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Stoichiometry of the photolysis of water by illuminated chloroplast fragments
S t o i c h i o m e t r y of t h e P h o t o l y s i s of W a t e r Chloroplast Fragments
by Illuminated
John D. Spikes From the Division of Biology, University of Utah, Salt Lake City, Utah
Received May 28, 1951 INTRODUCTION The first direct approach to the study of the component reactions of photosynthesis was made by Hill (1) who showed that the isolated chloroplasts of certain plants catalyzed the oxidation of water to oxygen gas and hydrogen ions upon illumination in the presence of a suitable electron acceptor (oxidant). The above process, which is apparently identical with the oxygen evolution reaction of photosynthesis (2), has been called the Hill reaction. The term photolysis has also been proposed, by analogy to such terms as photosynthesis, photoreduction and photooxidation (3). It should be pointed out that carbon dioxide is not fixed by these cell-free preparations (4). The over-all stoichiometry of the reaction has been studied by a number of workers. Hill (5), using potassium ferric oxalate as oxidant, showed that the oxygen evolved was proportional to the amount of ferric salt added to the system in the ratio of one molecule of oxygen'to each four ferric ions. On this basis he suggested the following equation for the over-all reaction: 4K~Fe(C~O4)3 + 2H~O + 4K + --~ 4K4Fe(C204)~ + 4H + + 05. (1) Hill later (6) measured the photochemical activity of isolated chloroplasts by two independent methods. The amount of oxygen produced was determined directly by its reaction with hemoglobin to form oxyhemoglobin, while the amount of ferrous iron produced was determined by its reaction with methemoglobin. The second method gave apparently lower rates than the first, but it was shown that this resulted from 1This work was supported by grants from the University of Utah Research Fund and the United States Atomic Energy Commission. 101
102
JOHN D. SPIKES
the slow rate of reaction between ferrous iron and methemoglobin. Differential effects with different inhibitors on the rate of reaction as measured in the two ways were also found. Urethan, for example, inhibited the rate of reaction more as measured by oxygen evolution than as measured by ferrous iron production. Again this discrepancy was apparently due to the secondary reactions used for measurement rather than to the photolysis reaction. Holt and French (7) obtained an average of 92.9% of the expected theoretical yield of oxygen in terms of the quantity of oxidant supplied to the system. They suggested that the reaction actually gave 100% yield, but that some of the oxygen produced was consumed in photooxidation reactions. They also measured the hydrogen-ion production, and obtained an average of 99% of the theoretical yield. Arnon and Whatley (3), with ferricyanide as the added oxidant, obtained 102% of the theoretical oxygen yield. With quinone as oxidant, oxygen yields of from 80 to 102% (3,8,9) have been obtained, while oxidation-reduction dyes such as phenolindophenol have resulted in oxygen yields of 98-107% (3,10). The 0nly anomalous yield reported in the literature is that for chromate which gave only 45-75% of the theoretical oxygen yield (10). Clendenning and Gotham (11) made simultaneous determinations at 20-min. intervals of both the oxygen evolution and the hydrogen-ion formation by illuminated spinach chloroplasts in unbuffered reaction mixtures using Hill's solution as oxidant. They found a stoiehiometric agreement which was unaffected by the length of the reaction period, by the chloroplast concentration, or by the composition of the oxidant solution. This previous work on the stoichiometry of the photolysis of water by isolated chloroplasts upon illumination was based on over-all results, or upon results at rather long intervals, rather than on the simultaneous and continuous determination of the compounds involved throughout the course of the reaction. It would be of interest to experimentally dissociate some of the constituent reactions from the over-all process for the purpose of study. This possibility was examined in the present work by simultaneous determination of the rate of the reaction by three different methods. Potassium ferricyanide was used as a convenient added oxidant since it reacts reversibly at electrode surfaces and does not seriously inhibit the reaction. The over-all equation for the photolysis of water with this
PHOTOLYSIS OF WATER
103
m a t e r i a l as o x i d a n t has b e e n suggested (7) as follows: ,4K3Fe(CN)6 -t- 4 K + + 2 H 2 0 --~ 4 K 4 F e ( C N ) 6 + 4 H + + 02.
(2)
T h e a b o v e r e a c t i o n was followed b y s i m u l t a n e o u s d e t e r m i n a t i o n of (a) t h e a m o u n t of o x y g e n evolved, (b) t h e a m o u n t of h y d r o g e n ion produced, a n d (c) t h e a m o u n t of f e r r i c y a n i d e r e d u c e d to f e r r o c y a n i d e . T h e s e factors were d e t e r m i n e d e i t h e r c o n t i n u o u s l y or a t short i n t e r v a l s t h r o u g h o u t t h e course of t h e reaction. METHODS
Preparation and Storage of Chloroplast Fragments Chloroplast fragments, rather than intact whole chloroplasts, were used in the present work. The fragments were prepared (3) by blending 50 g. of shredded spinach leaves (Spinaceaoleracea,obtained on the local market) for 3 min. in a Waring Blendor with 60 ml. of a solution 0.01 M in potassium chloride and 0.5 M in sucrose. All operations, unless otherwise specified, were carried out in a cold room at I~ under dim green light. The blend was filtered through cheesecloth and then centrifuged at 1000 • g for 10 min. to remove intact chloroplasts and larger fragments of leaf material. The supernatant was centrifuged for 15 rain. at 21,000 X g in an International size 2 centrifuge fitted with a Multispeed attachment. The sedimented chloroplast fragments were found to contain over 98% of the chlorophyll of the supernatant of the low speed centrifuging. The supernatant was poured off and discarded, and the centrifuge tube and packed fragments were rinsed several times with the KCl-sucrose solution. The fragments were resuspended in the KCl-sucrose solution with a motor-driven rubber policeman, and then were filtered through a wad of cotton to remove clumped material. The chlorophyll content (mixture of chlorophylls a and b) was determined with a Coleman Jr. spectrophotometer, and the preparation was diluted with KCl-sucrose solution to give a final concentration of 1.0 mg. chlorophyll/ ml. In practice, large quantities of chloroplast fragments were prepared at one time. These were dispensed in appropriate quantities into test tubes which were stoppered and stored in a Deepfreeze at -36~ Such preparations showed no change in photochemical activity during storage periods of 6 months. It has been reported that chloroplast materials may be stored almost indefinitely in this manner (12). This procedure permitted large numbers of experiments to be carried out on the same batch of material which is advantageous in view of the usual variability of biological systems. For use, tubes of frozen chloroplast fragments were thawed in the dark by immersion in beakers of water at 2~ The thawed material was used immediately, since it rapidly lost photochemical activity, even at 2~
Measuring Techniques An attachment, consisting of a stainless steel water bath with a plate glass bottom, was constructed to fit a Precision refrigerated rectangular Warburg apparatus. It was equipped with a circulating pump, a shaking arrangement, and a series of 150-w.
104
JOHN D. SPIKES
reflector-flood incandescant lamps as a light source. Gelatin projection filters were used to control the color of light used. Special rectangular Warburg vessels with bottom dimensions of 2 X 5 cm. were constructed. These were provided with a side arm, a vent, a ground-in platinum electrode, and a Beckman No. 270 saturated calomel electrode and a Beckman No. 290 glass electrode fitted into the vessel with rubber sleeves. The total volume of the system was a function of the position of the electrodes in the rubber sleeves. To avoid errors, the volume of each vessel was determined at the end of each run by means of a plunger-type calibrator similar to thaL described b y Scholander et al. (13). The electrodes were connected to the measuring instruments with shielded cables. Oxygen determinations were made in the usual manner using Brodie's fluid in the manometers. The density of the fluid was determined from time to time. Readings could be made with the shaking mechanism in operation. This permitted determinations to be made at very short intervals if necessary. Rates of oxygen evolution were calculated from the oxygen evolution data for the period from 2-10 rain. after the start of illumination and were expressed in terms of QC~ which is equal to cu. mm. oxygen/hr./mg, of chlorophyll present. Changes in the pH of the system were determined with a Beckman model G pH meter. Readings could be made at 10-sec. intervals and could be estimated to 0.01 pH units, if necessary, in order to give an almost continuous indication of p H changes. The electrodes were checked with standard buffer before and after each run. The amount of ferricyanide reduced a t any given time was determined potentiometrically (14). The output of the platinum and calomel electrode system was run into a Brown electronic strip-chart recorder-potentiometer which had a full-scale sensitivity of 2.5 mv. and a period for full scale movement of 12 sec. The electrodes were matched to the recorder by means of a battery-operated input device which had an input impedance of approximately 5 • l0 s ohms to prevent polarization of the electrodes, and an output impedance of approximately 1000 ohms to m a t c h the input of the recorder. This unit will be described in detail elsewhere. The potential vs. time curves obtained upon illumination of active photolytic systems had the shape of typical potentiometric titration curves. The amount of ferricyanide reduced at any given time was derived from this curve by an application of the Nernst equation as previously described (14). The reaction mixture had a total volume of 3.0 ml., and contained chloroplast fragments equivalent to 1.0 mg. of chlorophyll. This volume gave a layer of reaction mixture approximately 3 mm. deep in the reaction vessels, which permitted light saturation of the system with the light intensity used. All work was carried out with a gelatin filter which cut out wavelengths shorter t h a n 5800 A. The light intensity a t vessel level was approximately 8000 lux. All reactions were run in an atmosphere of tank nitrogen (99.5% N2). Photooxidation reactions were reduced by the use of this inert atmosphere (as were dark oxidation reactions) and by the use of red light for illumination as described. Reactions were carried out at a temperature of 15 ~ 0.1~ The shaking rate was 100 oscillations/min., and the amplitude of movement was 3 cm. at vessel level.
105
PHOTOLYSIS OF WATER CONTROL EXPERIMENTS
Effect of Initial Ferricyanide Concentration on Rate In order to obtain measurable volumes of oxygen, it was necessary to use higher concentrations of ferricyanide in the present work than had been used previously with the potentiometric technique (14). Thus it was (if interest to examine the effect of initial ferricyanide concentration on the rate of the reaction. The manometric technique was used for this determination in consideration of the range of ferricyanide concentration employed. The results of typical experiments are shown in Fig. 1. The curve indicates that the rate of oxygen evolution decreases
.~
0
5~
O.OOI M
INITIs
,
O.OI M
, I
O.I M
FERRIGYANIDE GONCENTRATION
Fie. 1. Curve showing the rate of oxygen evolution by illuminated chloroplast fragments as a function of initial ferricyanide concentration. Chloroplast fragment concentration equivalent to 1.0 mg. of chlorophyll. Potassium phosphate buffer concentration 0.10 M, p H 6.80. Sucrose concentration 0.17 M. Potassium chloride concentration 0.01 M. Initial potassium ferricyanide concentration before illumination as indicated. The points shown were taken from a number of different experiments.
as the initial ferricyanide concentration increases. The curve has the form, under the conditions employed, of: Rate = - A log rferricyanide~initi~l + B, where A and B are constants. On the basis of these results a standard concentration of 0.005 M ferricyanide was used for all experiments unless otherwise indicated. This concentration gave a rapid rate of reaction and still resulted in the formation of sufficient oxygen to permit accurate results.
106
JOI-i~- D. SPIKES
Effect of Bvffer Concentration on Rate In order to measure hydrogen-ion production in the system by means of pH changes, the buffer concentration must be such that the change in pH will be sufficient to permit precise determination of the hydrogenion production. The effect of different concentrations of potassium phosphate buffer on the rate of reaction as measured by oxygen evolution was investigated. It was found that, under the conditions employed, the rate of reaction is essentially zero order in buffer concentration. Clendenning and Gotham (11), using quinone as the oxidant, found the rate of oxygen evolution to be independent of phosphate buffer concentration from 0.04 to 0.4 M.
Determination of Hydrogen-Ion Production The constant-pH method of determining hydrogen-ion production (7) during the course of the reaction could not be used for the present work as potential and manometric measurements were to be made simultaneously. Instead, the pH changes during the course of the reaction were determined. The resulting data were then converted into hydrogen-ion production data by use of a curve prepared by titrating aliquots of the same reaction system with acid and observing the resulting pH changes. In using this technique, care must be taken that the pH changes will not result in an inactivation of the system (3,7,14). Titration experiments were carried out to determine the buffering capacity of systems containing different concentrations of potassium phosphate buffer. On the basis of this work it was found that hydrogen-ion production could be determined with an accuracy almost comparable to that of the oxygen determinations in systems containing 0.01 M buffer and the standard concentration of oxidant. The resulting pH changes were not great enough to produce significant inactivation of the system during the time periods employed.
Accuracy of Measurements The precision of the manometric technique for measuring oxygen evolution was studied by making simultaneous rate determinations on a series of six manometric systems containing the standard reaction mixture. The average standard deviation of such simultaneous determinations was less than 3%. The reproducibility of the potentiometric
107
PHOTOLYSIS OF WATER
method for measuring the rate of ferricyanide reduction was examined by making successive measurements on samples of the same batch of frozen chloroplast fragments in the standard reaction mixture. The average standard deviation in rate determinations using this technique was also less than 3%. The accuracy of the method used for measuring the rate of hydrogen-ion production was investigated by making successive rate determinations on standard systems using samples of the same frozen chloroplast fragment preparation. Rate determinations made with this technique were somewhat more variable than those made with the other methods, but the standard deviation was still no larger than 4%. 16
< ORET,OA.y.ELD T.
1/) Z 0 12 E Io ILl J W h
o
/
8
(/) LI.I .J 0 =E
/
@ FERRICYAN IDE
J
REDUCTION
4 OXYGEN
=E 0
0 0
EVOLUTION
He PRODUGTION
I
I
I0
20
TIME
IN MINUTES
I
30
Fza. 2. Curves showing the quantities of electrons transferred with respect to oxygen evolved, hydrogen ion produced and ferricyanide reduced in the photolysis of water by illuminated chloroplast fragments. Chloroplast fragment concentration equivalent to 1.0 mg. of chlorophyll. Potassium phosphate buffer concentration 0.01 M, pH 6.80. Sucrose concentration 0.17 M. Potassium chloride concentration 0.01 M. Initial potassium ferricyanide concentration before illumination 0.005 M.
108
JOHN D. SPIKES
Stoichiometry of the Reaction The stoichiometry of the photolysis of water by illuminated chloroplasts was determined by making simultaneous measurements on the oxygen evolution (at 2-rain. intervals), the potential change (recorded continuously) and the pH change (at 2-min. intervals) of the same system using the electrode vessels and techniques described. The potential change data were converted to the rate of ferricyanide reduction, while the pH change data were converted to the rate of hydrogen-ion production. Then all of these data were converted into terms of millimoles of electrons transferred as a function of time on the assumption that Eq. (2) represents the correct stoichiometry for the reaction. The results of a typical experiment of this sort are shown in Fig. 2. The points indicate that the reaction does follow the relations given in Eq. (2) within the accuracy of the methods used. Further, it shows that these relations are maintained throughout the course of the reaction. Under the conditions employed in the present work the various component reactions of the photolysis of water by illuminated chloroplast preparations (evolution of oxygen, reduction of added oxidant, and hydrogen-ion production) cannot be dissociated sufficiently to permit their study separately. These results further show that each of the three methods of measurement described can be used equally well to measure the rate of the reaction. SUMMARY
1. Simultaneous determinations of the rate of oxygen evolution, the rate of reduction of added oxidant, and the rate of hydrogen-ion production during the photolysis of water by illuminated chloroplast fragments were made continuously on the same system during the course of illumination. 2. The stoichiometry indicated in the following equation was found to hold within the experimental error throughout the course of the reaction: 4KsFe(CN)6 + 4K + + 2H~O --~ 4K4Fe(CN)e + 4H + + 02. 3. None of the component reactions of the photolysis of water could be dissociated under the conditions employed in order to permit separate study.
PHOTOLYSIS OF WATER
109
REFERENCES l. HILL, R., Nature 139, 881 (1937). 2. HOLT, A. S., AND FRENCH, C. S., in Photosynthesis in Plants, Chap. 14, Edited by J. FRANCK AND W. E. LooMIs. Iowa State College Press, Ames, Iowa, 1949. 3. ARNON,D. I., AND WHATLEY,F. R., Arch. Biochem. 23, 141 (1949). 4. BROWN,A. H., AND FRA~rCK,J., Arch. Biochem. 16, 55 (1948). 5. HILL, R., Proc. Roy Soc. (London) 127B, 192 (1939). 6. HILL, R., AND SCARISBRICK,R., Proc. Roy Soc. (London) 129B, 239 (1940). 7. HOLT, A. S., AND FRENCH, C. S., Arch. Biochem. 9, 25 (1946). 8. WARBURG,0., AND Lg~rrGENS, W., Naturwissenschaften 32, 161 (1944). 9. WARBURG,O., AND Lr.~TTGENS,W., Biokhimiya 11, 303 (1946). 10. HOLT, A. S., AND FRENCH, C. S., Arch. Bioehem. 19, 368 (1948). 11. CLENDENNING,K. A., AND GORHAM,P. R., Can. J. Research C28, 78 (1950). 12. G o m ~ , P. R., AND CLENDENNING,K. A., Can. J. Research C28, 513 (1950). 13. SCHOLANDER,P. F., NIEMEYER,H., ANDCLOFF, C. L.~ Science 112, 437 (1950). 14. SPIKES, J. D., LUMRY,R., EYRING,H., AND WAYRYNEN,R., Arch. Biochem. 28, 48 (1950).
by Illuminated
John D. Spikes From the Division of Biology, University of Utah, Salt Lake City, Utah
Received May 28, 1951 INTRODUCTION The first direct approach to the study of the component reactions of photosynthesis was made by Hill (1) who showed that the isolated chloroplasts of certain plants catalyzed the oxidation of water to oxygen gas and hydrogen ions upon illumination in the presence of a suitable electron acceptor (oxidant). The above process, which is apparently identical with the oxygen evolution reaction of photosynthesis (2), has been called the Hill reaction. The term photolysis has also been proposed, by analogy to such terms as photosynthesis, photoreduction and photooxidation (3). It should be pointed out that carbon dioxide is not fixed by these cell-free preparations (4). The over-all stoichiometry of the reaction has been studied by a number of workers. Hill (5), using potassium ferric oxalate as oxidant, showed that the oxygen evolved was proportional to the amount of ferric salt added to the system in the ratio of one molecule of oxygen'to each four ferric ions. On this basis he suggested the following equation for the over-all reaction: 4K~Fe(C~O4)3 + 2H~O + 4K + --~ 4K4Fe(C204)~ + 4H + + 05. (1) Hill later (6) measured the photochemical activity of isolated chloroplasts by two independent methods. The amount of oxygen produced was determined directly by its reaction with hemoglobin to form oxyhemoglobin, while the amount of ferrous iron produced was determined by its reaction with methemoglobin. The second method gave apparently lower rates than the first, but it was shown that this resulted from 1This work was supported by grants from the University of Utah Research Fund and the United States Atomic Energy Commission. 101
102
JOHN D. SPIKES
the slow rate of reaction between ferrous iron and methemoglobin. Differential effects with different inhibitors on the rate of reaction as measured in the two ways were also found. Urethan, for example, inhibited the rate of reaction more as measured by oxygen evolution than as measured by ferrous iron production. Again this discrepancy was apparently due to the secondary reactions used for measurement rather than to the photolysis reaction. Holt and French (7) obtained an average of 92.9% of the expected theoretical yield of oxygen in terms of the quantity of oxidant supplied to the system. They suggested that the reaction actually gave 100% yield, but that some of the oxygen produced was consumed in photooxidation reactions. They also measured the hydrogen-ion production, and obtained an average of 99% of the theoretical yield. Arnon and Whatley (3), with ferricyanide as the added oxidant, obtained 102% of the theoretical oxygen yield. With quinone as oxidant, oxygen yields of from 80 to 102% (3,8,9) have been obtained, while oxidation-reduction dyes such as phenolindophenol have resulted in oxygen yields of 98-107% (3,10). The 0nly anomalous yield reported in the literature is that for chromate which gave only 45-75% of the theoretical oxygen yield (10). Clendenning and Gotham (11) made simultaneous determinations at 20-min. intervals of both the oxygen evolution and the hydrogen-ion formation by illuminated spinach chloroplasts in unbuffered reaction mixtures using Hill's solution as oxidant. They found a stoiehiometric agreement which was unaffected by the length of the reaction period, by the chloroplast concentration, or by the composition of the oxidant solution. This previous work on the stoichiometry of the photolysis of water by isolated chloroplasts upon illumination was based on over-all results, or upon results at rather long intervals, rather than on the simultaneous and continuous determination of the compounds involved throughout the course of the reaction. It would be of interest to experimentally dissociate some of the constituent reactions from the over-all process for the purpose of study. This possibility was examined in the present work by simultaneous determination of the rate of the reaction by three different methods. Potassium ferricyanide was used as a convenient added oxidant since it reacts reversibly at electrode surfaces and does not seriously inhibit the reaction. The over-all equation for the photolysis of water with this
PHOTOLYSIS OF WATER
103
m a t e r i a l as o x i d a n t has b e e n suggested (7) as follows: ,4K3Fe(CN)6 -t- 4 K + + 2 H 2 0 --~ 4 K 4 F e ( C N ) 6 + 4 H + + 02.
(2)
T h e a b o v e r e a c t i o n was followed b y s i m u l t a n e o u s d e t e r m i n a t i o n of (a) t h e a m o u n t of o x y g e n evolved, (b) t h e a m o u n t of h y d r o g e n ion produced, a n d (c) t h e a m o u n t of f e r r i c y a n i d e r e d u c e d to f e r r o c y a n i d e . T h e s e factors were d e t e r m i n e d e i t h e r c o n t i n u o u s l y or a t short i n t e r v a l s t h r o u g h o u t t h e course of t h e reaction. METHODS
Preparation and Storage of Chloroplast Fragments Chloroplast fragments, rather than intact whole chloroplasts, were used in the present work. The fragments were prepared (3) by blending 50 g. of shredded spinach leaves (Spinaceaoleracea,obtained on the local market) for 3 min. in a Waring Blendor with 60 ml. of a solution 0.01 M in potassium chloride and 0.5 M in sucrose. All operations, unless otherwise specified, were carried out in a cold room at I~ under dim green light. The blend was filtered through cheesecloth and then centrifuged at 1000 • g for 10 min. to remove intact chloroplasts and larger fragments of leaf material. The supernatant was centrifuged for 15 rain. at 21,000 X g in an International size 2 centrifuge fitted with a Multispeed attachment. The sedimented chloroplast fragments were found to contain over 98% of the chlorophyll of the supernatant of the low speed centrifuging. The supernatant was poured off and discarded, and the centrifuge tube and packed fragments were rinsed several times with the KCl-sucrose solution. The fragments were resuspended in the KCl-sucrose solution with a motor-driven rubber policeman, and then were filtered through a wad of cotton to remove clumped material. The chlorophyll content (mixture of chlorophylls a and b) was determined with a Coleman Jr. spectrophotometer, and the preparation was diluted with KCl-sucrose solution to give a final concentration of 1.0 mg. chlorophyll/ ml. In practice, large quantities of chloroplast fragments were prepared at one time. These were dispensed in appropriate quantities into test tubes which were stoppered and stored in a Deepfreeze at -36~ Such preparations showed no change in photochemical activity during storage periods of 6 months. It has been reported that chloroplast materials may be stored almost indefinitely in this manner (12). This procedure permitted large numbers of experiments to be carried out on the same batch of material which is advantageous in view of the usual variability of biological systems. For use, tubes of frozen chloroplast fragments were thawed in the dark by immersion in beakers of water at 2~ The thawed material was used immediately, since it rapidly lost photochemical activity, even at 2~
Measuring Techniques An attachment, consisting of a stainless steel water bath with a plate glass bottom, was constructed to fit a Precision refrigerated rectangular Warburg apparatus. It was equipped with a circulating pump, a shaking arrangement, and a series of 150-w.
104
JOHN D. SPIKES
reflector-flood incandescant lamps as a light source. Gelatin projection filters were used to control the color of light used. Special rectangular Warburg vessels with bottom dimensions of 2 X 5 cm. were constructed. These were provided with a side arm, a vent, a ground-in platinum electrode, and a Beckman No. 270 saturated calomel electrode and a Beckman No. 290 glass electrode fitted into the vessel with rubber sleeves. The total volume of the system was a function of the position of the electrodes in the rubber sleeves. To avoid errors, the volume of each vessel was determined at the end of each run by means of a plunger-type calibrator similar to thaL described b y Scholander et al. (13). The electrodes were connected to the measuring instruments with shielded cables. Oxygen determinations were made in the usual manner using Brodie's fluid in the manometers. The density of the fluid was determined from time to time. Readings could be made with the shaking mechanism in operation. This permitted determinations to be made at very short intervals if necessary. Rates of oxygen evolution were calculated from the oxygen evolution data for the period from 2-10 rain. after the start of illumination and were expressed in terms of QC~ which is equal to cu. mm. oxygen/hr./mg, of chlorophyll present. Changes in the pH of the system were determined with a Beckman model G pH meter. Readings could be made at 10-sec. intervals and could be estimated to 0.01 pH units, if necessary, in order to give an almost continuous indication of p H changes. The electrodes were checked with standard buffer before and after each run. The amount of ferricyanide reduced a t any given time was determined potentiometrically (14). The output of the platinum and calomel electrode system was run into a Brown electronic strip-chart recorder-potentiometer which had a full-scale sensitivity of 2.5 mv. and a period for full scale movement of 12 sec. The electrodes were matched to the recorder by means of a battery-operated input device which had an input impedance of approximately 5 • l0 s ohms to prevent polarization of the electrodes, and an output impedance of approximately 1000 ohms to m a t c h the input of the recorder. This unit will be described in detail elsewhere. The potential vs. time curves obtained upon illumination of active photolytic systems had the shape of typical potentiometric titration curves. The amount of ferricyanide reduced at any given time was derived from this curve by an application of the Nernst equation as previously described (14). The reaction mixture had a total volume of 3.0 ml., and contained chloroplast fragments equivalent to 1.0 mg. of chlorophyll. This volume gave a layer of reaction mixture approximately 3 mm. deep in the reaction vessels, which permitted light saturation of the system with the light intensity used. All work was carried out with a gelatin filter which cut out wavelengths shorter t h a n 5800 A. The light intensity a t vessel level was approximately 8000 lux. All reactions were run in an atmosphere of tank nitrogen (99.5% N2). Photooxidation reactions were reduced by the use of this inert atmosphere (as were dark oxidation reactions) and by the use of red light for illumination as described. Reactions were carried out at a temperature of 15 ~ 0.1~ The shaking rate was 100 oscillations/min., and the amplitude of movement was 3 cm. at vessel level.
105
PHOTOLYSIS OF WATER CONTROL EXPERIMENTS
Effect of Initial Ferricyanide Concentration on Rate In order to obtain measurable volumes of oxygen, it was necessary to use higher concentrations of ferricyanide in the present work than had been used previously with the potentiometric technique (14). Thus it was (if interest to examine the effect of initial ferricyanide concentration on the rate of the reaction. The manometric technique was used for this determination in consideration of the range of ferricyanide concentration employed. The results of typical experiments are shown in Fig. 1. The curve indicates that the rate of oxygen evolution decreases
.~
0
5~
O.OOI M
INITIs
,
O.OI M
, I
O.I M
FERRIGYANIDE GONCENTRATION
Fie. 1. Curve showing the rate of oxygen evolution by illuminated chloroplast fragments as a function of initial ferricyanide concentration. Chloroplast fragment concentration equivalent to 1.0 mg. of chlorophyll. Potassium phosphate buffer concentration 0.10 M, p H 6.80. Sucrose concentration 0.17 M. Potassium chloride concentration 0.01 M. Initial potassium ferricyanide concentration before illumination as indicated. The points shown were taken from a number of different experiments.
as the initial ferricyanide concentration increases. The curve has the form, under the conditions employed, of: Rate = - A log rferricyanide~initi~l + B, where A and B are constants. On the basis of these results a standard concentration of 0.005 M ferricyanide was used for all experiments unless otherwise indicated. This concentration gave a rapid rate of reaction and still resulted in the formation of sufficient oxygen to permit accurate results.
106
JOI-i~- D. SPIKES
Effect of Bvffer Concentration on Rate In order to measure hydrogen-ion production in the system by means of pH changes, the buffer concentration must be such that the change in pH will be sufficient to permit precise determination of the hydrogenion production. The effect of different concentrations of potassium phosphate buffer on the rate of reaction as measured by oxygen evolution was investigated. It was found that, under the conditions employed, the rate of reaction is essentially zero order in buffer concentration. Clendenning and Gotham (11), using quinone as the oxidant, found the rate of oxygen evolution to be independent of phosphate buffer concentration from 0.04 to 0.4 M.
Determination of Hydrogen-Ion Production The constant-pH method of determining hydrogen-ion production (7) during the course of the reaction could not be used for the present work as potential and manometric measurements were to be made simultaneously. Instead, the pH changes during the course of the reaction were determined. The resulting data were then converted into hydrogen-ion production data by use of a curve prepared by titrating aliquots of the same reaction system with acid and observing the resulting pH changes. In using this technique, care must be taken that the pH changes will not result in an inactivation of the system (3,7,14). Titration experiments were carried out to determine the buffering capacity of systems containing different concentrations of potassium phosphate buffer. On the basis of this work it was found that hydrogen-ion production could be determined with an accuracy almost comparable to that of the oxygen determinations in systems containing 0.01 M buffer and the standard concentration of oxidant. The resulting pH changes were not great enough to produce significant inactivation of the system during the time periods employed.
Accuracy of Measurements The precision of the manometric technique for measuring oxygen evolution was studied by making simultaneous rate determinations on a series of six manometric systems containing the standard reaction mixture. The average standard deviation of such simultaneous determinations was less than 3%. The reproducibility of the potentiometric
107
PHOTOLYSIS OF WATER
method for measuring the rate of ferricyanide reduction was examined by making successive measurements on samples of the same batch of frozen chloroplast fragments in the standard reaction mixture. The average standard deviation in rate determinations using this technique was also less than 3%. The accuracy of the method used for measuring the rate of hydrogen-ion production was investigated by making successive rate determinations on standard systems using samples of the same frozen chloroplast fragment preparation. Rate determinations made with this technique were somewhat more variable than those made with the other methods, but the standard deviation was still no larger than 4%. 16
< ORET,OA.y.ELD T.
1/) Z 0 12 E Io ILl J W h
o
/
8
(/) LI.I .J 0 =E
/
@ FERRICYAN IDE
J
REDUCTION
4 OXYGEN
=E 0
0 0
EVOLUTION
He PRODUGTION
I
I
I0
20
TIME
IN MINUTES
I
30
Fza. 2. Curves showing the quantities of electrons transferred with respect to oxygen evolved, hydrogen ion produced and ferricyanide reduced in the photolysis of water by illuminated chloroplast fragments. Chloroplast fragment concentration equivalent to 1.0 mg. of chlorophyll. Potassium phosphate buffer concentration 0.01 M, pH 6.80. Sucrose concentration 0.17 M. Potassium chloride concentration 0.01 M. Initial potassium ferricyanide concentration before illumination 0.005 M.
108
JOHN D. SPIKES
Stoichiometry of the Reaction The stoichiometry of the photolysis of water by illuminated chloroplasts was determined by making simultaneous measurements on the oxygen evolution (at 2-rain. intervals), the potential change (recorded continuously) and the pH change (at 2-min. intervals) of the same system using the electrode vessels and techniques described. The potential change data were converted to the rate of ferricyanide reduction, while the pH change data were converted to the rate of hydrogen-ion production. Then all of these data were converted into terms of millimoles of electrons transferred as a function of time on the assumption that Eq. (2) represents the correct stoichiometry for the reaction. The results of a typical experiment of this sort are shown in Fig. 2. The points indicate that the reaction does follow the relations given in Eq. (2) within the accuracy of the methods used. Further, it shows that these relations are maintained throughout the course of the reaction. Under the conditions employed in the present work the various component reactions of the photolysis of water by illuminated chloroplast preparations (evolution of oxygen, reduction of added oxidant, and hydrogen-ion production) cannot be dissociated sufficiently to permit their study separately. These results further show that each of the three methods of measurement described can be used equally well to measure the rate of the reaction. SUMMARY
1. Simultaneous determinations of the rate of oxygen evolution, the rate of reduction of added oxidant, and the rate of hydrogen-ion production during the photolysis of water by illuminated chloroplast fragments were made continuously on the same system during the course of illumination. 2. The stoichiometry indicated in the following equation was found to hold within the experimental error throughout the course of the reaction: 4KsFe(CN)6 + 4K + + 2H~O --~ 4K4Fe(CN)e + 4H + + 02. 3. None of the component reactions of the photolysis of water could be dissociated under the conditions employed in order to permit separate study.
PHOTOLYSIS OF WATER
109
REFERENCES l. HILL, R., Nature 139, 881 (1937). 2. HOLT, A. S., AND FRENCH, C. S., in Photosynthesis in Plants, Chap. 14, Edited by J. FRANCK AND W. E. LooMIs. Iowa State College Press, Ames, Iowa, 1949. 3. ARNON,D. I., AND WHATLEY,F. R., Arch. Biochem. 23, 141 (1949). 4. BROWN,A. H., AND FRA~rCK,J., Arch. Biochem. 16, 55 (1948). 5. HILL, R., Proc. Roy Soc. (London) 127B, 192 (1939). 6. HILL, R., AND SCARISBRICK,R., Proc. Roy Soc. (London) 129B, 239 (1940). 7. HOLT, A. S., AND FRENCH, C. S., Arch. Biochem. 9, 25 (1946). 8. WARBURG,0., AND Lg~rrGENS, W., Naturwissenschaften 32, 161 (1944). 9. WARBURG,O., AND Lr.~TTGENS,W., Biokhimiya 11, 303 (1946). 10. HOLT, A. S., AND FRENCH, C. S., Arch. Bioehem. 19, 368 (1948). 11. CLENDENNING,K. A., AND GORHAM,P. R., Can. J. Research C28, 78 (1950). 12. G o m ~ , P. R., AND CLENDENNING,K. A., Can. J. Research C28, 513 (1950). 13. SCHOLANDER,P. F., NIEMEYER,H., ANDCLOFF, C. L.~ Science 112, 437 (1950). 14. SPIKES, J. D., LUMRY,R., EYRING,H., AND WAYRYNEN,R., Arch. Biochem. 28, 48 (1950).