Photophosphorylation in Halobacterium Halobium

Proc. Nat. Acad. Sci. USA Vol. 71, No. 4, pp. 1234-1238, April 1974

Photophosphorylation in Halobacterium halobium (Halobacteria/photosynthesis/bacteriorhodopsin/chemiosmotic theory/active transport) ARLETTE DANON* AND WALTHER STOECKENIUSt * The Weizmann Institute of Science, Rehovot, Israel; and t Cardiovascular Research Institute and Department of Biochemistry and Biophysics, University of California, San Francisco, Calif. 94143 Communicated by Daniel I. Arnon, November 14, 1973

ABSTRACT Halobacterium halobium cells grown un- We show here that apparently purple membrane-containing der semi-anaerobic conditions convert part of their cell H. halobium cells are capable of photophosphorylation using a membrane into "purple membrane" which contains a which is different in its first energy conversion rhodopsin-like protein, bacteriorhodopsin. Under anaero- mechanism bic conditions in the dark the ATP content of such cells steps from that used by chlorophyll-containing organisms. decreases sharply. Either light or oxygen restores the ATP MATERIALS AND METHODS content to the original level. The light effect is mediated by the purple membrane. Inhibitors of the respiratory Cell Strain and Growth Conditions. For all experiments chain abolish the oxygen response but do not affect the Halobacterium halobium R, was used (5). Cells were grown at light response. Uncouplers, which function as proton on a gyratory shaker in a synthetic medium (6) supple- translocators, abolish the light response. These results in- 370 dicate that the purple membrane functions as a light- mented with 2% malate. The amount of purple membrane driven proton pump and the cells use the resulting chemi- formed depends on illumination (7, 8, 3). Illumination was osmotic gradient for ATP synthesis. provided by cool white fluorescent lamps and kept constant at 5 to 6 X 104 ergs/cm2 per sec. Aeration was controlled by The extreme halophile Halobacterium halobium, when grown varying the amount of growth medium in the culture vessels. at low 02 concentrations in the light, synthesizes a rhodop- At a medium to vessel volume ratio of 1:15, the formation of sin-like protein-bacteriorhodopsin-which forms distinct the purple membrane was suppressed; at the ratio of 1:2, patches in the surface membrane of the cell and may occupy optimal purple membrane yields were obtained. The cultures about 50% of the total membrane area (1-3). Bacteriorho- a cell density of about 108 cells per ml and nm were inoculated at dopsin has a broad absorption maximum around 560 grown until the cell density of 6 to 10 X 108 had been reached. which lends a deep purple' color to the isolated membrane patches. The patches have been termed tlih purple membrane. Analytical Techniques. Purple membrane content of the Bacteriorhodopsin is the' only protein found in the purple cells was determined on 40-ml aliquots of the culture. Cells membrane which, in addition, contains only 25% lipids. were harvested by centrifugation and lysed by dialysis against When the purple membrane is exposed to a flash of visible distilled water (5). The lysate was centrifuged at 50,000 X g light, the absorption maximum shifts to 415 nm with a fast for 30 min and the pellet resuspended in 2-5 ml of distilled return to the long wavelength form in the dark (Cone, R. A., water; any remaining turbidity was removed by centrifuga- Fein, A., and Stoeckenius, W., unpublished). This transient tion at 700 X g for 5 min. To one-half of the sample 0.1 M bleaching is accompanied by a cyclic release and uptake of cetyltrimethylammonium bromide (CTAB) pH 8.0 was added protons. Under continuous illumination the pigment ap- to give a final concentration of 0.01 M; this shifts the purple parently oscillates rapidly between the long and short wave- membrane absorption maximum to 369 nm (1). The difference length form and, when it is incorporated into the surface spectrum between the CTAB-treated and the untreated membrane of the cell, the concomitant release and uptake of sample was obtained on a Cary 14 spectrophotormeter protons occurs as a vectorial reaction, resulting in a net out- equipped with the accessory for scattering samples. The purple ward translocation of protons from the cells (3). The resultant membrane concentration is expressed as AOD 570 nm/mg of electrochemical gradient can presumably be used to satisfy cell protein. Protein was determined by the Lowry technique. the energy requirements of the cell according to Mitchell's ATP was determined in 0.2-ml samples of the cell suspen- chemiosmotic theory for energy coupling (4). sion, which were rapidly diluted into 1.8 ml of 0.02 M boiling Tris - HCl buffer (pH 7.4 at room temperature). Boiling was continued for an additional 5 min, and the sample cooled in ice. The luciferin-luciferase assay was used according to ATP concentrations Abbreviations: ATPase, adenosine triphosphatase; dC'CP, keto- Stanley and Williams (9). Negligible malononitrile 3-chlorophenylhydrazone(carbonylcyanide 3-chlo- were found in the suspension medium after separation from rophenylhydrazone); QTAB, cetyltrimethylammonium bromide; the cells. DCCD, N,N'-dicyclohexylcarbodiimide; DCMU, 3-(3,4-dichlo- Assay Conditions. Cells were harvested by centrifugation rophenyl)-1,1-dimethylurea; DNP, 2-dinitrophenol; FCCP, and resuspended to an OD of 0.5 at 640 nm in a salt solution ketomalononitrile 4-trifluoromethoxyphenylhydrazone(carbonyl- but without the nutrients. 4-trifiuoromethoxyphenylhydrazone); NQNO, 2-n-nonyl- identical to the growth medium cyanide The cells were kept under aeration for at least 2 hr at 370 be- 4-hydroxyquinoline-N-oxide; PMS, N-methylphenazonium This methosulfate (phenazine methosulfate). fore 20-ml samples were transferred to the assay vessel. 1234 Proc. Nat. Acad. Sci. USA 71 (1974) Photophosphorylation in Halobacterium halobium 1235

OA_ 570 N2 N2 02 I 11l w A B 12 0

02_ b a a 0 0. 6mMM 011;* -- a 4 KCN 0~~~~KC

0 10- I~~ I I -0.2 -V , As _ _. _ _ 4 5 369 5 '10' 60 80 1O 60 80 350 450 550 Minutes nm FIG. 1. Difference spectrum of unbleached minus CTAB- FIG. 3. Effect of an electron transport inhibitor on the light bleached membrane preparation of vigorously aerated (a) and (A) and 02 (B) response of cells. Only the 02 response is affected. poorly aerated cells (b). The maximum at 570 nm and minimum ATP per mg of protein at time 0 = 15.6 nmol. AOD 570 nm/mg at 369 nm, which are characteristic for the purple membrane, are of protein = 0.101. present only in the preparation from poorly aerated cells. RESULTS magnetically stirred glass vessel had a capacity of 50 ml and Fig. 1 shows an example of the difference spectra used for was surrounded by a water bath kept at 37°. For some experi- determination of purple membrane content of the cells. It ments it was equipped with a pH electrode. Provisions for should, however, be noted that the total amount of material bubbling the cell suspension with N2 or 02 and for the removal in the preparations from cells containing little purple mem- of samples were made. In some experiments cells were sparged brane is small because the centrifugation at 50,000 X g for 30 with N2 in a larger vessel and then transferred anaerobically min will almost exclusively sediment the purple membrane to the assay vessel flushed with N2. from the cell lysate and most of the other surface membrane Light sources were either a 250-W flood lamp or a 500-W fragments-the red membrane (5, 10)-remain in suspension. tungsten filament lamp in a slide projector. Balzers broad- Samples from cells containing high concentrations of purple band and narrow-band filters (Filtraflex K 1-7 and B10) were membrane therefore scatter considerably more light, and the used. A 5-cm thick flat-sided flask filled with water and cooled OD measured becomes dependent on the geometry of the spec- by a fan served as a heat filter. Light intensity was measured trophotometer used. Differences in scattering between CTAB- with a Yellow Springs light meter (YSI model 65). treated and untreated reference also become significant. Nevertheless, the technique may be used for a rough comparison of the amount of purple membrane in the cultures, provided the same spectrophotometer is used in all experiments and care is taken to minimize the effects due to differences in light scattering. The highest values of AOD 570 nm/mg of protein for cells grown at low PO2 as described under Methods are about 0.4. At this concentration, a large part of the cell surface membrane consists of purple membrane (3). The chilling and centrifugation during harvesting sharply reduces the ATP content of cells. The loss is recovered when

N2 02 120 B

80 -h-v IC - ~~~~~~~~~~~~~~~I 4e 0 40 -0~~~~~0 - +h-v

0 20 40 60 80 Minutes FIG. 2. Cells incubated in the dark under N2 show a fast drop in ATP content. Only the poorly aerated cells, which contain purple membrane, respond to light (h.av) with an increase of Minutes ATP content (A). Both vigorously aerated (B) and poorly aerated FIG. 4. Effect of phenazine methosulfate (PMS) on the 02 and cells (A) increase their ATP content when 02 is admitted. ATP light response of cells. Only the 02 response is affected. ATP per per mg of protein at time 0: (A) 14.4 nmol; (B) 14.9 nmol. AOD mg of protein at time 0 = 32.5 nmol. AOD 570 nm/mg of protein 570 nm/mg of protein: (A) 0.155; (B) 0.009. = 0.208. 1236 Cell Biology: Danon and Stoeckenius Proc. Nat. Acad. Sci. USA 71 (1974)

TABLE 1. Effects ofinhibitors on ATP increase in N2 H. halobium cells 12 20-Dlo-9 25 jg Light/anaerobic Dark/aerobic DNP2.5 X 10-4M + + FCCP10M ++ ++ at CCCP 10 M ++ N Dio-9 5 ,g/ml + + + DCCD 10-6M ++ ++ L PMS 10-3 M 0 ++ 0to + h V KCN6 X 10-3M 0 ++ o NQNO 5 &g/ml 0 + 20 1 40 I 60o so Antimycin 10 Mg/ml 0 + M] Nes DCMU2X1OM 0 + FIG. 6. Effect of an AT~ase inhibitor on the anaerobic light Valinomycin 5 jug/ml 0 0 response of cells. ATP per mg of protein at time 0 = 23.9 nmol. AOD 570 nm/mg of protein = 0.101. 0 = no inhibition + + = complete inhibition + = >50% inhibition N = not tested For meaning of abbreviations, see first page of this article. The light response apparently does not depend on a func- tioning electron transport chain. Inhibitors of respiration and oxidative phosphorylation such as KCN or 2-n-nonyl-4- the cells are resuspended in basal salt solution and aerated. hydroxyquinoline-N-oxide (NQNO) have no effect on the Similar observations have been made with other bacteria light response of the cells but inhibit the oxygen-dependent (11). In salt solution the cells apparently respire on endog- ATP increase (Figs. 3 and 4 and Table 1). The light response is enous substrate. Respiration in basal salt may continue for sensitive to uncouplers of respiration and adenosine triphos- 20 hr or longer (Bogomolni, R. A., Baker, R. A. & Stoeck- phatase (ATPase) inhibitors. Carbonylcyanide 3-chloro- enius, W., in preparation). phenylhydrazone (CCCP) and other uncouplers, which in- When cells in salt solution are transferred to the assay vessel crease the permeability of membranes and lipid bilayers to and sparged with N2 in the dark, the suspension rapidly be- protons (12-14), abolish the light and the oxygen response comes anaerobic, and within 10-20 min the ATP content of (Fig. 5 and Table 1). Dio-9 and NN'-dicyclohexylcarbodi- the cells drops to approximately 30% of the initial level. It imide (DCCD), inhibitors of the ATPases in chloroplasts, then remains nearly constant with only a small further de- mitochondria and prokaryotic cells (15), also abolish the crease detectable over several hours (Fig. 2). This reduction in light and the oxygen response (Fig. 6 and Table 1). If cells ATP content does not occur when the vessel is illuminated. respiring in the dark are illuminated, respiration is at least After ATP depletion in the dark, illumination with white partially inhibited (3, 16) and a transient 10-20% decrease light at about 106 erg/cm2 per sec causes a rapid rise in the in ATP level is observed (Fig. 7). Switching off the light again ATP level to the original value or slightly higher. This new causes a similar transient decrease in ATP content (not level is maintained until the light is turned off. The ATP con- shown). The ATP level of cells respiring in the light is usually tent then rapidly drops again to the dark value (Fig. 2A). slightly higher than in aerobic dark or anaerobic light cells. This effect of alternating light and dark conditions can be Valinomycin has no effect on either the respiration or the repeated several times without change in the size of the re- light-induced ATP increase. This is not surprising because sponse. Essentially the same increase in ATP content of the the permeability of the cell membrane to K+ has been found cells is observed when oxygen is admitted to the suspension to be high, even in the absence of valinomycin (Passow, H. instead of light. The light but not the 02 effect is dependent and Stoeckenius, W., unpublished). on the presence of purple membrane in the cells (Fig. 2B). N2 02 N2 160I-

N2 02 120t 120 1'"M CCCP

< 80 + h-v 80 0 0 40 40

+h!v ° 2WV 100 120 140 0 20 40 60 so) Minutes Mkiutes FIG. 7. Transition from aerobic dark to aerobic light condi- FIG. 5. Effect of an uncoupler on the light and 02 response of tions and subsequently anaerobic light conditions causes transient cells. Both are inhibited. ATP per mg of protein at time 0 = 25.5 decreases in ATP level of cells. ATP per mg of protein at time 0 = nmol. AOD 570 nm/mg of protein = 0.101. 32.5 nmol. zOD 570 nm/mg of protein = 0.208. Proc. Nat. Acad. Sci. USA 71 (1974) Photophosphorylation in Halobacterium halobium 1237

The light-induced increase in ATP content depends on the presence of purple membrane in the cells and on light intensity. Fig. 8 shows the effect of narrow-band interference filters with a half-width of 10 nm. Intensity of the transmitted light was kept constant for all wavelengths tested at about 3.0 X in ATP content is 103 erg/cm2 per sec. The increase compared 0 to the absorption spectrum of the lysate of whole cells and 0 the absorption spectrum of the purple membrane fraction prepared from the lysate. The ATP levels attained by the iaXI- *6 cells are roughly proportional to the amount of light energy E absorbed by the purple fraction and not to the absorption of the whole cell lysate. The absorption spectrum of the latter is dominated by bacterioruberin, the main carotenoid pig- 450 500 550 600 650 700 ment present in these cells (17). This shows that only the nm in increas- FIG. 8. Comparison of the absorption spectrum of cell sus- light absorbed by the purple membrane is effective of ATP at is as pensions of H. halobium R1 with the amount produced ing the ATP level in the cells. The correspondence good different wavelengths. Curve a: absorption spectrum of the cell as can be expected from spectroscopic measurements on a suspension after dialysis against distilled water to lyse the cells strongly scattering suspension which also contains large and reduce light scattering. Curve b: absorption spectrum of the amounts of other pigments with overlapping absorption purple membrane fraction prepared from the cell lysate by cen- bands. trifugation at 50,000 X g for 30 min. The stippled bars indicate the increase in ATP content of the cells after illumination for 10 min DISCUSSION at the wavelength indicated. H. halobium has been known as an obligate aerobic organism (18). Under anaerobic conditions in the dark, the cells rapidly use the remaining 02 (8, 3) and deplete their energy reserve We, therefore, tentatively conclude that H. halobium can to a limiting value of about 5 nmol of ATP per mg of protein. use light energy to synthesize ATP-in other words, that it can Either 02 or light will restore the ATP content of the cells carry out photophosphorylation. to the same or a slightly higher level than found under aerobic The results reported here are remarkably similar to those conditions at the beginning of the experiment. This presum- obtained with other photosynthetic organisms; however, ably indicates ATP synthesis with either respiration or light H. halobium does not contain chlorophyll and photophos- as alternative sources of energy. The extent of the ATP phorylation is mediated by the purple membrane. Only cells synthesis cannot, of course, be estimated from the ATP con- which contain purple membrane show the increase in ATP tent of the cells because the rate of ATP use by the cells is content in the light and only light absorbed by the purple unknown and is probably different under light and dark, membrane is effective. aerobic and anaerobic conditions. The fast decrease of the It has been shown earlier (3) that purple membrane-con- ATP level under anaerobic conditions in the dark indicates a taining cells of H. halobium, in the absence of other energy high rate of use. However, the nearly constant level reached sources, can generate a chemiosmotic gradient across their after a few minutes, which typically amounts to <30% of cell membrane when they are exposed to light. Evidence has the original level, implies that the cells shut off most of their been provided that this is due to a rapid light-driven cycling energy-requiring functions before the ATP reserve is com- of b cteriorhodopsin between a long and short wavelength pletely exhausted. These functions are apparently reactivated form with concomitant transport of protons across the cell when either 02 or light becomes again available as energy membrane. Alternatively, the cells can generate such a source, because return to anaerobiosis or darkness again gradient through respiration when oxidizable substrate and leads to a rapid decline of ATP to the 30% level. The general 02 are available. Further evidence for the function of bac- phenomenon that cells maintain an ATP reserve by drasti- teriorhodopsin as a light-driven proton pump is provided cally reducing metabolic functions when their energy supply by the incorporation of the purple membrane into lipid vesicles is cut off has been observed in a wide variety of prokaryotic and the demonstration that this model system generates a and eukaryotic organisms (19). The other possible inter- proton gradient in the light (23). We have postulated (3) pretation of our observations that light and/or oxygen sharply that the gradient in H. halobium drives metabolic processes decrease the use of ATP appears highly unlikely, simply such as ion translocation and ATP synthesis in accordance because there is no apparent energy source for ATP synthesis with Mitchell's chemiosmotic theory of energy coupling under anaerobic conditions in the dark. Very similar changes (4). The experiments reported here bear out part of this in ATP level under anaerobiosis and aerobiosis in the light prediction. and in the dark have been reported for the facultative photo- The action of specific inhibitors of electron transport and troph Rhodospirillum rubrum and for Chromatium D (20-22). phosphorylation and of uncouplers contributes further evi- The time resolution in our experiments is not sufficient to dence for the postulated mechanism. The effect of the un- quantitatively compare the rates of change in ATP content; couplers DNP, FCCP, and CCCP is explained by their action they appear to be approximately four times slower in H. as proton translocators which collapse the gradient and inhibit halobium. The steady state amounts of ATP per mg of protein both photophosphorylation and oxidative phosphorylation. are comparable and so are the light intensities used. More- Dio-9 and DCCD, known as inhibitors of the ATP-synthesiz- over, in H. halobium the effect of 02 and light on the rate of ing enzyme in bacteria, chloroplasts and mitochondria (15, ATP increase and the level reached are virtually the same. 24), as expected abolish both the 02 and light effect on ATP 1238 Cell Biology: Danon and Stoeckenius Proc. Nat. Acad. Sci. USA 71 (1974)

content of the cells; the proton gradient, however, is not converting system containing only one new protein. This affected (Bogomolni, R. A., Baker, R. A., and Stoeckenius, will have to be further explored. The advantage for the in- W., in preparation). This may indicate that both the light- vestigating scientist is that this system allows one to easily and the Orgenerated gradients use the same phosphorylating and cleanly separate not only conceptually but also prepara- enzyme; however, two separate light- and 02-controlled tively the first energy conversion mechanism from the rest ATPases which are both inhibited by Dio-9 and DCCD of the cell's energy converting systems. cannot be ruled out. DCMU, a specific inhibitor of an early event in the chain of redox reactions in Photosystem II (25), We are grateful to Drs. Mordechai Avron, Elisha Tel-Or, Michel Revel, Henri Atlan and Zippora Elchanan for critical discussion as expected, has no effect on the light-driven ATP increase and help with equipment and chemicals. This work was supported in H. halobium. (The partial inhibition of the 02 effect at the by NIH Program Project Grant HL 06285; it was begun at the low concentration used here remains unexplained.) This University of California, San Francisco, and continued at the indicates that the early events in purple membrane-mediated Weizmann Institute of Science. W.S. also thanks the NASA for photophosphorylation are different from the noncyclic path- financial support to visit the Weizmann Institute during the way of chlorophyll-mediated light energy conversion. We course of this work. also exclude cyclic electron flow because inhibitors of the 1. Oesterhelt, D. & Stoeckenius, W. (1971) Nature New Biol. electron transport chain such as NQNO and antimycin have 233, 149-152. no effect on the photophosphorylation, but inhibit oxida- 2. Blaurock, A. E. & Stoeckenius, W. (1971) Nature New Biol. tive phosphorylation. Also the purple membrane contains 233, 152-155. 3. Oesterhelt, D. & Stoeckenius, W. (1973) Proc. Nat. Acad. only one protein, bacteriorhodopsin. Therefore, all observa- Sci. USA 70, 2853-2857. tions so far argue against participation of redox reactions and 4. Mitchell, P. (1972) J. Bioenerget. 3, 5-24. an electron transport chain in photophosphorylation by H. 5. Stoeckenius, W. & Kunau, W. H. (1968) J. Cell. Biol. 38, halobium. 337-357. The reactions of light-driven ATP synthesis are thus very 6. Onishi, H., McCance, M. E. & Gibbons, N. E. (1965) Can. J. Microbiol. 11, 365-373. different from those observed in chlorophyll-containing or- 7. Oesterhelt, D. (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, ganisms (26). They are, however, easily understood if we 1554-1555. assume that a rapid light-induced and dark-reversible con- 8. Danon, A. & Stoeckenius, W. (1972) NASA Symp. Extreme formational change in bacteriorhodopsin transports protons Environments. Mechanisms of Microbial Adaption (Ames Research Center, Moffett Field, California, June 1972), across the membrane and thus converts light energy into a p. 25. chemiosmotic gradient which can drive ATP synthesis. 9. Stanley, P. E. & Williams, S. G. (1969) Anal. Biochem. 29, Further support for this model is derived from the lipid vesi- 381-392. cles with incorporated purple membrane. Addition of mito- 10. Oesterhelt, D. & Stoeckenius, W. in Biomembranes, eds. chondrial ATPase and hydrophobic proteins to these vesicles Fleischer, S., Packer, L. & Estabrook, R. W., Methods in Enzymology (Academic Press, New York), Vol. 31, in results ina a model system exhibiting light-driven ATP syn- press. thesis (23). 11. Cole, H. A., Wimpenny, J. W. T. & Hughes, D. E. (1967) The natural habitats of Halobacteria are salt flats and stag- Biochim. Biophys. Acta 143, 445-453. nant puddles at the edge of tropical seas where salt concen- 12. Mitchell, P. & Moyle, J. (1967) Biochem. J. 105, 1147- 1162. trations close to saturation are maintained (18). Temperature 13. Hopfer, U., Lehninger, A. L. & Thompson, T. E. (1968) and solar radiation density are high and the water is rich Proc. Nat. Acad. Sci. USA 59, 484-490. in organic materials resulting from the decay of organisms 14. Liberman, E. A. & Topaly, V. P. (1968) Biochim. Biophyjs. which died when their salt tolerance was exceeded. The P02 Acta 163, 125-136. must be low in such an environment. Halobacteria are pro- 15. Harold, F. M. (1972) Bacteriol. Rev. 36, 172-230. 16. Oesterhelt, D. & Krippahl, G. (1973) FEBS Lett. 36, 72-76. tected against the high radiation density by a high content 17. Kelly, M., Norgard, S., & Liaaen-Jensen, S. (1970) Acta of carotenoids. They are often present in such large numbers Chem. Scand. 24, 2169-2182. that the water acquires a deep orange or red color. Any oxygen 18. Larsen, H. (1967) Advan. Microbial Physiol. 1, 97-132. diffusing in from the surface will be used up in the topmost 19. Chapman, A. G., Fall, L., & Atkinson, D. E. (1971) J. Bacteriol. 108, 1072-1086. layer. Halobacteria have apparently adapted to this environ- 20. Sch6n, G. (1969) Arch. Mikrobiol. 66, 348-364. ment by incorporating a pigment into their cell membrane. 21. Welsch, F. & Smith, L. (1969) Biochemistry 8, 3403-3408. that converts light energy and thus provides an alternative 22. Gibson, J. & Morita, S. (1967) J. Bacteriol. 93, 1544-1550. to oxidative mechanisms for energy production. It uses the 23. Racker, E. & Stoeckenius, W. (1974) J. Biol. Chem. 249, most energy-rich part of the prevailing long wavelength 662-663. 24. McCarty, R. E., Guillory, R. J. & Racker, E. (1965) J. Biol. radiation for this purpose and converts it to a proton gradient Chem. 240, PC4822-PC4823. which forms the link to the oxidative energy metabolism 25. Izawa, S. & Good, N. E. (1972) in Photosynthesis and Nitro- of the cell. It should be pointed out that chlorophyll-contain- gen Fixation, ed. San Pietro, A., Methods in Enzymology ing halophile prokaryotes have been isolated from the same (Academic Press, New York), Vol. 24, part B, pp. 355-377. 26. Arnon, D. I., Tsujimoto, H. Y. & McSwain, B. D. (1967) habitat (27). One wonders what advantage bacteriorhodopsin- Nature 214, 562-566. mediated photophosphorylation has that allows it to compete. 27. Raymond, J. C. & Sistrom, W. R. (1967) Arch. Mikrobiol. The reason could be the extreme simplicity of the energy 59, 255-268.