Photoconduction and photosynthesis

9°

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 4 5 1 4 5

PHOTOCONDUCTION AND PHOTOSYNTHESIS I. T H E PHOTOCONDUCTIVITY OF C H L O R O P H Y L L MONOLAYERS K. J. M c C R E E

Physics and Engineering Laboratory, D.S.I.R., Lower Hurt (New Zealand) (Received A u g u s t 3rd, 1964)

SUMMARY

Pure chlorophyll has been laid down as a stack of monomolecular layers on a photoconductivity grid. With gold electrodes, the photocurrent responded instantly to the light, whereas with aluminium, silver and colloidal graphite it did not. The photocurrent was very small, and not significantly greater than with a chlorophyll film which had been deposited by evaporation from a solution in acetone. One electron per sec was collected for every lO9 quanta per sec absorbed.

INTRODUCTION

A modern theory of photosynthesis must explain how several hundred chlorophyll molecules channel the energy which they receive from light quanta to one reaction centre, where it is to be converted into chemical energy and then used by the plant for its synthesising activities. According to CALVIN1,s the energy is carried by free electrons and holes. If the electrons and holes were to migrate to different points, which could be the opposite sides of a chlorophyll monolayer, it might be possible to explain how oxidizing and reducing reaction centres could be physically separated. This theory follows the suggestion first put forward by KATZ~ in 1949, that the very successful theories of solid-state physics might be applied to living organisms. Though some doubt has been expressed by physicists about the relevance of the band theory of conduction in inorganic crystals to biological materials~, 7, there is sufficient experimental evidence of electrical conduction and photoconduction for such suggestions still to be considered seriously ~ 6. Among the plant materials which have shown conduction are proteins 9-12, pigments3,13-2°, chloroplasts1~, zl and leaves 22. In most cases the materials were in a very unphysiological state (dried, in a vacuum). Also, where a photocurrent was observed, the quantum efficiency was not measured. It is perhaps not surprising that biologists have remained somewhat sceptical 23-29. The new experiments reported here are concerned firstly with the dark-current photoconductivity of chlorophyll (Part I), and secondly with photoconduction in more physiologically active materials (Part II). Among the previous measurements on chlorophyll are those of NELSON3, who found that chlorophyll and methyl chlorophyllide when deposited from an organic Biochim. Biophys..~lcta, lO2 (1965) 90-95

P H O T O C O N D U C T I O N A N D P H O T O S Y N T H E S I S . I.

91

solvent would conduct a small dark-current photocurrent across a platinum grid on a glass plate. The current did not respond instantly to the light, but took several minutes to settle down to a steady value. The same effect was found by ARNOLD AND M A c L A Y ls, who used a colloidal graphite grid. ROSENBERGAND CAMISCOL116compressed chlorophyll crystals between electrodes of monel metal and conductive glass, and measured the variation of current with temperature. They do not mention whether or not the currents were instantaneous. PUTZEIKO AND T E R E N I N 17-2° measured the Dember photovoltage of chlorophyll evaporated from organic solvents, and found that water vapour increased the signal. In these measurements the chlorophyll was either crystalline or deposited from an organic solvent. In the green plant, it is probably in the form of a monomolecular layer, bound on one side to fats and on the other side to water and proteins. Monomolecular layers of chlorophyll can be produced by the technique of LANGMUIRAND BLODGETT27-3a. The chlorophyll is spread from acetone solution on the clean surface of a trough of water. With proper control it forms a monomolecular layer, which can then be deposited on a glass plate by dipping the plate through the layer. A stack of layers can be built up by repeated dipping. Photoconductivity in chlorophyll monolayers has been reported in passing by ARNOLD AND MACLAY 15, but there appears to have been no systematic study of chlorophyll in this form. In most of the previous measurements the photocurrent did not respond immediately when the light was switched on and off. This may have been due to the wrong electrode material being useda4, 35. In the present measurements, the electrode material was varied and it was found that this had a great effect on the photocurrent. The amount of light absorbed by the sample was measured, so that the quantum efficiency could be calculated. It is necessary to know this before discussing the connection between photocurrents and photosynthesis. METHOD

Chlorophyll was extracted from fresh plants and purified by chromatography. It was laid down as a monolayer on water and picked up on a fused-silica slide. The conductivity was measured immediately in a vacuum.

Preparation of chlorophyll Small quantities of chlorophyll were extracted from New Zealand spinach purified immediately before use by paper chromatography.

(Tetragonia expansa) and TABLE

I

SPECTROSCOPIC PROPERTIES OF CHLOROPHYLL SAMPLES (IN ACETONE)

Sample No.

Red peak~blue minimum

Blue peak~red peak

Chlorophyll a i 2 3 4

56 33 41 3°

1. 5 1.6 1.5 1.6

Chlorophyll b i 2

13 16

3.3 3.3

Biockim. Biophys..4cta, lO2 (1965) 9 0 - 9 5

92

I~. j. MCCREE

T h e spectroscopic p r o p e r t i e s of r e p r e s e n t a t i v e samples are given in T a b l e I. These samples were as p u r e as a n y which h a v e been p r e p a r e d 36, b u t their p u r i t y was not at all c o m p a r a b l e with t h a t used in p r e s e n t - d a y solid-state physics. To show t h a t c h l o r o p h y l l was an intrinsic p h o t o c o n d u c t o r it w o u l d be necessary to use a perfect single crystal. H o w e v e r , this is an a c a d e m i c p o i n t in a b i o p h y s i c a l investigation, which m u s t be concerned more w i t h the relation b e t w e e n the p h o t o c u r r e n t a n d the p l a n t t h a n w i t h the origin of t h e p h o t o c u r r e n t .

Deposition of monomolecular layers T h e t e c h n i q u e for d e p o s i t i n g m o n o l a y e r s on w a t e r a n d picking t h e m up on slides is reviewed in the b o o k s b y SOBOTKA37 a n d ADAM~, a n d TRURNIT AND COLMANO39 h a v e described t h e p r e p a r a t i o n a n d p r o p e r t i e s of chlorophyll monolayers. Our films were l a i d on glass-distilled de-ionized w a t e r m a d e lO -4 M in CaCO 3 (pH 7.5-8.0). T h e y were c o m p r e s s e d to 16 d y n e s / c m w i t h c a s t o r oil a n d d e p o s i t e d at t h e r a t e of one l a y e r p e r rain on to fused-silica microscope slides w i t h c o n d u c t i v i t y grids on t h e m (see n e x t section). U p to 50 l a y e r s could be p u t on to one slide from one film. The slide was i m m e d i a t e l y t a k e n to t h e m e a s u r i n g cell, where the c o n d u c t i v i t y was m e a s u r e d under vacuum.

Conductivity measurements T h e c o n d u c t i v i t y grid was f o r m e d b y v a c u u m e v a p o r a t i o n of m e t a l on to a fused-silica p l a t e which h a d been cleaned w i t h cerium oxide, d e t e r g e n t a n d water. F o r t h e deposition of gold, the surface was m a d e h y d r o p h o b i c with d i m e t h y l - d i c h l o r o silane (General Electric SC 87 " D r i - F i l m " ) , as r e c o m m e n d e d b y I-IoLLAND4°. The h y d r o p h o b i c surface was an a d v a n t a g e also in the deposition of the monolayers. D u r i n g t h e m e t a l deposition, a length of fine copper wire w o u n d into a zigzag was p l a c e d over t h e p l a t e to form t h e g a p of 0.25 m m across which the c o n d u c t i v i t y of t h e chlorophyll was to be measured. I n t h e m e a s u r i n g cell c o n t a c t was m a d e to the two sides of the grid with p l a t i n u m - t i p p e d wires which pressed on to t h e e v a p o r a t e d m e t a l a t the end r e m o t e from t h e chlorophyll. I t was found a d v i s a b l e to p a i n t a small a r e a of t h e m e t a l a r o u n d the c o n t a c t w i t h colloidal graphite. The cell was e v a c u a t e d to a few microns of m e r c u r y w i t h a r o t a r y v a c u u m p u m p . If this was not done the d a r k c u r r e n t was m a n y orders g r e a t e r t h a n t h e p h o t o c u r r e n t . All m e a s u r e m e n t s were m a d e at r o o m t e m p e r a t u r e . A field of up to 14000 V/cm (360 V across 0.025 cm) was a p p l i e d to t h e grid from d r y batteries. The current was passed t h r o u g h a lO TM f2 resistor, across which was connected a v i b r a t i n g - c o n d e n s e r e l e c t r o m e t e r v o l t m e t e r . The limit of s e n s i t i v i t y of this v o l t m e t e r was lO -4 V, corresponding to a c u r r e n t of lO -14 A. L i g h t was p r o v i d e d b y a I 5 o - W reflector s p o t l a m p , run from 5o-cycle mains. T h e l a m p was switched on for 15 sec at a time. I n f r a r e d r a d i a t i o n was r e m o v e d w i t h filters, a n d t h e r e m a i n i n g visible r a d i a t i o n , as m e a s u r e d w i t h a thermopile, was 9~ W per m z (about 15oo ftcandles). RESULTS

W i t h no chlorophyll p r e s e n t the c u r r e n t g r a d u a l l y fell, until it reached a s t e a d y value of less t h a n IO-1~ A (Fig. I). This t y p e of b e h a v i o u r is c o m m o n w i t h dielectrics. Biochim. Biophys. Acta, lO2 (1965) 9 0 - 9 5

PEOTOCONDUCTION AND PHOTOSYNTHESIS. I.

93

The same behaviour was found in the dark when the chlorophyll films were present; the dark current of chlorophyll must be very small. Without the chlorophyll, there was no photocurrent. With it, there was a photocurrent of i o - l L I O -t3 A. It was quite different with different materials as the grid. With aluminium, silver and colloidal graphite the photocurrent dropped a great deal

2.4I 2.2

20 1.8 1.6 [ 1.4 Ax10-121.2 1.0 0.8 0.6 0.4 0.2

oc

IO 1'5 2'0 2'5 30 35 z~O 4:5 50 Time (min) fnom applying voltage (36QV)

5'5

60

Fig. I. Recorder trace of c u r r e n t a g a i n s t t i m e for a film of 3 monolayers of chlorophyll a on a gold grid. The pulses s h o w where the light was p u t on (for 15 sec in all b u t one case).

\ \\ \ i0-I-~

Monoloyers \

\ o

4.0

"\

film

\

3.5 3.C

\\\\

o

I

\

E°voporated"

(A)

D~rK

t

i0-I"

I 2.5 A x 10-12 2.0

1.5 1.0 b

0.5

10-14!

0

0 5'0 160 1~0 260 250 360 350 Potential difference (V acposs 0.025 cm) Fig. 2. P h o t o c u r r e n t plotted a g a i n s t t i m e for various n u m b e r s of chlorophyll a monolayers. The p h o t o c u r r e n t f r o m a film oI chlorophyll sufficient for a b o u t 3 o monolayers b u t deposited b y e v a p o r a t i o n of acetone solvent is s h o w n for comparison. The " d a r k - c u r r e n t " curve is an average one for the I-, 3- a n d 7-monolayer films (36o V).

I'0

Time (rain)

100

Fig. 3. P h o t o c u r r e n t p l o t t e d a g a i n s t voltage for a 5o-monolayer film of chlorophyll a on a gold grid.

Biochim. Biophys. Acta, IO2 (1965) 90-95

94

I<. j. ~CCREE

from its initial value a n d t o o k a long t i m e to reach equilibrium, whereas w i t h gold it r e s p o n d e d i n s t a n t a n e o u s l y a n d u n a m b i g u o u s l y to the light. The b e h a v i o u r was not p e r f e c t l y simple, however. T h e size of t h e p h o t o c u r r e n t pulse d e p e n d e d on t h e t i m e which h a d elapsed since the electric field h a d been a p p l i e d (Fig. I). There was a t e n d e n c y for the p h o t o c u r r e n t to decrease less with time, as the n u m b e r of m o n o l a y e r s was increased. W i t h a s t a c k of 50 m o n o l a y e r s t h e p h o t o c u r r e n t was almost indep e n d e n t of time. T h e reason for this b e h a v i o u r is unknown. T h e results o b t a i n e d w i t h various n u m b e r s of chlorophyll a m o n o l a y e r s on gold grids are shown in Fig. 2. P h o t o c u r r e n t s were o b t a i n e d from b o t h chlorophyll a a n d chlorophyll b, b u t m o s t of the m e a s u r e m e n t s were m a d e on chlorophyll a. T h e p h o t o c u r r e n t was found to be p r o p o r t i o n a l to the electric field, up to 14ooo V/cm, in a series of m e a s u r e m e n t s w i t h the 5o-layer film (Fig. 3). DISCUSSION

The p h o t o c u r r e n t s are v e r y small. Measurements showed t h a t 5o m o n o l a y e r s a b s o r b a b o u t IO % of the incident light. T h u s a c u r r e n t of lO -12 A (6. lO 6 electrons/sec) is p r o d u c e d from t h e a b s o r p t i o n of IO % of IOO W / m 2 of r a d i a t i o n (3" lO15 q u a n t a / s e c at a w a v e l e n g t h of 5oo m/~). I t would seem t h a t s o m e t h i n g like lO 9 q u a n t a are needed to p r o d u c e one electron. There m a y , of course, be m a n y electrons p r o d u c e d in the film which are not collected b y the electrodes, b u t even so, it is e x t r e m e l y u n l i k e l y t h a t there are enough p h o t o e l e c t r o n s to a c t i v a t e t h e p h o t o s y n t h e t i c process, which has a q u a n t u m efficiency of n e a r l y one. The p h o t o c u r r e n t from m o n o l a y e r s was a p p r o x i m a t e l y the same as t h a t from an equal q u a n t i t y of chlorophyll e v a p o r a t e d s t r a i g h t on to the grid from an acetone solution. W h e t h e r oriented in m o n o l a y e r s or not, therefore, c h l o r o p h y l l could never be called an efficient p r o d u c e r of photoelectrons. I t is a c t u a l l y a b o u t as efficient as t h e o t h e r organic p h o t o c o n d u c t o r s which h a v e been t e s t e d 41. This does not necessarily m e a n t h a t p h o t o c o n d u c t i v e effects are i r r e l e v a n t to p h o t o s y n t h e s i s . A s t a c k of m o n o l a y e r s in a v a c u u m is still a long w a y from chlorophyll in its n a t u r a l state. F o r instance, it m a y be necessary to h a v e w a t e r present, in o r d e r to keep the molecules oriented. The m e a s u r e m e n t s of PUTZEIKO AND T E R E N I N 19,2° on e v a p o r a t e d films show t h a t the p h o t o c u r r e n t can be m u c h g r e a t e r in a moist a t m o s phere. I n order to r e p e a t their m e a s u r e m e n t s with monolayers, it has been necessary to change to their m e t h o d of m e a s u r i n g the p h o t o c u r r e n t . W e t films could n~t be used on the p h o t o c o n d u c t i v i t y grid, because t h e y b r o k e down electrically. I n t h e P u t z e i k o m e t h o d the chlorophyll forms the dielectric of a condenser which is illumin a t e d t h r o u g h one p l a t e with i n t e r r u p t e d light. A small p o t e n t i a l difference is developed in the direction of the light as a result of the diffusion of electrons or holes. The results of some new m e a s u r e m e n t s m a d e b y this m e t h o d are given in P a r t I I . REFERENCES

I D. F. BRADLEY AND M. CALVIN, 2;1t'0C. Natl. Acad. Sci. U.S., 41 (I955) 5032 E. KATZ, in J. FRAm< AND \V. E. LOOMIS, Photosynthesis in Planls, l o w a State College Press, Ames., 1949, p. 287. 3 R. C. NELSON, J. Chem. Phys., 27 (1957) 864. 4 M. NASHA, Rev. l~Iod. Phys., 31 (1959) 162. 5 ]3. ROSENBERG, J . Opt. Soc. Am., 51 (1961) 238.

Biochim. Biopl~ys. Acla, lO2 (1965) 9 °. 95

PHOTOCONDUCTION AND PHOTOSYNTHESIS. I.

95

6 C. G. ]3. GARRETT, Radiation Res. (Suppl.), 2 (196o) 34 o. 7 M. SUARD, G. BERTHIER AND B. PULLMAN, Biochim. Biophys. :Jcta, 52 (1961) 2548 M. CALVIN, J. Theoret. Biol., 2 (1961) 258. 9 D. D. ELEY AND D. I. SPIVEY, Nature, 188 (196o) 725 . i o B. I~OSENBERG, Nature, 193 (1962) 364 • i i C. P. S. TAYLOR, Nature, 189 (1961) 388. 12 N. I~IEHL, Kolloid-Z., 151 (1957) 66. 13 I{. C. NELSON, J. Opt. Soc. Am., 46 (1956) io. 14 B. ][{OSENBERG, J. Chem. Phys., 31 (1959) 238. 15 W. ARNOLD AND H. K. MACLAY, Brookhaven Syrup. Biol., 11 (1958) I. 16 ]3. 1ROSENBERG AND J. F. CAMISCOLI, J. Chem. Phys., 35 (1961) 982. 17 A. N. TERENIN, A. I{. PLITZEIKO AND I. AKIMOV, Discussions Faraday Soc., 27 (I959) 8318 A. N. TERENIN, A. I~. PUTZEIKO AND I. AKIMOV, J. Chim. Phys., 54 (1957) 716. 19 A. N. TERENIN AND A. K. PUTZEIKO, J. Chim. Phys., 55 (1958) 681. 20 A. K. PUTZEIKO AND A. N. TERENIN, Dokl. Akad. Nauk SSSR, 136 (1961) 1223. 21 W. ARNOLD AND H. Z . SHERWOOD, Proc. Natl. Acad. Sci. U.S., 43 (1957) lO5. 22 E. l ~ o u x AND L. FOISSAC, Compt. Rend., 246 (1958) 2677. 23 A. T. JAGENDORF, Surv. Biol. Progr., 4 (1962) 181. 24 R. K. CLAYTON, Ann. Rev. Plant Physiol., 14 (1963) 159. 25 E. RABINOWITCH, Plant Physiol., 34 (1959) 213" 26 E. t¢-ABI~OWlTCH, Discussions Faraday Sot., 27 (1959) I 6 I . 27 K. t3. ]DLODGETT, J. Am. Chem. Soc., 57 (1935) lOO7. 28 K. t3. BLODGETT AND I. LANGMUIR, Phys. Rev., 51 (1937) 964. 29 W. D. t-IARKINS AND T. F. ANDERSON, J. ,4m. Chem. Soc., 59 (1937) 2189. 3 ° M. F. E. NICOLAI AND C, WEURMAN, Koninkl. Ned. Akad. Welenschap. Proc., 41 (1938) 904. 31 E. E. JACOBS, A. E. VATTER AND A. S. HOLT, Arch. Biochem. Biophys., 53 (1954) 228. 32 A. HUGHES, Proc. Roy. Soc. London, Ser. A, 155 (1936) 71o. 33 I. LANGMUIR AND V. J. SCHAEFER, J. Am. Chem. Soc., 59 (1937) 2075. 34 R. H. tDUBE, Photoeonductivity of Solids, J o h n W i l e y , N e w Y ork, I96O, p. IiO. 35 D. M. J. COMPTON, W. G. SCHNEIDER AND T. C. WADDINGTON, J. Chem. Phys., 27 (1957) 16o. 36 F. I°. ZCHEILE ANn C. L. COMAR, Botan. Gaz., lO2 (1941) 463 . 37 H. SDBOTKA, 2VIongmglecular layers, A m e r i c a n A s s o c i a t i o n for t h e A d v a n c e m e n t of Science, W a s h i n g t o n , 19.54 . 38 N. t(. ADAM, Physics and Chemistry of Surfaces, 3rd E d . , O xford U n i v . Press, L o n d o n , 1946, p. 17. 39 H. J. TRI:RNIT AND G. COLMANO, Biochim. Bioph*'s. ,4eta, 31 (1959) 434. 4 ° L. HOLLAND, Vacuum Deposition of Thin Films, C h a p m a n a n d H a l l , L o n d o n , 1956, p. IOi. 41 L. E. L Y o n s AND G. C. MORRIS, J. Chem. Soe., (1957) 3648.

Biochim. Biophys. Acta, lO2 (1965) 9 0 - 9 5