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Photophosphorylation by isolated chromatophores of the purple sulfur bacteria
ARCHIVES
OF
BIOCHEMISTRY
AND
76, 168-179 (b-68)
BIOPHYSICS
Photophosphorylation by Isolated Chromatophores of the Purple Sulfur Bacterial I. C. Anderson From the Department
of Biology,
and R. C. Fuller
Brookhaven
Received
National
November
Laboratory,
Upton, New York
3, 1957
The anaerobic light-dependent formation of adenosine triphosphate (ATP) in plants has been shown to be associated with chloroplasts and/or chloroplast fragments by Arnon and co-workers (1,2). Frenkel(3) has also reported a light-dependent formation of ATP by a particulate fraction of the purple nonsulfur photosynthetic bacteria, Rhodospirillum rub-urn. The photosynthetic bacteria offer a fertile field for the investigation of the mechanism of the conversion of light energy into fixed ATP. All the photochemical pigments of the bacteria are contained in very small particles (300-600 A.) that have been referred to as chromatophores (4). These particles are not as complex as the higher plant chloroplast since they do not contain either the carbon-fixing system nor starch-storage facilities as does the chloroplast. Bacterial chromatophores appear to function solely as photochemical units for the production of reducing power and formation of ATP (5). The whole problem of the requirement for various cofactors in photosynthetic phosphorylation has been discussed by Arnon (6); however, there is some doubt as to the exact role of these cofactors, and their function in bacterial systems is completely unknown (7, 8). Therefore, work was undertaken with isolated and purified chromatophores from the sulfur purple photosynthetic bacterium, Chromatium, strain D, to elucidate the process of photosynthetic phosphorylation. EXPERIMENTAL Preparation of Plastids Chromatium collected from
strain D was grown as described by Hendley (9). Bacteria were 1 1. of media by centrifugation after 4-7 days of growth and sus-
1 Research carried out at Brookhaven National of the U. S. Atomic Energy Commission. 168
Laboratory
under the auspices
PHOTOSYNTHESIS
BY
ISOLATED
CHROMATOPHORES
169
pended in 40 ml. of a solution containing 0.4 M glucose, 0.1 M tris(hydroxymethyl)aminomethane (Tris) buffer at pH 7.8, and 4 g. of levigated alumina. Cells were ruptured in this mixture at 96°C. by 90 sec. of sonic vibration in a lo-kc. Raytheon oscillator. Other methods of disruption, such as alumina grinding, were used, but more efficient breaking was effected by sonic treatment, and the cellular fragments from both of the treatments were similar. Whole cells and debris were collected and discarded by centrifugation at 65,000 X g for 20 min. in a Spinco preparative centrifuge. The dark-red solution was then centrifuged at 144,000 X g for 90 min. The resulting particulate-free cellular fluid will The particles which sedimented at be referred to in the text as “supernatant.” Physical-chemical studies and this speed are referred to as “chromatophores.” electron microscopy indicate that the chromatophores are of uniform size with a diameter of approximately 300 A. (10). The chromatophores were washed in 20 ml. of a solution containing 0.4 M glucose and 0.1 Tris buffer at pH 7.8. The glucose and Tris supernatant from the above washing will be referred to as “chromatophore washings.” The washed chromatophores were then suspended in 10 ml. of the above glucose-Tris solution, and 0.5 ml. of this suspension was used in each photophosphorylation reaction mixture.
Photophosphorylation The experiments were carried out in an illuminated Warburg apparatus at 20°C. Light intensity was approximately 1500 ft.-candles at the surface of the water. Warburg flasks containing 3 ml. of reaction mixture (see Table I) were flushed with Ns for 10 min. in the dark, adenosine diphosphate (ADP) was tipped in from the side arm, and lights were turned on to initiate photophosphorylation. Flasks were shaken for 30 min., and aliquots were removed for inorganic phosphate analyses (11) both before initiation of photophosphorylation and immediately after the lights had been turned off at the termination of the experiment.
BacteriochlorophyEl Determination Because of its instability to light in organic solvents, bacteriochlorophyll is usually converted to the more stable bacteriopheophytin for quantitative determination. The bacteriochlorophyll content of reaction mixtures was determined as pheophytin as described by French (12). Also, measurements were made of the amount of absorption of reaction mixtures at 810 mF, which is an absorption maximum of in vivo bacteriochlorophyll. After a number of such measurements it became apparent that an optical density of one at 810 rnp (l-cm. light path) of a 3-ml. reaction mixture was equivalent to 0.06 mg. bacteriochlorophyll. Thereafter, the absorption at 810 rnp was measured, and bacteriochlorophyll was calculated from the above relationship.
Autoradiography
and IdentiJication
In order to identify the products formed, take place in the presence of P3a-labeled stopped by the addition of 12 ml. of boiling chromatographed in two dimensions using
of ATP
photophosphorylation was allowed to orthophosphate. The reactions were ethanol, and the resulting extract was phenol-water and butanol-propionic
170
ANDERSON AND FULLER
acid as the solvents (13). X-ray film was placed on the papers, and the film was developed after suitable exposure. Compounds on the paper were identified by cochromatography with known material.
RESULTS
Chromatophores were capable of a light-dependent esterification of ADP by inorganic phosphate with the concomitant formation of ATP as indicated in Eq. (1). Frequently hexokinase was added to the reaction ADP+Pi
chromatophores light
hexokinase
ATP + glucose _____f
--t ATP
glucose 6-phosphate
+ ADP
(2)
mixture in order to regenerate the phosphate acceptor [Eq. (2)]. Figure 1 is an autoradiograph of photophosphorylation in the presence of Pa*labeled inorganic phosphate. The only product formed in the absence of the “hexokinase trap” is ATP. Components of Photophosphorylution The components of photophosphorylation are indicated in Table I. The omission of light, ADP, Mg++ ions, supernatant, and the anaerobic condition all strikingly reduce the amount of photophosphorylation. Whereas there is an absolute requirement for light and ADP, the chromatophore preparation probably contains trace amounts of Mg++ ions, and supernatant; therefore, the data show only a stimulation by, rather than an absolute requirement for these two latter components. Phosphorylation occurred at maximum capacity with as little as 1 rmole ADP in the presence of hexokinase and glucose. Charaetmistics of Photophosphorylatio A curve demonstrating the effect of pH on the activity of photophosphorylation is shown in Fig. 2. Phosphorylation occurs over a wide pH range with maximum activity at pH 8.0. This is similar to the pH optimum that has been obtained for photophosphorylation by spinach chloroplasts (6, 14). Light saturation was obtained between 200 and 400 ft.-candles. This is in contrast to the 1500 ft.-candles required for light saturation in the Hill reaction of chloroplasts (15). Jagendorf and Avron (7) have found
Chromatium LParticles
Pia* 60 min. in light particles (144,000 x G)
Fro. 1. Autoradiograph of photophosphorylation. The reaction was carried out by adding 1.5 X 10’ counts/min. PS* to a reaction mixture which contained 0.28 mg. bacteriochlorophyll and allowed to incubate in the light for 60 min. Aliquot (0.01) applied at lower right-hand corner. Nine thousand counts were found in ATP and 5000 in orthophosphate. TABLE I Components of Photophosphorylation Each Warburg vessel contained the following additions except where indicated: 7.5 &moles ADP at pH 7.8, 10 pmoles MgC12 , 20 amoles phosphate at pH 7.8, 150 pmoIes glucose, 100 pmoles Tris buffer at 7.8, 0.3 ml. supernatant, 4 mg. of crude hexokinase (Sigma Chemical Co.), and 0.5 ml. of chromatophore preparationcontaining0.42 mg. bacteriochlorophyll. Illuminated for 30 min. at 20°C. in a nitrogen atm. Micromoles of Pi esterified/30 min.
Component omitted
None Supernatant Q++ 1 instead of 7.5 pmoles of ADP ADP Light Anaerobic conditions 171
12 5 3 11 1 0 5
172
ANDERSON
pH
OPTIMUM
AND
FULLER
OF PHOTOPHOSPHORYLATION
Fra. 2. pH optimum of photophosphorylation. Assays were made as described in Table I except pH was varied by suspending chromatophores in 0.4 M glucose and 150 pmoles of either Tris or glycylglycine buffer at various pH values. Vessels contained 0.22 mg. bacteriochlorophyll, and the reaction was allowed to proceed for 1 hr.
a requirement of up to 4000 ft.-candles to saturate photophosphorylation by spinach chloroplasts. The proportionality of the amount of chromatophores and rate of phosphorylation is shown in Fig. 3. Phosphorylation is a linear function of chromatophore addition over the range studied. Rates generally ranged between 50 and 100 pmoles of phosphate esterified/hr./mg. bacteriochlorophyll. In vitro phosphorylation for spinach chloroplasts has been reported as high as 400 ccmoles/mg. chlorophyll/hr. (6, 7). The slow rate of phosphorylation with these chromatophores may be simply a reflection of the slower rate of over-all photosynthesis in these cells. Rupture of cells and isolation of chromatophores in a solution containing 0.4 M glucose and 0.1 M Tris at pH 7.8 allows a better separation of cellular components and produces more active chromatophores with higher phosphorylation activity per unit of bacteriochlorophyll than is
PHOTOSYNTHESIS
BY
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CHROMATOPHORES
173
,-
mg
OF BACTERIOCHLOROPHYLL
3. Proportionality of photophosphorylation with chromatophore concentration. Assay as described in Table I except bacteriochlorophyll content varied as indicated. Reaction time was 1 hr. FIG.
obtained by sonication in dilute buffer solutions. After isolation, the chromatophores can be washed as many as three times with distilled water and stored at 0°C. for several days without any loss of activity. The isolated chromatophores retain up to 75% of their activity after lyophilization and storage over P206 for several months. E$ect of Supernatant on Photophosphorylation Frenkel (8) has reported that photophosphorylation by particulates from Rhodospirdlum rubrum was stimulated by the addition of catalytic amounts of reduced diphosphopyridine nucleotide (DPNH) or succinate. Phosphorylation by chromatophores of Chromatium also is stimulated by DPNH and succinate (see Table II). The addition of particulate-free supernatant from ruptured cells, however, causes a greater stimulation than is obtained with DPNH or succinate. Boiling this supernatant for 90 sec. decreased its stimulatory effect to the level shown by DPNH and succinate additions. This indicates that the supernatant might contain at least two stimulatory substances: one of these being heat-stable and acting in a manner similar to DPNH or succinate; the other a heatlabile protein. A confirmation of this proposal is demonstrated by the data in Tables III and IV. A difference in the origin of the two stimulatory factors of the super-
174
ANDERSON
AND
FULLER
natant is indicated by the data of Table V. The first four lines present the effects of succinate, supernatant, and boiled supernatant on photophosphorylation. The fifth line illustrates the stimulation obtained upon addition of chromatophore washings. A further increase of photophosphorylation with chromatophore washings was obtained upon addition of succinate. The stimulatory effect of chromatophore washings was TABLE Supernatant
II E$ect
Assay as described in Table I except that supernatant was omitted indicated in the table. Vessels contained 0.37 mg. bacteriochlorophyll. Additions
Micromoles of Pi esterified/30 min.
No additions 0.1 pmole DPNH 1.O pmole succinate 0.3 ml. supernatant 0.3 ml. boiled supernatant
7 11 12 16 11
unless
TABLE III on Photophosphorylation Assay as described in Table I except as indicated in the table. Vessels contained 0.31 mg. bacteriochlorophyll. Efect
of Supematant
Milliliters
Micromoles of succinate
0.00
0.0 1.0
4.2 6.4
of supernatant
0.10 0.30 0.50 Micromoles of Pi esteriiied/dO min.
7.5 7.7
12.7 11.9
12.0 12.3
0.50 (boiled)
8.2 7.7
TABLE IV Sulfate Fractionated Supernatant on Photophosphorylatim Assay as described in Table I except the supernatant was fractionated with ammonium sulfate. A 3345yo ammonium sulfate fraction was collected, resuspendedin 10 ml. of 0.1 M Tris at pH 7.8, and dialyzed against 0.001 M Tris buffer at pH 7.8. Vessels contained 0.31 mg. bacteriochlorophyll.
Efect
of Ammonium
Milliliters
of (NH&SO4 fraction
Micromoles of succinate
0.00
0.25 0.50 1.04 1.4 Micmmoles of Pi esteriIicd/30 min.
0.0
3.8 5.8 65
5.4 7.3 74
1.0 Per cent activity succinate
without
8.7 11.0 79
9.5 12.6 75
9.0 12.5 72
(bzd)
3.4 5.7 60
PHOTOSYNTHESIS
BY
ISOLATED
TABLE
V
of Supernatant
Origin
Factors
Assay as described in Table I except supernatant Vessels contained 0.39 mg. bacteriochlorophyll. Additions
None 1 pmole succinate 0.3 ml. supernatant 0.3 ml. boiled supernatant 1.0 ml. chromatophore washings* 1.0 ml. chromatophore washings plus 1 #mole succinate 7. 1.0 ml. boiled chromatophore washings Experimental
section
omitted
unless indicated.
Micromoles of Pi esterilied/30 min.
1. 2. 3. 4. 5. 6.
a he
175
CHROMATOPHORES
6.0 9.0 17.5 10.0 9.5 15.0 5.8
for definition.
completely destroyed by 90 sec. of boiling. These data indicate that the heat-labile protein was indeed coming from the chromatophores, but that the heat-stable factor is apparently not bound to these chromatophores and may be originating from other cytoplasmic sources. Several protein factors which participate in photosynthesis have been isolated from spinach chloroplasts. Among these are an enzyme which permits a net reduction of diphosphopyridine nucleotide (DPN) by grana (16), a TPNH diaphorase (17)) and a factor stimulating photophosphorylation (14). The protein factor from spinach chloroplasts which stimulates photosynthetic phosphorylation was prepared as described by Avron and Jagendorf (14). This factor from spinach chloroplasts failed to stimulate photophosphorylation by isolated chromatophores of Chromatium.2 Nucleotide Specificity The results in Table VI are a summary of some experiments on nucleotide specificity. Phosphorylation with ADP is stimulated by supernatant as was expected. There was no phosphorylation with adenylic acid (AMP) together with a small amount of ATP. This indicates the absence of adenylic kinase in the chromatophores. The addition of supernatant to reaction mixtures of chromatophore containing AMP, together with a small amount of ATP, allowed for as much esterification as was obtained with ADP, indicating the presence of adenylic kinase in the supernatant. Other assays for adenylic kinase demonstrated the ability of the * Anderson,
I. C., and Fuller,
R. C., unpublished.
176
ANDERSON AND FULLER
TABLE VI Nucleotide Specificity Assay as described in Table I except hexokinase was omitted and ADP, other nucleotides, and supernatant were added’ as indicated below. Vessels contained 0.38 mg. bacteriochlorophyll. Additions p??WleS
20 ADP 20 ADP 10 AMP 10 AMP 20 IDP 20 IDP
Micromoles
+ 0.3 ml. supernatant + 1 ATP + 1 ATP + 0.3 ml. supernatant + 0.3 ml. supernatant
of Pi &&&d/30
min.
4.5 17.2 0.5 17.4 4.0 9.0
supernatant to convert mixtures of AMP and ATP to ADP, whereas the chromatophores lacked this ability. The last two lines of the table illustrate that inosine diphosphate (IDP) can serve as a source of phosphate acceptor. Inosine triphosphate was identified as the product by paper chromatographic and autoradiographic methods. Inosine diphosphate is not as good a phosphate acceptor as is ADP. DISCUSSION
Short periods of sonic oscillation in the presence of levigated alumina ruptured Chromatium cells. Small pigmented particles were liberated from the cells during this process. These particles were of uniform size with a diameter of 300 A. and have been referred to as chromatophores. Electron micrographs of whole cells have indicated that there is variation in the size of in vivo particles. However, most of the particles present in whole cells are of the size found in the chromatophore preparation (10). Further morphological and physiological studies of the particles of Chromalium are under way at this laboratory. These small particles of Chromutium carry out a light-dependent formation of ATP from ADP and inorganic phosphate, and this esterification requires Mg++ ions. Photophosphorylation by the chromatophores was stimulated by the addition of small amounts of the particulate-free supernatant which was obtained from the ruptured cells. It was shown that the supernatant contained at least two stimulatory substances. One of these substances was heat-stable and could be replaced by catalytic amounts of either DPNH or succinate. Frenkel (8) also has reported the stimulation of photophosphorylation by catalytic amounts
PHOTOSYNTHESIS
BY
ISOLATED
CHROMATOPHORES
177
of DPNH or succinate with particulate preparations from R. rubrum. The other stimulatory substance of the supernatant was nondialysable and, therefore, it was assumed to be a protein. The two stimulatory substances of the supernatant had different intracellular origins. The protein factor appeared to come from the chromatophores during their isolation and washing. The heat-stable component did not come from the chromatophores, but originated from some other cellular source. These observations give an indication of the function of the two stimulatory components. The protein factor apparently is associated with the chromatophore in viva. It may be an enzyme which is involved in photophosphorylation and which is partially solubilized during isolation of chromatophores. The heat-stable component of the supernatant presumably functions as an oxidation-reduction balancing agent, since it may be replaced by catalytic amounts of DPNH or succinate. A generalized outline of the steps of photophosphorylation is presented by the following equations (8). The primary photochemical acceptor of the reducing portion of water is represented by X and the acceptor of the oxidizing portion of water is represented by Y. Hz0 + X (ox.) + Y (red.) H.X-tH0.Y
Concomitant
light chromatophores
) H.X + HO-Y
Electron carriers > X (ox.) + Y (red.) + HOH in chromatophores
(3) (4)
with Eq. (4): ADP + Pi
Coupling between electron carriers and phosphorylation
+ ATP
(5)
Frenkel (8) postulated from his studies with R. rubrum that succinate and DPNH have a “sparking” effect by reducing some intermediate of the cyclic electron-carrier system, Eq. (4). An alternative explanation is that DPNH and succinate act by balancing the primary photochemical acceptors X and Y. In viva, X and Y are balanced; that is, if X is mainly oxidized then Y is mainly reduced and vice versa. This balance would be unpoised if, during the process of isolation of chromatophores, the photochemical acceptor of the oxidizing portion of water (Y) were to become oxidized. Under these conditions a certain portion of the photochemical acceptors would be unable to accept the reducing and oxidizing
178
ANDERSON AND FULLER
parts of water. The present data appear to support this alternative explanation. Oxygen is a potent inhibitor of photophosphorylation, and it is possible that the primary photochemical acceptor of the oxidizing portion of water would be susceptible to autoxidation. Succinate and DPNH appear to be nonspecific substances which are capable of acting on (or balancing) some oxidation-reduction system. Also, as seen in Table IV, the effect of succinate was independent of the degree of saturation of the chromatophores by the protein factor. This would indicate that a certain constant proportion of some process of photophosphorylation was nonfunctional in the absence of succinate. Succinate and DPNH in the bacterial systems, and such compounds as ascorbate in higher plant chloroplasts, may function, therefore, only as redox balancing agents to protect a photochemical acceptor from autoxidation in vitro and have no significant function as cofactors in wivo. SUMMARY
Chromatophores were isolated from the photosynthetic purple sulfur bacterium, Chromatium strain D. These particles were capable of a lightdependent esterification of ADP and inorganic phosphate with the resulting formation of ATP. Phosphorylation was stimulated by the supernatant from the ruptured cells. The supernatant contained two stimulatory substances: one a heat-stable factor such as DPNH or succinate, and a second labile protein. No additional cofactors were required. ADDENDUM After this paper had been submitted, a paper by J. Newton and M. Kamen, Biochim et Biophys. Acta 26, 462 (1957), on photophosphorylation by Chromatium particles appeared. The same sized pigmented particle appeared to have been studied in both laboratories, and, therefore, some of the results presented in this paper become repetitive with those of Newton and Kamen. There is, however, a great difference in the photophosphorylative capacity of the particulates studied. Particulate8 isolated as described in this paper were capable of a minimum of 20 times as much photophosphorylation per unit of bacteriochlorophyll as those of Newton and Kamen. This observation becomes important when considering the necessary components of any electron-transport system for photophosphorylation.
REFERENCES 1. ARNON, D. I., WHATLEY, F. R., AND ALLEN, M. B., J. Am. Chem. Sot. 76, 6324 (1954).
2. WHATLEY, F.R., ALLEN, M.B., ROSENBERG,L. L., CAPINDALE,J. B., AND ARNON, D. I., Biochim. et Biophys. Acta 20, 462 (1956).
PHOTOSYNTHESIS
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179
3. FRENKEL, A. W., J. Am. Chem. Sot. 76, 5568 (1954). 4. SCHACHMAN, H. K., PARDEE, A. B., AND STANIER, R. Y., Arch. Biochem. Biophys 33, 245 (1952). 5. FULLER, R. C., AND ANDERSON, I. C., Plant Physiol. 32, Suppl. xvi (1957). 6. ARNON, D. I., WHATLEY, F. R., AND ALLEN, M. B., Nature 27, 182 (1957). 7. JAGENDORF, A. T., AND AVRON, M., Plant Physiol. 32, Suppl. iv (1957). 8. FRENKEL, A. W., J. Biol. Chem. 222, 823 (1956). 9. HENDLEY, D. D., J. Bacterial. 70, 625 (1955). 10. BERGERON, J. A., ANDERSON, I. C., AND FULLER, R. C., Plant Physiol. 32 Suppl. xvi (1957). 11. LOWRY, O., AND LOPEZ, J., J. Biol. Chem. 162, 421 (1946). 12. FRENCH, C. S., J. Gen. Physiol. 23, 483 (1940). 13. BENSON, A. A., BASSHAM, J. A., CALVIN, M., GOODALE, T. C., HAAS, V. A., AND STEPKA, W., J. Am. Chem. Sot. 72, 1710 (1950). 14. AVRON, M., AND JAGENDORF, A. T., Nature 179, 4% (1957). 15. HILL, R., AND SCARISBRICK, R., Nature 146, 61 (1940). 16. SAN PIETRO, A., AND LANGE, H. M., Science 124, 118 (1956). 17. AVRON, M., AND JAGENDORF, A. T., Arch. Biochem. Biophys. 66, 475 (1956).
OF
BIOCHEMISTRY
AND
76, 168-179 (b-68)
BIOPHYSICS
Photophosphorylation by Isolated Chromatophores of the Purple Sulfur Bacterial I. C. Anderson From the Department
of Biology,
and R. C. Fuller
Brookhaven
Received
National
November
Laboratory,
Upton, New York
3, 1957
The anaerobic light-dependent formation of adenosine triphosphate (ATP) in plants has been shown to be associated with chloroplasts and/or chloroplast fragments by Arnon and co-workers (1,2). Frenkel(3) has also reported a light-dependent formation of ATP by a particulate fraction of the purple nonsulfur photosynthetic bacteria, Rhodospirillum rub-urn. The photosynthetic bacteria offer a fertile field for the investigation of the mechanism of the conversion of light energy into fixed ATP. All the photochemical pigments of the bacteria are contained in very small particles (300-600 A.) that have been referred to as chromatophores (4). These particles are not as complex as the higher plant chloroplast since they do not contain either the carbon-fixing system nor starch-storage facilities as does the chloroplast. Bacterial chromatophores appear to function solely as photochemical units for the production of reducing power and formation of ATP (5). The whole problem of the requirement for various cofactors in photosynthetic phosphorylation has been discussed by Arnon (6); however, there is some doubt as to the exact role of these cofactors, and their function in bacterial systems is completely unknown (7, 8). Therefore, work was undertaken with isolated and purified chromatophores from the sulfur purple photosynthetic bacterium, Chromatium, strain D, to elucidate the process of photosynthetic phosphorylation. EXPERIMENTAL Preparation of Plastids Chromatium collected from
strain D was grown as described by Hendley (9). Bacteria were 1 1. of media by centrifugation after 4-7 days of growth and sus-
1 Research carried out at Brookhaven National of the U. S. Atomic Energy Commission. 168
Laboratory
under the auspices
PHOTOSYNTHESIS
BY
ISOLATED
CHROMATOPHORES
169
pended in 40 ml. of a solution containing 0.4 M glucose, 0.1 M tris(hydroxymethyl)aminomethane (Tris) buffer at pH 7.8, and 4 g. of levigated alumina. Cells were ruptured in this mixture at 96°C. by 90 sec. of sonic vibration in a lo-kc. Raytheon oscillator. Other methods of disruption, such as alumina grinding, were used, but more efficient breaking was effected by sonic treatment, and the cellular fragments from both of the treatments were similar. Whole cells and debris were collected and discarded by centrifugation at 65,000 X g for 20 min. in a Spinco preparative centrifuge. The dark-red solution was then centrifuged at 144,000 X g for 90 min. The resulting particulate-free cellular fluid will The particles which sedimented at be referred to in the text as “supernatant.” Physical-chemical studies and this speed are referred to as “chromatophores.” electron microscopy indicate that the chromatophores are of uniform size with a diameter of approximately 300 A. (10). The chromatophores were washed in 20 ml. of a solution containing 0.4 M glucose and 0.1 Tris buffer at pH 7.8. The glucose and Tris supernatant from the above washing will be referred to as “chromatophore washings.” The washed chromatophores were then suspended in 10 ml. of the above glucose-Tris solution, and 0.5 ml. of this suspension was used in each photophosphorylation reaction mixture.
Photophosphorylation The experiments were carried out in an illuminated Warburg apparatus at 20°C. Light intensity was approximately 1500 ft.-candles at the surface of the water. Warburg flasks containing 3 ml. of reaction mixture (see Table I) were flushed with Ns for 10 min. in the dark, adenosine diphosphate (ADP) was tipped in from the side arm, and lights were turned on to initiate photophosphorylation. Flasks were shaken for 30 min., and aliquots were removed for inorganic phosphate analyses (11) both before initiation of photophosphorylation and immediately after the lights had been turned off at the termination of the experiment.
BacteriochlorophyEl Determination Because of its instability to light in organic solvents, bacteriochlorophyll is usually converted to the more stable bacteriopheophytin for quantitative determination. The bacteriochlorophyll content of reaction mixtures was determined as pheophytin as described by French (12). Also, measurements were made of the amount of absorption of reaction mixtures at 810 mF, which is an absorption maximum of in vivo bacteriochlorophyll. After a number of such measurements it became apparent that an optical density of one at 810 rnp (l-cm. light path) of a 3-ml. reaction mixture was equivalent to 0.06 mg. bacteriochlorophyll. Thereafter, the absorption at 810 rnp was measured, and bacteriochlorophyll was calculated from the above relationship.
Autoradiography
and IdentiJication
In order to identify the products formed, take place in the presence of P3a-labeled stopped by the addition of 12 ml. of boiling chromatographed in two dimensions using
of ATP
photophosphorylation was allowed to orthophosphate. The reactions were ethanol, and the resulting extract was phenol-water and butanol-propionic
170
ANDERSON AND FULLER
acid as the solvents (13). X-ray film was placed on the papers, and the film was developed after suitable exposure. Compounds on the paper were identified by cochromatography with known material.
RESULTS
Chromatophores were capable of a light-dependent esterification of ADP by inorganic phosphate with the concomitant formation of ATP as indicated in Eq. (1). Frequently hexokinase was added to the reaction ADP+Pi
chromatophores light
hexokinase
ATP + glucose _____f
--t ATP
glucose 6-phosphate
+ ADP
(2)
mixture in order to regenerate the phosphate acceptor [Eq. (2)]. Figure 1 is an autoradiograph of photophosphorylation in the presence of Pa*labeled inorganic phosphate. The only product formed in the absence of the “hexokinase trap” is ATP. Components of Photophosphorylution The components of photophosphorylation are indicated in Table I. The omission of light, ADP, Mg++ ions, supernatant, and the anaerobic condition all strikingly reduce the amount of photophosphorylation. Whereas there is an absolute requirement for light and ADP, the chromatophore preparation probably contains trace amounts of Mg++ ions, and supernatant; therefore, the data show only a stimulation by, rather than an absolute requirement for these two latter components. Phosphorylation occurred at maximum capacity with as little as 1 rmole ADP in the presence of hexokinase and glucose. Charaetmistics of Photophosphorylatio A curve demonstrating the effect of pH on the activity of photophosphorylation is shown in Fig. 2. Phosphorylation occurs over a wide pH range with maximum activity at pH 8.0. This is similar to the pH optimum that has been obtained for photophosphorylation by spinach chloroplasts (6, 14). Light saturation was obtained between 200 and 400 ft.-candles. This is in contrast to the 1500 ft.-candles required for light saturation in the Hill reaction of chloroplasts (15). Jagendorf and Avron (7) have found
Chromatium LParticles
Pia* 60 min. in light particles (144,000 x G)
Fro. 1. Autoradiograph of photophosphorylation. The reaction was carried out by adding 1.5 X 10’ counts/min. PS* to a reaction mixture which contained 0.28 mg. bacteriochlorophyll and allowed to incubate in the light for 60 min. Aliquot (0.01) applied at lower right-hand corner. Nine thousand counts were found in ATP and 5000 in orthophosphate. TABLE I Components of Photophosphorylation Each Warburg vessel contained the following additions except where indicated: 7.5 &moles ADP at pH 7.8, 10 pmoles MgC12 , 20 amoles phosphate at pH 7.8, 150 pmoIes glucose, 100 pmoles Tris buffer at 7.8, 0.3 ml. supernatant, 4 mg. of crude hexokinase (Sigma Chemical Co.), and 0.5 ml. of chromatophore preparationcontaining0.42 mg. bacteriochlorophyll. Illuminated for 30 min. at 20°C. in a nitrogen atm. Micromoles of Pi esterified/30 min.
Component omitted
None Supernatant Q++ 1 instead of 7.5 pmoles of ADP ADP Light Anaerobic conditions 171
12 5 3 11 1 0 5
172
ANDERSON
pH
OPTIMUM
AND
FULLER
OF PHOTOPHOSPHORYLATION
Fra. 2. pH optimum of photophosphorylation. Assays were made as described in Table I except pH was varied by suspending chromatophores in 0.4 M glucose and 150 pmoles of either Tris or glycylglycine buffer at various pH values. Vessels contained 0.22 mg. bacteriochlorophyll, and the reaction was allowed to proceed for 1 hr.
a requirement of up to 4000 ft.-candles to saturate photophosphorylation by spinach chloroplasts. The proportionality of the amount of chromatophores and rate of phosphorylation is shown in Fig. 3. Phosphorylation is a linear function of chromatophore addition over the range studied. Rates generally ranged between 50 and 100 pmoles of phosphate esterified/hr./mg. bacteriochlorophyll. In vitro phosphorylation for spinach chloroplasts has been reported as high as 400 ccmoles/mg. chlorophyll/hr. (6, 7). The slow rate of phosphorylation with these chromatophores may be simply a reflection of the slower rate of over-all photosynthesis in these cells. Rupture of cells and isolation of chromatophores in a solution containing 0.4 M glucose and 0.1 M Tris at pH 7.8 allows a better separation of cellular components and produces more active chromatophores with higher phosphorylation activity per unit of bacteriochlorophyll than is
PHOTOSYNTHESIS
BY
ISOLATED
CHROMATOPHORES
173
,-
mg
OF BACTERIOCHLOROPHYLL
3. Proportionality of photophosphorylation with chromatophore concentration. Assay as described in Table I except bacteriochlorophyll content varied as indicated. Reaction time was 1 hr. FIG.
obtained by sonication in dilute buffer solutions. After isolation, the chromatophores can be washed as many as three times with distilled water and stored at 0°C. for several days without any loss of activity. The isolated chromatophores retain up to 75% of their activity after lyophilization and storage over P206 for several months. E$ect of Supernatant on Photophosphorylation Frenkel (8) has reported that photophosphorylation by particulates from Rhodospirdlum rubrum was stimulated by the addition of catalytic amounts of reduced diphosphopyridine nucleotide (DPNH) or succinate. Phosphorylation by chromatophores of Chromatium also is stimulated by DPNH and succinate (see Table II). The addition of particulate-free supernatant from ruptured cells, however, causes a greater stimulation than is obtained with DPNH or succinate. Boiling this supernatant for 90 sec. decreased its stimulatory effect to the level shown by DPNH and succinate additions. This indicates that the supernatant might contain at least two stimulatory substances: one of these being heat-stable and acting in a manner similar to DPNH or succinate; the other a heatlabile protein. A confirmation of this proposal is demonstrated by the data in Tables III and IV. A difference in the origin of the two stimulatory factors of the super-
174
ANDERSON
AND
FULLER
natant is indicated by the data of Table V. The first four lines present the effects of succinate, supernatant, and boiled supernatant on photophosphorylation. The fifth line illustrates the stimulation obtained upon addition of chromatophore washings. A further increase of photophosphorylation with chromatophore washings was obtained upon addition of succinate. The stimulatory effect of chromatophore washings was TABLE Supernatant
II E$ect
Assay as described in Table I except that supernatant was omitted indicated in the table. Vessels contained 0.37 mg. bacteriochlorophyll. Additions
Micromoles of Pi esterified/30 min.
No additions 0.1 pmole DPNH 1.O pmole succinate 0.3 ml. supernatant 0.3 ml. boiled supernatant
7 11 12 16 11
unless
TABLE III on Photophosphorylation Assay as described in Table I except as indicated in the table. Vessels contained 0.31 mg. bacteriochlorophyll. Efect
of Supematant
Milliliters
Micromoles of succinate
0.00
0.0 1.0
4.2 6.4
of supernatant
0.10 0.30 0.50 Micromoles of Pi esteriiied/dO min.
7.5 7.7
12.7 11.9
12.0 12.3
0.50 (boiled)
8.2 7.7
TABLE IV Sulfate Fractionated Supernatant on Photophosphorylatim Assay as described in Table I except the supernatant was fractionated with ammonium sulfate. A 3345yo ammonium sulfate fraction was collected, resuspendedin 10 ml. of 0.1 M Tris at pH 7.8, and dialyzed against 0.001 M Tris buffer at pH 7.8. Vessels contained 0.31 mg. bacteriochlorophyll.
Efect
of Ammonium
Milliliters
of (NH&SO4 fraction
Micromoles of succinate
0.00
0.25 0.50 1.04 1.4 Micmmoles of Pi esteriIicd/30 min.
0.0
3.8 5.8 65
5.4 7.3 74
1.0 Per cent activity succinate
without
8.7 11.0 79
9.5 12.6 75
9.0 12.5 72
(bzd)
3.4 5.7 60
PHOTOSYNTHESIS
BY
ISOLATED
TABLE
V
of Supernatant
Origin
Factors
Assay as described in Table I except supernatant Vessels contained 0.39 mg. bacteriochlorophyll. Additions
None 1 pmole succinate 0.3 ml. supernatant 0.3 ml. boiled supernatant 1.0 ml. chromatophore washings* 1.0 ml. chromatophore washings plus 1 #mole succinate 7. 1.0 ml. boiled chromatophore washings Experimental
section
omitted
unless indicated.
Micromoles of Pi esterilied/30 min.
1. 2. 3. 4. 5. 6.
a he
175
CHROMATOPHORES
6.0 9.0 17.5 10.0 9.5 15.0 5.8
for definition.
completely destroyed by 90 sec. of boiling. These data indicate that the heat-labile protein was indeed coming from the chromatophores, but that the heat-stable factor is apparently not bound to these chromatophores and may be originating from other cytoplasmic sources. Several protein factors which participate in photosynthesis have been isolated from spinach chloroplasts. Among these are an enzyme which permits a net reduction of diphosphopyridine nucleotide (DPN) by grana (16), a TPNH diaphorase (17)) and a factor stimulating photophosphorylation (14). The protein factor from spinach chloroplasts which stimulates photosynthetic phosphorylation was prepared as described by Avron and Jagendorf (14). This factor from spinach chloroplasts failed to stimulate photophosphorylation by isolated chromatophores of Chromatium.2 Nucleotide Specificity The results in Table VI are a summary of some experiments on nucleotide specificity. Phosphorylation with ADP is stimulated by supernatant as was expected. There was no phosphorylation with adenylic acid (AMP) together with a small amount of ATP. This indicates the absence of adenylic kinase in the chromatophores. The addition of supernatant to reaction mixtures of chromatophore containing AMP, together with a small amount of ATP, allowed for as much esterification as was obtained with ADP, indicating the presence of adenylic kinase in the supernatant. Other assays for adenylic kinase demonstrated the ability of the * Anderson,
I. C., and Fuller,
R. C., unpublished.
176
ANDERSON AND FULLER
TABLE VI Nucleotide Specificity Assay as described in Table I except hexokinase was omitted and ADP, other nucleotides, and supernatant were added’ as indicated below. Vessels contained 0.38 mg. bacteriochlorophyll. Additions p??WleS
20 ADP 20 ADP 10 AMP 10 AMP 20 IDP 20 IDP
Micromoles
+ 0.3 ml. supernatant + 1 ATP + 1 ATP + 0.3 ml. supernatant + 0.3 ml. supernatant
of Pi &&&d/30
min.
4.5 17.2 0.5 17.4 4.0 9.0
supernatant to convert mixtures of AMP and ATP to ADP, whereas the chromatophores lacked this ability. The last two lines of the table illustrate that inosine diphosphate (IDP) can serve as a source of phosphate acceptor. Inosine triphosphate was identified as the product by paper chromatographic and autoradiographic methods. Inosine diphosphate is not as good a phosphate acceptor as is ADP. DISCUSSION
Short periods of sonic oscillation in the presence of levigated alumina ruptured Chromatium cells. Small pigmented particles were liberated from the cells during this process. These particles were of uniform size with a diameter of 300 A. and have been referred to as chromatophores. Electron micrographs of whole cells have indicated that there is variation in the size of in vivo particles. However, most of the particles present in whole cells are of the size found in the chromatophore preparation (10). Further morphological and physiological studies of the particles of Chromalium are under way at this laboratory. These small particles of Chromutium carry out a light-dependent formation of ATP from ADP and inorganic phosphate, and this esterification requires Mg++ ions. Photophosphorylation by the chromatophores was stimulated by the addition of small amounts of the particulate-free supernatant which was obtained from the ruptured cells. It was shown that the supernatant contained at least two stimulatory substances. One of these substances was heat-stable and could be replaced by catalytic amounts of either DPNH or succinate. Frenkel (8) also has reported the stimulation of photophosphorylation by catalytic amounts
PHOTOSYNTHESIS
BY
ISOLATED
CHROMATOPHORES
177
of DPNH or succinate with particulate preparations from R. rubrum. The other stimulatory substance of the supernatant was nondialysable and, therefore, it was assumed to be a protein. The two stimulatory substances of the supernatant had different intracellular origins. The protein factor appeared to come from the chromatophores during their isolation and washing. The heat-stable component did not come from the chromatophores, but originated from some other cellular source. These observations give an indication of the function of the two stimulatory components. The protein factor apparently is associated with the chromatophore in viva. It may be an enzyme which is involved in photophosphorylation and which is partially solubilized during isolation of chromatophores. The heat-stable component of the supernatant presumably functions as an oxidation-reduction balancing agent, since it may be replaced by catalytic amounts of DPNH or succinate. A generalized outline of the steps of photophosphorylation is presented by the following equations (8). The primary photochemical acceptor of the reducing portion of water is represented by X and the acceptor of the oxidizing portion of water is represented by Y. Hz0 + X (ox.) + Y (red.) H.X-tH0.Y
Concomitant
light chromatophores
) H.X + HO-Y
Electron carriers > X (ox.) + Y (red.) + HOH in chromatophores
(3) (4)
with Eq. (4): ADP + Pi
Coupling between electron carriers and phosphorylation
+ ATP
(5)
Frenkel (8) postulated from his studies with R. rubrum that succinate and DPNH have a “sparking” effect by reducing some intermediate of the cyclic electron-carrier system, Eq. (4). An alternative explanation is that DPNH and succinate act by balancing the primary photochemical acceptors X and Y. In viva, X and Y are balanced; that is, if X is mainly oxidized then Y is mainly reduced and vice versa. This balance would be unpoised if, during the process of isolation of chromatophores, the photochemical acceptor of the oxidizing portion of water (Y) were to become oxidized. Under these conditions a certain portion of the photochemical acceptors would be unable to accept the reducing and oxidizing
178
ANDERSON AND FULLER
parts of water. The present data appear to support this alternative explanation. Oxygen is a potent inhibitor of photophosphorylation, and it is possible that the primary photochemical acceptor of the oxidizing portion of water would be susceptible to autoxidation. Succinate and DPNH appear to be nonspecific substances which are capable of acting on (or balancing) some oxidation-reduction system. Also, as seen in Table IV, the effect of succinate was independent of the degree of saturation of the chromatophores by the protein factor. This would indicate that a certain constant proportion of some process of photophosphorylation was nonfunctional in the absence of succinate. Succinate and DPNH in the bacterial systems, and such compounds as ascorbate in higher plant chloroplasts, may function, therefore, only as redox balancing agents to protect a photochemical acceptor from autoxidation in vitro and have no significant function as cofactors in wivo. SUMMARY
Chromatophores were isolated from the photosynthetic purple sulfur bacterium, Chromatium strain D. These particles were capable of a lightdependent esterification of ADP and inorganic phosphate with the resulting formation of ATP. Phosphorylation was stimulated by the supernatant from the ruptured cells. The supernatant contained two stimulatory substances: one a heat-stable factor such as DPNH or succinate, and a second labile protein. No additional cofactors were required. ADDENDUM After this paper had been submitted, a paper by J. Newton and M. Kamen, Biochim et Biophys. Acta 26, 462 (1957), on photophosphorylation by Chromatium particles appeared. The same sized pigmented particle appeared to have been studied in both laboratories, and, therefore, some of the results presented in this paper become repetitive with those of Newton and Kamen. There is, however, a great difference in the photophosphorylative capacity of the particulates studied. Particulate8 isolated as described in this paper were capable of a minimum of 20 times as much photophosphorylation per unit of bacteriochlorophyll as those of Newton and Kamen. This observation becomes important when considering the necessary components of any electron-transport system for photophosphorylation.
REFERENCES 1. ARNON, D. I., WHATLEY, F. R., AND ALLEN, M. B., J. Am. Chem. Sot. 76, 6324 (1954).
2. WHATLEY, F.R., ALLEN, M.B., ROSENBERG,L. L., CAPINDALE,J. B., AND ARNON, D. I., Biochim. et Biophys. Acta 20, 462 (1956).
PHOTOSYNTHESIS
BY
ISOLATED
CHROMATOPHORES
179
3. FRENKEL, A. W., J. Am. Chem. Sot. 76, 5568 (1954). 4. SCHACHMAN, H. K., PARDEE, A. B., AND STANIER, R. Y., Arch. Biochem. Biophys 33, 245 (1952). 5. FULLER, R. C., AND ANDERSON, I. C., Plant Physiol. 32, Suppl. xvi (1957). 6. ARNON, D. I., WHATLEY, F. R., AND ALLEN, M. B., Nature 27, 182 (1957). 7. JAGENDORF, A. T., AND AVRON, M., Plant Physiol. 32, Suppl. iv (1957). 8. FRENKEL, A. W., J. Biol. Chem. 222, 823 (1956). 9. HENDLEY, D. D., J. Bacterial. 70, 625 (1955). 10. BERGERON, J. A., ANDERSON, I. C., AND FULLER, R. C., Plant Physiol. 32 Suppl. xvi (1957). 11. LOWRY, O., AND LOPEZ, J., J. Biol. Chem. 162, 421 (1946). 12. FRENCH, C. S., J. Gen. Physiol. 23, 483 (1940). 13. BENSON, A. A., BASSHAM, J. A., CALVIN, M., GOODALE, T. C., HAAS, V. A., AND STEPKA, W., J. Am. Chem. Sot. 72, 1710 (1950). 14. AVRON, M., AND JAGENDORF, A. T., Nature 179, 4% (1957). 15. HILL, R., AND SCARISBRICK, R., Nature 146, 61 (1940). 16. SAN PIETRO, A., AND LANGE, H. M., Science 124, 118 (1956). 17. AVRON, M., AND JAGENDORF, A. T., Arch. Biochem. Biophys. 66, 475 (1956).