Proc. Nat. Acad. Sci. USAVol. 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 andBiophysics, University of California, San Francisco, Calif. 94143

Communicated by Daniel I. Arnon, November 14, 1973

ABSTRACT Halobacterium halobium cells grown un-der semi-anaerobic conditions convert part of their cellmembrane into "purple membrane" which contains arhodopsin-like protein, bacteriorhodopsin. Under anaero-bic conditions in the dark the ATP content of such cellsdecreases sharply. Either light or oxygen restores the ATPcontent to the original level. The light effect is mediatedby the purple membrane. Inhibitors of the respiratorychain abolish the oxygen response but do not affect thelight response. Uncouplers, which function as protontranslocators, abolish the light response. These results in-dicate that the purple membrane functions as a light-driven proton pump and the cells use the resulting chemi-osmotic gradient for ATP synthesis.

The extreme halophile Halobacterium halobium, when grownat low 02 concentrations in the light, synthesizes a rhodop-sin-like protein-bacteriorhodopsin-which forms distinctpatches in the surface membrane of the cell and may occupyabout 50% of the total membrane area (1-3). Bacteriorho-dopsin has a broad absorption maximum around 560 nmwhich lends a deep purple' color to the isolated membranepatches. The patches have been termed tlih purple membrane.Bacteriorhodopsin is the' only protein found in the purplemembrane which, in addition, contains only 25% lipids.When the purple membrane is exposed to a flash of visiblelight, the absorption maximum shifts to 415 nm with a fastreturn to the long wavelength form in the dark (Cone, R. A.,Fein, A., and Stoeckenius, W., unpublished). This transientbleaching is accompanied by a cyclic release and uptake ofprotons. Under continuous illumination the pigment ap-parently oscillates rapidly between the long and short wave-length form and, when it is incorporated into the surfacemembrane of the cell, the concomitant release and uptake ofprotons occurs as a vectorial reaction, resulting in a net out-ward translocation of protons from the cells (3). The resultantelectrochemical gradient can presumably be used to satisfythe energy requirements of the cell according to Mitchell'schemiosmotic theory for energy coupling (4).

Abbreviations: ATPase, adenosine triphosphatase; dC'CP, keto-

malononitrile 3-chlorophenylhydrazone(carbonylcyanide 3-chlo-

rophenylhydrazone); QTAB, cetyltrimethylammonium bromide;DCCD, N,N'-dicyclohexylcarbodiimide; DCMU, 3-(3,4-dichlo-rophenyl)-1,1-dimethylurea; DNP, 2-dinitrophenol; FCCP,ketomalononitrile 4-trifluoromethoxyphenylhydrazone(carbonyl-cyanide 4-trifiuoromethoxyphenylhydrazone); NQNO, 2-n-nonyl-4-hydroxyquinoline-N-oxide; PMS, N-methylphenazoniummethosulfate (phenazine methosulfate).

We show here that apparently purple membrane-containingH. halobium cells are capable of photophosphorylation using amechanism which is different in its first energy conversionsteps from that used by chlorophyll-containing organisms.

MATERIALS AND METHODS

Cell Strain and Growth Conditions. For all experimentsHalobacterium halobium R, was used (5). Cells were grown at370 on a gyratory shaker in a synthetic medium (6) supple-mented with 2% malate. The amount of purple membraneformed depends on illumination (7, 8, 3). Illumination wasprovided by cool white fluorescent lamps and kept constantat 5 to 6 X 104 ergs/cm2 per sec. Aeration was controlled byvarying the amount of growth medium in the culture vessels.At a medium to vessel volume ratio of 1:15, the formation ofthe purple membrane was suppressed; at the ratio of 1:2,optimal purple membrane yields were obtained. The cultureswere inoculated at a cell density of about 108 cells per ml andgrown until the cell density of 6 to 10 X 108 had been reached.

Analytical Techniques. Purple membrane content of thecells was determined on 40-ml aliquots of the culture. Cellswere harvested by centrifugation and lysed by dialysis againstdistilled water (5). The lysate was centrifuged at 50,000 X gfor 30 min and the pellet resuspended in 2-5 ml of distilledwater; any remaining turbidity was removed by centrifuga-tion at 700 X g for 5 min. To one-half of the sample 0.1 Mcetyltrimethylammonium bromide (CTAB) pH 8.0 was addedto give a final concentration of 0.01 M; this shifts the purplemembrane absorption maximum to 369 nm (1). The differencespectrum between the CTAB-treated and the untreatedsample was obtained on a Cary 14 spectrophotormeterequipped with the accessory for scattering samples. The purplemembrane concentration is expressed as AOD 570 nm/mg ofcell protein. Protein was determined by the Lowry technique.ATP was determined in 0.2-ml samples of the cell suspen-

sion, which were rapidly diluted into 1.8 ml of 0.02 M boilingTris - HCl buffer (pH 7.4 at room temperature). Boiling was

continued for an additional 5 min, and the sample cooled inice. The luciferin-luciferase assay was used according to

Stanley and Williams (9). Negligible ATP concentrationswere found in the suspension medium after separation fromthe cells.

Assay Conditions. Cells were harvested by centrifugationand resuspended to an OD of 0.5 at 640 nm in a salt solutionidentical to the growth medium but without the nutrients.The cells were kept under aeration for at least 2 hr at 370 be-fore 20-ml samples were transferred to the assay vessel. This

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Photophosphorylation in Halobacterium halobium 1235

OA_ 570

b02_

a0

-0.

011;* -- a

2-V4 5 369 5

350 450 550nm

FIG. 1. Difference spectrum of unbleached minus CTAB-bleached membrane preparation of vigorously aerated (a) andpoorly aerated cells (b). The maximum at 570 nm and minimumat 369 nm, which are characteristic for the purple membrane, arepresent only in the preparation from poorly aerated cells.

magnetically stirred glass vessel had a capacity of 50 ml andwas surrounded by a water bath kept at 37°. For some experi-ments it was equipped with a pH electrode. Provisions forbubbling the cell suspension with N2 or 02 and for the removalof samples were made. In some experiments cells were spargedwith N2 in a larger vessel and then transferred anaerobicallyto the assay vessel flushed with N2.

Light sources were either a 250-W flood lamp or a 500-Wtungsten filament lamp in a slide projector. Balzers broad-band and narrow-band filters (Filtraflex K 1-7 and B10) wereused. A 5-cm thick flat-sided flask filled with water and cooledby a fan served as a heat filter. Light intensity was measuredwith a Yellow Springs light meter (YSI model 65).

120

80IC4e40

N2 02B

-h-v- ~~~~~~~~~~~~~~~I

0

-0~~~~~0- +h-v

0 20 40 60 80Minutes

FIG. 2. Cells incubated in the dark under N2 show a fast dropin ATP content. Only the poorly aerated cells, which containpurple membrane, respond to light (h.av) with an increase ofATP content (A). Both vigorously aerated (B) and poorly aeratedcells (A) increase their ATP content when 02 is admitted. ATPper mg of protein at time 0: (A) 14.4 nmol; (B) 14.9 nmol. AOD570 nm/mg of protein: (A) 0.155; (B) 0.009.

N2

a

0.4

N2 02I 11l w

A B0

6mMMKCN 0~~~~KC

10- I~~ I I 0

, As _ _. _ _

'10' 60 80Minutes

1O 60 80

FIG. 3. Effect of an electron transport inhibitor on the light(A) and 02 (B) response of cells. Only the 02 response is affected.ATP per mg of protein at time 0 = 15.6 nmol. AOD 570 nm/mgof protein = 0.101.

RESULTS

Fig. 1 shows an example of the difference spectra used fordetermination of purple membrane content of the cells. Itshould, however, be noted that the total amount of materialin the preparations from cells containing little purple mem-brane is small because the centrifugation at 50,000 X g for 30min will almost exclusively sediment the purple membranefrom the cell lysate and most of the other surface membranefragments-the red membrane (5, 10)-remain in suspension.Samples from cells containing high concentrations of purplemembrane therefore scatter considerably more light, and theOD measured becomes dependent on the geometry of the spec-trophotometer used. Differences in scattering between CTAB-treated and untreated reference also become significant.Nevertheless, the technique may be used for a rough com-parison of the amount of purple membrane in the cultures,provided the same spectrophotometer is used in all experi-ments and care is taken to minimize the effects due to dif-ferences in light scattering. The highest values of AOD 570nm/mg of protein for cells grown at low PO2 as described underMethods are about 0.4. At this concentration, a large part ofthe 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

MinutesFIG. 4. Effect of phenazine methosulfate (PMS) on the 02 and

light response of cells. Only the 02 response is affected. ATP permg of protein at time 0 = 32.5 nmol. AOD 570 nm/mg of protein= 0.208.

Proc. Nat. Acad. Sci. USA 71 (1974)

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1236 Cell Biology: Danon and Stoeckenius

TABLE 1 . Effects of inhibitors on ATP increase inH. halobium cells

Light/anaerobic Dark/aerobic

DNP2.5 X 10-4M + +FCCP10M ++ ++CCCP 10 M ++ NDio-9 5 ,g/ml ++ +DCCD 10-6M ++ ++PMS 10-3M 0 ++KCN6 X 10-3M 0 ++NQNO 5 &g/ml 0 +Antimycin 10 Mg/ml 0 +DCMU2X1OM 0 +Valinomycin 5 jug/ml 0 0

0 = no inhibition ++ = complete inhibition+ = >50% inhibition N = not testedFor meaning of abbreviations, see first page of this article.

the cells are resuspended in basal salt solution and aerated.Similar observations have been made with other bacteria(11). In salt solution the cells apparently respire on endog-enous substrate. Respiration in basal salt may continue for20 hr or longer (Bogomolni, R. A., Baker, R. A. & Stoeck-enius, W., in preparation).When cells in salt solution are transferred to the assay vessel

and sparged with N2 in the dark, the suspension rapidly be-comes anaerobic, and within 10-20 min the ATP content ofthe cells drops to approximately 30% of the initial level. Itthen remains nearly constant with only a small further de-crease detectable over several hours (Fig. 2). This reduction inATP content does not occur when the vessel is illuminated.After ATP depletion in the dark, illumination with whitelight at about 106 erg/cm2 per sec causes a rapid rise in theATP level to the original value or slightly higher. This newlevel is maintained until the light is turned off. The ATP con-tent then rapidly drops again to the dark value (Fig. 2A).This effect of alternating light and dark conditions can berepeated several times without change in the size of the re-sponse. Essentially the same increase in ATP content of thecells is observed when oxygen is admitted to the suspensioninstead of light. The light but not the 02 effect is dependenton the presence of purple membrane in the cells (Fig. 2B).

N2

12

at

20-Dlo-9 25 jg

0Lto+ h V

o20 1 40

M] Nes

FIG. 6. Effect of an AT~ase inhibitor on the anaerobic lightresponse of cells. ATP per mg of protein at time 0 = 23.9 nmol.AOD 570 nm/mg of protein = 0.101.

The light response apparently does not depend on a func-tioning electron transport chain. Inhibitors of respiration andoxidative phosphorylation such as KCN or 2-n-nonyl-4-hydroxyquinoline-N-oxide (NQNO) have no effect on thelight response of the cells but inhibit the oxygen-dependentATP increase (Figs. 3 and 4 and Table 1). The light response issensitive to uncouplers of respiration and adenosine triphos-phatase (ATPase) inhibitors. Carbonylcyanide 3-chloro-phenylhydrazone (CCCP) and other uncouplers, which in-crease the permeability of membranes and lipid bilayers toprotons (12-14), abolish the light and the oxygen response(Fig. 5 and Table 1). Dio-9 and NN'-dicyclohexylcarbodi-imide (DCCD), inhibitors of the ATPases in chloroplasts,mitochondria and prokaryotic cells (15), also abolish thelight and the oxygen response (Fig. 6 and Table 1). If cellsrespiring in the dark are illuminated, respiration is at leastpartially inhibited (3, 16) and a transient 10-20% decreasein ATP level is observed (Fig. 7). Switching off the light againcauses a similar transient decrease in ATP content (notshown). The ATP level of cells respiring in the light is usuallyslightly higher than in aerobic dark or anaerobic light cells.Valinomycin has no effect on either the respiration or the

light-induced ATP increase. This is not surprising becausethe permeability of the cell membrane to K+ has been foundto be high, even in the absence of valinomycin (Passow, H.and Stoeckenius, W., unpublished).

N2 02 N2160I-

N2

120

80

40

1'"M CCCP

+h!v

0 20 40 60 so)Mkiutes

FIG. 5. Effect of an uncoupler on the light and 02 response ofcells. Both are inhibited. ATP per mg of protein at time 0 = 25.5nmol. AOD 570 nm/mg of protein = 0.101.

02120t

< 80

40

+ h-v

00

° 2WV 100 120 140Minutes

FIG. 7. Transition from aerobic dark to aerobic light condi-tions and subsequently anaerobic light conditions causes transientdecreases in ATP level of cells. ATP per mg of protein at time 0 =32.5 nmol. zOD 570 nm/mg of protein = 0.208.

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Photophosphorylation in Halobacterium halobium 1237

The light-induced increase in ATP content depends on thepresence of purple membrane in the cells and on light inten-sity. Fig. 8 shows the effect of narrow-band interference filterswith a half-width of 10 nm. Intensity of the transmitted lightwas kept constant for all wavelengths tested at about 3.0 X103 erg/cm2 per sec. The increase in ATP content is comparedto the absorption spectrum of the lysate of whole cells andthe absorption spectrum of the purple membrane fractionprepared from the lysate. The ATP levels attained by thecells are roughly proportional to the amount of light energyabsorbed by the purple fraction and not to the absorptionof the whole cell lysate. The absorption spectrum of the latteris dominated by bacterioruberin, the main carotenoid pig-ment present in these cells (17). This shows that only thelight absorbed by the purple membrane is effective in increas-ing the ATP level in the cells. The correspondence is as goodas can be expected from spectroscopic measurements on astrongly scattering suspension which also contains largeamounts of other pigments with overlapping absorptionbands.

DISCUSSIONH. halobium has been known as an obligate aerobic organism(18). Under anaerobic conditions in the dark, the cells rapidlyuse the remaining 02 (8, 3) and deplete their energy reserveto a limiting value of about 5 nmol of ATP per mg of protein.Either 02 or light will restore the ATP content of the cellsto the same or a slightly higher level than found under aerobicconditions at the beginning of the experiment. This presum-ably indicates ATP synthesis with either respiration or lightas alternative sources of energy. The extent of the ATPsynthesis cannot, of course, be estimated from the ATP con-tent of the cells because the rate of ATP use by the cells isunknown and is probably different under light and dark,aerobic and anaerobic conditions. The fast decrease of theATP level under anaerobic conditions in the dark indicates ahigh rate of use. However, the nearly constant level reachedafter a few minutes, which typically amounts to <30% ofthe original level, implies that the cells shut off most of theirenergy-requiring functions before the ATP reserve is com-pletely exhausted. These functions are apparently reactivatedwhen either 02 or light becomes again available as energysource, because return to anaerobiosis or darkness againleads to a rapid decline of ATP to the 30% level. The generalphenomenon that cells maintain an ATP reserve by drasti-cally reducing metabolic functions when their energy supplyis cut off has been observed in a wide variety of prokaryoticand eukaryotic organisms (19). The other possible inter-pretation of our observations that light and/or oxygen sharplydecrease the use of ATP appears highly unlikely, simplybecause there is no apparent energy source for ATP synthesisunder anaerobic conditions in the dark. Very similar changesin ATP level under anaerobiosis and aerobiosis in the lightand in the dark have been reported for the facultative photo-troph Rhodospirillum rubrum and for Chromatium D (20-22).The time resolution in our experiments is not sufficient toquantitatively compare the rates of change in ATP content;they appear to be approximately four times slower in H.halobium. The steady state amounts of ATP per mg of proteinare comparable and so are the light intensities used. More-over, in H. halobium the effect of 02 and light on the rate ofATP increase and the level reached are virtually the same.

00

I-iaX

*6E

450 500 550 600 650 700nm

FIG. 8. Comparison of the absorption spectrum of cell sus-pensions of H. halobium R1 with the amount of ATP produced atdifferent wavelengths. Curve a: absorption spectrum of the cellsuspension after dialysis against distilled water to lyse the cellsand reduce light scattering. Curve b: absorption spectrum of thepurple membrane fraction prepared from the cell lysate by cen-trifugation at 50,000 X g for 30 min. The stippled bars indicate theincrease in ATP content of the cells after illumination for 10 minat the wavelength indicated.

We, therefore, tentatively conclude that H. halobium canuse light energy to synthesize ATP-in other words, that it cancarry out photophosphorylation.The results reported here are remarkably similar to those

obtained with other photosynthetic organisms; however,H. halobium does not contain chlorophyll and photophos-phorylation is mediated by the purple membrane. Only cellswhich contain purple membrane show the increase in ATPcontent in the light and only light absorbed by the purplemembrane is effective.

It has been shown earlier (3) that purple membrane-con-taining cells of H. halobium, in the absence of other energysources, can generate a chemiosmotic gradient across theircell membrane when they are exposed to light. Evidence hasbeen provided that this is due to a rapid light-driven cyclingof b cteriorhodopsin between a long and short wavelengthform with concomitant transport of protons across the cellmembrane. Alternatively, the cells can generate such agradient through respiration when oxidizable substrate and02 are available. Further evidence for the function of bac-teriorhodopsin as a light-driven proton pump is providedby the incorporation of the purple membrane into lipid vesiclesand the demonstration that this model system generates aproton gradient in the light (23). We have postulated (3)that the gradient in H. halobium drives metabolic processessuch as ion translocation and ATP synthesis in accordancewith Mitchell's chemiosmotic theory of energy coupling(4). The experiments reported here bear out part of thisprediction.The action of specific inhibitors of electron transport and

phosphorylation and of uncouplers contributes further evi-dence for the postulated mechanism. The effect of the un-couplers DNP, FCCP, and CCCP is explained by their actionas proton translocators which collapse the gradient and inhibitboth photophosphorylation and oxidative phosphorylation.Dio-9 and DCCD, known as inhibitors of the ATP-synthesiz-ing enzyme in bacteria, chloroplasts and mitochondria (15,24), as expected abolish both the 02 and light effect on ATP

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1238 Cell Biology: Danon and Stoeckenius

content of the cells; the proton gradient, however, is notaffected (Bogomolni, R. A., Baker, R. A., and Stoeckenius,W., in preparation). This may indicate that both the light-and the Orgenerated gradients use the same phosphorylatingenzyme; however, two separate light- and 02-controlledATPases which are both inhibited by Dio-9 and DCCDcannot be ruled out. DCMU, a specific inhibitor of an earlyevent in the chain of redox reactions in Photosystem II (25),as expected, has no effect on the light-driven ATP increasein H. halobium. (The partial inhibition of the 02 effect at thelow concentration used here remains unexplained.) Thisindicates that the early events in purple membrane-mediatedphotophosphorylation are different from the noncyclic path-way of chlorophyll-mediated light energy conversion. Wealso exclude cyclic electron flow because inhibitors of theelectron transport chain such as NQNO and antimycin haveno effect on the photophosphorylation, but inhibit oxida-tive phosphorylation. Also the purple membrane containsonly one protein, bacteriorhodopsin. Therefore, all observa-tions so far argue against participation of redox reactions andan electron transport chain in photophosphorylation by H.halobium.The reactions of light-driven ATP synthesis are thus very

different from those observed in chlorophyll-containing or-ganisms (26). They are, however, easily understood if weassume that a rapid light-induced and dark-reversible con-formational change in bacteriorhodopsin transports protonsacross the membrane and thus converts light energy into achemiosmotic gradient which can drive ATP synthesis.Further support for this model is derived from the lipid vesi-cles with incorporated purple membrane. Addition of mito-chondrial ATPase and hydrophobic proteins to these vesiclesresults ina a model system exhibiting light-driven ATP syn-thesis (23).The natural habitats of Halobacteria are salt flats and stag-

nant puddles at the edge of tropical seas where salt concen-trations close to saturation are maintained (18). Temperatureand solar radiation density are high and the water is richin organic materials resulting from the decay of organismswhich died when their salt tolerance was exceeded. The P02must be low in such an environment. Halobacteria are pro-tected against the high radiation density by a high contentof carotenoids. They are often present in such large numbersthat the water acquires a deep orange or red color. Any oxygendiffusing in from the surface will be used up in the topmostlayer. Halobacteria have apparently adapted to this environ-ment by incorporating a pigment into their cell membrane.that converts light energy and thus provides an alternativeto oxidative mechanisms for energy production. It uses themost energy-rich part of the prevailing long wavelengthradiation for this purpose and converts it to a proton gradientwhich forms the link to the oxidative energy metabolismof the cell. It should be pointed out that chlorophyll-contain-ing halophile prokaryotes have been isolated from the samehabitat (27). One wonders what advantage bacteriorhodopsin-mediated photophosphorylation has that allows it to compete.The reason could be the extreme simplicity of the energy

converting system containing only one new protein. Thiswill have to be further explored. The advantage for the in-vestigating scientist is that this system allows one to easilyand cleanly separate not only conceptually but also prepara-tively the first energy conversion mechanism from the restof the cell's energy converting systems.

We are grateful to Drs. Mordechai Avron, Elisha Tel-Or, MichelRevel, Henri Atlan and Zippora Elchanan for critical discussionand help with equipment and chemicals. This work was supportedby NIH Program Project Grant HL 06285; it was begun at theUniversity of California, San Francisco, and continued at theWeizmann Institute of Science. W.S. also thanks the NASA forfinancial support to visit the Weizmann Institute during thecourse of this work.

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