure. Also, the direction of flow of

photocurrent was independent of the direction of illumination of the assembly.

sis [19]. In this model, the orientation of the photosynthetic reaction centers in the chloroplast membranes is such that electrons move from the inner (lumenal) surface to the outer (stromal) surface under the influence of light. Therefore, the electrical potential of the external surface of chloroplast membranes swings negative with respect to the internal surface upon illumination. The polarity of the flow of photocurrent in Figs. 3 and 4 is such that the platinum gauze electrode in contact with the platinized chloroplasts is negative with respect to the counter electrode. From the method of preparation of the platinized chloroplasts and the detailed view of Fig. 2, it is clear that the gauze electrode that makes pressure contact with the platinum colloid particles also makes contact with at least part of the reducing ends of Photosystem I.

A model for the interfacial reactions and flow of photocurrent follows in a straightforward manner from the previous discussion. Electrons that emerge at the surface of the thylakoid membrane and contact the precipitated platinum can flow into the gauze electrode. These electrons flow to the counter electrode, which in the case of the silver/silver chloride electrode, cause the reaction AgCl + e- + Ag + Cl-. (Presumably, an analogous reaction occurs in the case of the platinum counter electrode, although the identity of the species being reduced is less clear.) Electric continuity in the electrolyte-impregnated filter paper and platinized chloroplasts is maintained by the movement of ions. A local positive potential is created in the intra-thylakoid (luminal) space as a result of the Photosystem II water-splitting reaction: 2 H,O + hv + 0, + 4 H+ + 4 e-. The four electrons are taken up by the electrodes in contact with the reducing end of Photosystem I, whereas the four hydrogen ions are left behind in the lumenal space, creating a local positive potential. This local positive potential is neutralized by the movement of chloride ions that are created at the interface of silver/silver chloride electrode and electro- lyte-impregnated filter paper. Chloride ions are permeable to biological membranes

1201.

177

In conclusion, a composite metal-photosynthetic membrane material has been developed and entrapped on electrolyte-impregnated fiberglass filter paper. Ori- ented photocurrents, which are consistent with the prevailing vectorial model of photosynthesis, have been observed, and provide a simple macroscopic demonstra- tion of the validity of this molecular model.

ACKNOWLEDGEMENTS

The author thanks J.P. Eubanks for technical support and P.S. Mattie for secretarial support. He also thanks T.M. Cotton, S.W. Feldberg, R.S. Knox, N.M. Kostic, and M. Seibert for valuable discussions. This research was funded by the Office of Basic Energy Sciences, U.S. Department of Energy, and Materials Labora- tory, Wright Aeronautical Laboratories. Oak Ridge National Laboratory is operated by Martin Marietta Energy Systems, Inc., under contract DE-AC05-840R21400 for the U.S. Department of Energy.

REFERENCES

1 H. Ochiai, H. Shibata, A. Fujishima and K. Honda, Agric. Biol. Chem., 43 (1979) 881.

2 J.O’M. Bockris and M.S. Tunulh in H. Keyzer and F. Gutmann (Eds.), Bioelectrochemistry, Plenum

Press, New York, 1980, p. 19.

3 T. Iida, H. Shiozawa, H. Kobayashi and T. Mitamura, Agric. Biol. Chem., 46 (1982) 275.

4 T. Iida, H. Kobayashi, T. Mitamura and K. Suzuki, Agric. Biol. Chem., 47 (1983) 175.

5 A. Agostiano, A. Ceghe and M. Della Monica, Bioelectrochem. Bioenerg. 10 (1983) 377.

6 M. Okano, T. Iida, H. Shinohara, H. Kobayashi and T. Mitamura, Agric. Biol. Chem., 48 (1984) 48.

7 A. Agostiano and F.K. Fong, Bioelectrochem. Bioenerg., 17 (1987) 325.

8 M. Seibert and M.W. Kendall-Tobia, B&him. Biophys. Acta, 681 (1982) 504.

9 H. Ochiai, H. Shibata, Y. Sawa and T. Katoh, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 2442.

10 T. Haehnel, A. Heupel and D. Hengstermann, Z. Naturforsch., 33c (1978) 392.

11 W. Haehnel and H.J. Hochhmeier, Bioelectrochem. Bioenerg., 6 (1979) 563.

12 R. Bhardwaj, R.L. Pan and E.L. Gross, Nature, 289 (1981) 369.

13 D.G. Sanderson, E.L. Gross and M. Seibert, Appl. Biochem. Biotechnol., 14 (1987) 1.

14 R. Carpentier and M. Mimeault, Biotechnol. Lett., 9 (1987) 111.

15 J.F. Becker, N.E. Geacintov and C.E. Swenberg, Biochim. Biophys. Acta, 503 (1978) 545.

16 E. Greenbaum, Science, 230 (1985) 1373.

17 S.G. Reeves and D.O. Hall, Methods Enzymol., 69 (1980) 85.

18 D.A. Walker, Methods Enzymol., 69 (1980) 94.

19 J. Barber, Plant Ceil Environ., 6 (1983) 311.

20 R.D. Keynes and J.C. EIIory (Eds.), The Binding and Transport of Anions in Living Tissues, Philos.

Trans. R. Sot. London B, 299 (1982) 435.