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Bioelectrochemistry tind Bimergefics, 21(1989) 171-177 A section of J. EIectromaL Chem, and constituting Vol. 275 (1989) Ekevier Sequoia S.A., Lausanne - printed in The Netherlands

Biomokcuku electronics: observation of oriented by enlmpped platinized chl~roph~~ts

E. Greenbaum

photocurzents

Chemical Technology Diubion, Oak Ridge Nation-d Laboratory, Oak Ridge, TN 37831-6194 (U.S.A.)

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

ABSTRACT

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 membrrme material was immobii on filter paper and sandwiched between metal gauze electrodes. Upon irradiation, oriented photocurxn ts were observed, The direction of the Row 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. OrientLd reaction centers are embedded in the photosynthetic membranes hat are composed of grana stacks and stroma lamellae, structures that are topolo@ally equivalent to spheres. Therefore, irrespective of the rotational orientation of the thylakoid mem- br;llles, 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

0302498/89/$03.50 Q 1989 Ebcvier Sequoia S.A.

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phot.ocurrents from platinized chloroplast membrsmes that have been entrapped on fiberglass filter paper. What distinguishes these expziments 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 f&us of chloroplasts or chloroplast particles [l-7], bacterial membrane vesicles [8] and even living micrbor&m [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. 1151, 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 expedients were performed in which ccuntzr electrodes of either platinum or silver/silver chloride were used.

EXPERMENTAL

Type C chloroplasts were prepared according to the procedure described by Reeves and Hall [17]. In this preparation, the chloroplast envelope is rupturd 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, waq prepared separately. One milliliter of this solution was combined with 5 ml of chlo:oplast suspension (containing 3 mg of chlorophyll) in Walke&i assay medium [18j. The 6-ml volume was placed iu a temperature-controlled, water-jacketed chamber fitted with O-ring connectors to provide a hermetic seal and with i&t 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 21OC.

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 mm were typically used. As indicated schematically in Fig. 1, after incubation,

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Fig 1. Schematic illustration of entrapped platinizcd cbloropb~~ts. 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. Where control experiments without hydrogen incubation produced a chloroplast mixture that filtered immediately, the platinized chloroplast required a considerably longer time, typically 5 to 30 mm. 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 platied-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 disk5 had a diameter of approximately 1 cm. In order to avoid &xtrochemical reactions with atmospheric oxygen, all of the experiments were performed in a helium atmosphere. Two platinized cblosoplast 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 41 platinized cbloroplast disk. The uss of the red cut-off filter served as a control and precautionary measure to ensure that the photocurrents observed were

PLATINUtd CONTACT

Fig. 2. Schematic iUustration of interface detail of outer membrane interface Electrical contact with the reducing etd 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 +&e 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 ckzber. This dowed for &CtriC~ contact tith the platinized-cbloroplasts-electrode assembly through an external measuring sys?em, while maintaining the assembkj in a helium atmosphere. The outputs of the electrometers were used to drive a two-pen chart recorder, which documented the

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data. Electrical contact between tile platinized chioroplasts 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 intc=sity. Figrue 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 platinum/p~atinum de&rode structure. There are several roteworthy 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 platinum/platinum and platinum/silver 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 tiadiated from the “back” side, the light was somewhat attenuated. However, the elecolyteimpregnated filter paper is by no means opaque, and photocurrents were easily se=n in this mode of irradiation. The key result of this control experiment was that the direction of the flow of photocurrent was the samk, 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 orient&ion of the m=- sured photocurrent is consistent with the prevailing vectorial model of photosynthe-

I I I I I I I I

I 0 2 4 6 * 8 10 12 14 1% 18

INCIDENT LIGHT/W m+ Fig. 3. Phot- t vs. incident light intensity (PI/A~ electrode assembly). The direction of flow of photocurrent was consistent with the generally accepted vectorial model of photosynthesis.

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Fig. 4. Siidtanmus @I tocments as a fmxion of cmntcr-elcctrode. The direction of flow of photocurrent was preserved with a .5ymmetfic ektrode structure. Also, the direction of flow of pbotocumnr 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 cau 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 pl&uu.n counter &ctrode, 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 pokntial is created in the intra-thylakoid @uninalj 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 neutralkd 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 I20].

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In conchsioxq a composite metal-photosynthetic membrane material has been developed and entrapped on electrolyteimpregnated fiberglass filter paper. Ori- ented ph&xxrrents, which zre 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 secre^tial support. He also th.?nks T.M. Cotton, S.W. Feldberg, RS. 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 hergy, and Materials Labora- tory, Wright Aeronautical Laboratories. Oak Ridge National Laboratory is operated by Martin Marietta Energy Systems, Inc., under contract D&AC05840R22400 for the U.S. Department of Energy.

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