Eur. J. Biochem. 77, 325 - 335 (1977)

Bacteriorhodopsin-Mediated Photophosphorylation in Halobacterium halobium Rainer HARTMANN and Dieter OESTERHELT Institut fur Bjochemie der Universitat Wiirzburg

(Received December 3, 1976)

The rate of halobacterial photophosphorylation was found to be a linear function of light intensity over a wide range (between 1 and 20 mW/cm2). At higher light intensities (above 25 mW/cm2) the ATP-synthesizing system itself limits the maximal rate of photophosphorylation. The optimal external pH range for this type of photophosphorylation is between pH 6.2 and 7.2 external. The photophosphorylation rate is directly proportional to the bacteriorhodopsin content of the cells. The quantum requirement for photophosphorylation was found to be 22 f 5 photons per ATP molecule synthesized. According to Mitchell’s chemiosmotic hypothesis of energy coupling phosphorylation can be driven by a membrane potential or a pH gradient or a combination of both. From the results of experiments with drugs which abolish or reduce either one of the two components we conclude that the major driving force for photophosphorylation above an external pH value of 6.5 is the membrane potential, while at more acidic pH value the pH gradient becomes dominating. We did not observe a correlation between a transient alkalinization of the medium and ATP-synthesis upon illumination under certain conditions. Halobacteria growing under oxygen-limiting conditions synthesize the retinal protein complex bacteriorhodopsin [ l ] which, with lipids, forms the purple membrane within the cytoplasmic membrane of the cell [2]. Bacteriorhodopsin has been demonstrated to function as a light-driven proton pump creating an electrochemical proton gradient which can be used for ATP synthesis [1,3]. Thus photophosphorylation in H. halobium is mediated by bacteriorhodopsin and not by chlorophyll as in higher plants and photosynthetic microorganisms [3- 51. Studies in vivo with inhibitors of electron transport and studies with lipid vesicles containing only the components of an ATP-synthase and bacteriorhodopsin clearly demonstrated that no electron carriers are involved in this type of photophosphorylation [3,6,7]. Thus, the light energy converting system of Halobacteria appears to be a relatively simple one: first an electrochemical proton gradient is created by a cyclic photochemical reaction of bacteriorhodopsin and second, the ATP-synthase of the cell membrane catalyses the conversion of the gradient energy into the chemical bond energy of the ATP molecule. The simplicity of the halobacterial system has made it to an appealing model system for testing the validity of the chemiosmotic hypothesis of energy coupling

[8,9]. This paper describes characteristic features of halobacterial photophosphorylation.

MATERIALS AND METHODS Strain, Culture Conditions and Determination of Bacteriorhodopsin Halobacterium halobium RIM1 [lo] containing no gas vacuoles and no bacterioruberine was grown in complete medium 1111 in shake cultures at 40 “C. Aeration was limiting for growth, and this limitation was achieved when 700ml medium plus 35 ml inoculum were used in 2 1 flasks and the rotary shaker was operated at 105 rev./min. Cells were harvested after 5 days when the culture had reached a turbidity of about 0.9 to 1 at 578 nm (Photometer Eppendorf model 1101 M). The bacteriorhodopsin content of the cells was determined by difference spectroscopy of a sample of lysed cells against a standard sample where bacteriorhodopsin was bleached with hydroxylamine in the light [12]. The protein concentration of sample and standard were adjusted to 5.5 mgiml. The spectrum was taken with a double-beam spectrophotometer Perkin-Elmer 124 (Hitachi) equipped with an

326

integrating sphere attachment. For calculation of the bacteriorhodopsin concentration an absorption coefficient of 63000 cm-l M-' was used. Protein was determined by the biuret method [13]. The bacteriorhodopsin content of the cells grown under the described conditions was found to vary between 0.9 and 1.4 nmol/mg protein. To obtain cells with different bacteriorhodopsin content 500 ml cultures were shaken in 1-1 conical flasks under the same conditions as above. Cells were harvested after 1, 2, 3 or 5 days and the bacteriorhodopsin content was determined directly in the whole cells by difference spectroscopy of hydroxylamine-bleached cells against unbleached cells [ 121. Cells could be kept in the medium at 4 "C for 2 or 3 days without affecting the rate and extent of photophosphorylation. Monitoring cf p H and Determination qf A T P Cells were spun down at 13000 x g for 5 min and resuspended in basal salt containing 250 g NaCI, 20 g MgS04. 7 H 2 0 and 2 g KCl per liter. The cell suspension was adjusted to a turbidity of 4.0 or 5.5 at 578 nm corresponding to a protein concentration of 2 or 2.75 mg/ml. In some cases buffered basal salt was used. Buffering with Tris-HC1 and Tris-maleate up to the highest concentrations used (12.5 mM) had no influence on the rate and extent of photophosphorylation in contrast to other buffering substances like citrate and imidazole. For the determination of ATP 10 ml of a cell suspension in basal salt were stirred in a cylindric glass chamber (2 cm diameter) with a water jacket at 25 "C. For anaerobic conditions nitrogen was passed over the surface of the suspension and the glass chamber was closed with parafilm (American Can Comp. Neenah, Wis.) except for a small hole for sampling and another one for the pH electrode. For maintaining dark conditions it was sufficient to use dimmed room lights (>0.1 mW/cm2, no fluorescent tubes). Illumination was provided by a 150-W slide projector (Rollei), with a OG 515 cut-off filter (Schott) at a light intensity of 50 mW/cm2 at the position of the sample. In some cases a 250-W projector (Noris Trumpf Halogen 250) at a light intensity of 300 mW/ cm2 was used. The light intensity was measured with a bolometer (Kipp and Zonen) connected to a Knick Microvoltmeter. Lower light intensities were achieved by using neutral density glass filters (Schott). The turbidity of the cell suspensions was determined at 578 nm before and after the experiments in order to check for cell lysis. Experiments were taken into account only where the change in absorbance was smaller than 10 %. The pH was monitored with a Radiometer glass electrode (model GK 230iC) attached to a Knick pH

Bacteriorhodopsin-Mediated Photophosphorylation

meter (type 27) and a pen recorder (Servogor S, Metrawatt). For ATP determination 0.1-ml samples were rapidly pipetted into 5 ml of a 10 mM phosphate buffer pH 7.4 with 0.1 mM EDTA at 0 "C. Cells were lysed immediately and no loss of ATP was found within 48 h at 4 "C. The luciferin-luciferase assay was run in a SKAN x P 2000 bioluminescence device [4]. The crude luciferase extract was prepared from 1 g firefly lanterns (desiccated tails purchased from Sigma) by extraction at 0 "C with 100 ml 0.1 M phosphate buffer pH 7.4 containing 1 mM EDTA. This preparation was kept overnight at 4-8 "C to consume endogenous ATP. The extract could be stored at -20 "C for some weeks without a significant loss of activity. A white-yellowish sediment appearing upon rethawing was removed by centrifugation. Determination of the Action Spectrum and the Quantum Requirement For the determination of the action spectrum a 900-W Xenon lamp was used. The light beam was passed through a 20-cm water bath, 400-nm cut-off filter and then focussed through a light guide on to the glass chamber with the cell suspension. The desired wavelength was selected by use of interference filters with a band width of 5 nm (Anders, Diendorf, G.F.R.). Anaerobic conditions were maintained as described above. Cell suspensions were always preilluminated with light filtered through OG 51 5 filter (Schott) for 10 min and then kept in the dark for 10 min before the experiment was started. In all experiments the rate of ATP synthesis was a linear function of the light intensity. The absolute light intensity was determined by a Radiometer detector head (model 580-25A) equipped with a narrow-beam attachment (model 580-00-11). The same instrument was used to determine light absorption of cell suspensions in an integrating sphere (50-cm diameter, see also [14]). Absorption of untreated cells and cells bleached with hydroxylamine in the light was compared. A correction for secondary absorption was not found to be necessary. From the absolute amount of light absorbed per unit time and the amount of ATP synthesized per unit time, the quantum requirement for ATP-synthesis was calculated. Chemicals ATP was from Boehringer, carbonylcyanide-ptrifluoromethoxyphenylhydrazone and 5,Sdimethyloxazolidine-2,4-dione were from Sigma, triphenylmethylphosphonium ion from K & K Fine Chemicals and phloretin was a gift of Dr Grell (Gottingen). Other chemicals used were from Merck (Darmstadt), and of analytical grade.

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R. Hartmann and D. Oesterhelt

RESULTS Changes in Cellular ATP Level Under anaerobic conditions in the dark the cellular ATP level drops to 25 to 30 % of that obtained in the light or by aeration [3 - 51. If the cells are kept anaerobically in the dark overnight at room temperature the ATP level decreases further to 5 to 10%. For a more detailed investigation of halobacterial photophosphorylation the analysis of the change in ATP levels is not a very sensitive method. A more suitable way of analysis is based on the rates of changes in ATP level. As can be seen in Fig. 1 the initial rate of photophosphorylation does not depend on the preexisting ATP level (10-50 usually). Therefore this initial rate directly reflects changes in the size of the driving force for phosphorylation as long as the ATPsynthesizing system itself does not become ratelimiting.

light intensity is increased from 25 to 100mW/cm2 [15]. Therefore we conclude that at 25 mW/cm2 the maximal rate of photophosphorylation under our conditions is limited by the ATP synthesizing system itself. The obtained maximal rate of photophosphorylation was 0.33 f0.04 nmol ATP x sC1 x mg protein-’. This result was calculated out of 8 experiments with 6 different cultures. It should be mentioned that the saturating light intensity of 25 mW/cm2 is easily provided by sunshine

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3 -X-

Rate o j Photophosphorylation as a Function of Light Intensity

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The rate of photophosphorylation depends linearily on light intensity between 3 and 20 mW/cm2 (Fig.2). Below 0.25 mW/cm2 no change of the ATP level is observed. Whether this is due to the limited accuracy of our ATP determination or to a true lag phase cannot be decided at present. At light intensities above 25 mW/cm2 no further acceleration of the rate of photophosphorylation can be observed. This intensity hardly saturates the proton-pumping process because an increase of light intensity to 50 mW/cm2 still leads to an increasing acidification of the medium (not shown). Furthermore the lightdependent uptake of K + can still be accelerated if the

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1

25

10

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Light intensity (rnwicrn’)

Fig. 2, Rate of pho tophosphorylu t ion at d$fLerent light in rensiiics. For each light intensity a new cell suspension (10 ml basal salt with 1 m M Tris-HCI pH 7.2, 1.8 mg proteiniml) was used. The final p H of the suspensions was between 6.5 and 6.8. Experiments were carried out under nitrogen. The cell suspension was first illuminated (12.5 mW/cm2) then kept in the dark for 20 min. To determine the dark level of ATP three samples were taken during the last minute before illumination. Then samples were taken at intervals depending on light intensity. At intensities below 1 mWlcmZ at 30-s intervals below 10 mW/cmz at 5 - 10-s intervals and above 10 mW/cmz light intensity at 3-s intervals. The time dependence of the increase in ATP level was linear up to 15 s at the highest light intensity used. Its slooe is taken as the initial velocity of phosphorylation and plotted against intensity

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Fig. I . Photophosphorylation at dlfferent light intensitie.s and rfiffiwni A T P levels. For each experiment 10 ml buflered cell suspension (basal salt with 12.5 mM Tris-maleate pH 6.5, 2 mg protein/ml) were used. Cell suspensions were pre-illuminated for 10 min at 50 mW/cmz, then kept in the dark for 15 min ( x , 0)or 4 h (0,A). At zero time cells were again illuminated at a light intensity of 2.5 mW/cmz (A, x ) or 12.5 mW/cmz ( 0 ,0). Samples for ATP determination were taken at indicated times. Each experiment was performed twice

328

Bacteriorhodopsin-Mediated Photophosphorylation

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565

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Fig. 3. Action spectrum of photophosphorylution and ab.sorption spectrum qfhacteriorhodopsin. Experimental conditions as in Fig. 3 ; 8 ml cell suspension (1.4 mg protein/ml). Pre-illumination and dark interval were 10 min. Three samples were taken during the last minute before illumination. Then samples were taken at 5 - 15-s intervals depending on the wavelength used. Changes in ATP content were plotted against time and the slope taken as the initial rate of photophosphorylation. The measured rates ( x ) were proportional to light intensity at all wavelengths and normalized to a light flux of 200 nano Einstein,% The light absorption of bacteriorhodopsin ( 0 ) in the cells was determined as described in Materials and Methods

in the natural habitats of Hulohucterium. The sunlight in the slightly smoggy atmosphere of a European city was determined to be 30-35 mW/cm2 when filtered through the same cut-off filter as the light of the projector. Action Spectra of Photophosphorylation and Correlation between ATP-Synthesis and Bacteriorhodopsin Content of the Cells The generally accepted assumption that photophosphorylation in Halobacteria is mediated by bacteriorhodopsin demands two conditions. Firstly, the action spectrum of photophosphorylation should be identical with the absorption spectrum of bacteriorhodopsin (Fig. 3) as was partly already shown [3]. Secondly, the initial rate of photophosphorylation should be proportional to the bacteriorhodopsin content of the cells under non-saturating illumination (Fig. 4). The quantum requirement for photophosphorylation is determined by comparison of the amount of ATP synthesized per unit time with the amount of light absorbed per unit time. It was found to be wavelength-independent . The average number obtained in five independent experiments was 22 f 5 photons per mol ATP synthesized. This figure appears to be rather high compared to the corresponding values in photosynthesis. One has to consider however that the

Fig.4. Correlution between the rate o f photophospl?orylation and hucteriorhodopsin content of the cells. Experimental conditions as in Fig. 3. Light intensity was 12.5 mW/cm2, protein concentration was 1.7 mg/ml. Each point is the average of two separate experiments

electrochemical proton gradient created by light does not only drive ATP synthesis but additionally drives transport processes like K + uptake and Na’ extrusion [15- 181. Furthermore, the energy for flagella movement may also be provided by the proton gradient. This large quantum requirement will be discussed together with the quantum requirement for H + ejection elsewhere. Dependence of Photophosphorylution on the p H of the Medium Between pH 4.5 and 8.7 light-mediated synthesis of ATP leads finally to the same cellular level of 7 k 1 nmol ATP/mg protein. Below pH 4 and above pH 9.5 no photophosphorylation can be detected. In contrast to the ATP level, the rate of photophosphorylation shows a more pronounced pH dependence (Fig. 5 ) . By comparing the rate of photophosphorylation as a function of light intensity at different pH values the following information is obtained. The linear part of the curves reflects the pH dependence of the proton-motive force available for ATP synthesis. This parameter includes the pH dependence of the proton pump bacteriorhodopsin as well as the pH dependence of the coupling process between proton pump and ATP synthesis (see Discussion). At higher light intensities a maximal rate of photophosphorylation is reached but its size depends on pH. The maximal rate expresses the pH dependence of the ATP-synthesizing system itself, if the rate is not limited by the energy-providing system. Limitation by the ATP-synthesizing system is very likely on the grounds of arguments we used before. Increasing acidification of the medium and acceleration of the

329

R. Hartmann and D. Oesterhelt 0.4

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Light intensity (rnW/crn2)

Fig. 5. Rate of'photophosphor~.lation at dvjerent external p H as a function of light intensity. Experimental conditions as described in Fig. 3 except for the following changes. Cells (2 mg protein/ml) were resuspended in basal salt with 12.5 mM Tris-maleate to keep the external pH constant in the range of 0.05 pH units at pH 8, 6.5 and 5. At pH 4.5 and 4.0 unbuffered basal salt was used, because the buffer substances citrate and acetate affected the rate of phosphorylation. Cells were kept in the dark for 2 to 4 h under nitrogen at room temperature (22 f 2 "C) to reach an equilibrium between internal and external pH and the cell suspensions were not pre-illuminated

uptake of K + [15] can still be observed upon increasing light intensities above 50 mW/cm2. This is found for all pH values in the experiments in Fig.5 and clearly indicates that not the energy supply but the ATP-synthesizing system is the limiting factor at higher light intensities. The data shown in Fig.5 together with additional experiments established that halobacterial photophosphorylation occurs most effectively between pH 6.2 and 7.2. In this pH interval the phosphorylating as well as the energy-providing system are in their optimal pH range. Contribution of Membrane Potential a n d p H Gradient to the Driving Force of Photophosphorylation According to Mitchell's chemiosmotic hypothesis the proton-motive force, the key element in energy coupling, consists of two related elements : an electrical component due to the membrane potential and a chemical component due to the pH gradient. By the use of drugs which abolish or strongly reduce either one of the two components it might be possible to decide whether the membrane potential or the pH difference is more important for bacteriorhodopsinmediated photophosphorylation. Drugs used for this purpose were 5,5-dimethyloxazolidine-2,4-dioneto diminish the pH gradient and triphenylmethylphosphonium ion to dissipate the membrane potential. 5,5-Dimethyloxazolidine-2,4-dioneis a weak acid which in its undissociated form can penetrate the

membrane and reduces a pH gradient (interior alkaline) if used at high concentrations. Triphenylmethylphosphonium ion is a lipophilic cation which can freely pass through the membrane and can abolish a membrane potential (interior negative) if used at high concentrations [19,20]. Fig.6 clearly demonstrates that 0.1 M 5,5-dimethyloxazolidine-2,4-dione has no influence on the rate and extent of photophosphorylation at pH 6.5, while 2 mM triphenylmethylphosphonium ion diminishes it significantly. In this case a small increase of ATP at a relative rate of 10% compared with the control cells is observed during the first 10- 15 s. Then the rate of photophosphorylation drops further to about 1%. The final ATP level reaches about 60 - 70 % of the control after 10 - 15 min (not shown). The sensitivity of photophosphorylation towards triphenylmethylphosphonium ion points to the membrane potential as the main component in the protonmotive force at pH 6.5. The observed slow increase in ATP level in the presence of triphenylmethylphosphonium ion can be understood by a consideration of the interrelation of the membrane potential and the pH gradient. A reduction of membrane potential leads to an increase of the pH gradient and vice versa [21]. In the experiment of Fig.6 the slow increase in ATP level may therefore be caused by the slowly developing pH gradient. In accordance with this a and combination of 5,5-dimethyloxazolidine-2,4-dione triphenylmethylphosphonium ion completely abolishes photophosphorylation (Fig. 6). It should be mentioned, however, that cells are not very stable if exposed to a combination of both drugs and lyse easily (see Materials and Methods). A possible argument against the above interpretation is that triphenylmethylphosphonium ion at the concentrations used might inhibit the ATP synthase itself. This can be ruled out by a Jagendorftype experiment [22]. If ATP synthesis is driven by an artificial pH gradient, no significant difference in the amount of ATP synthesized between cells with or without triphenylmethylphosphonium ion is observed (Fig. 7). The rate of phosphorylation appears to be even faster in the presence of triphenylmethylphosphonium ion. Experiments with tetrabutylammonium ion another lipophilic cation confirm the results obtained with triphenylmethylphosphonium ion: Photophosphorylation occurs at a greatly reduced rate while ATP synthesis induced by an artifically imposed pH gradient is not affected. The dominating contribution of the membrane potential to the driving force of photophosphorylation is not necessarily independent of the experimental conditions, e.g. the external pH. To answer this question the rate of ATP synthesis was compared at different pH values with and without triphenyl-

Bacteriorhodopsin-Mediated Photophosphorylation

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Fig. 6. Influence of 5,5-dimethq~loxazolidine-2,4-clionearid triphenylmethl.lpho.sphoniui~i ion on photophosphorylation. For each experiment 10 ml of a cell suspension in basal salt under nitrogen (1.9 mg protein/ml) were used: ( 0 )control, ( x ) with 100 m M 5,5-dimethyloxazolidine2,4-dione, (A) with 2 mM triphenylmethylphosphonium ion, (a)with 100 mM 5,5-dimethyloxazolidine-2,4-dione and 2 mM triphenylmethylphosphonium ion. The cell suspensions were first illuminated at a light intensity of 12.5 mW/cm2 then kept in the dark for 15 min. Samples were taken at indicated times

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Table 1. Influence of triphenylmethylpho.sphonium ion on the rate of ATP synthesis at dyjerent external p H The rate of ATP-synthesis was determined in 2 independent experiments at 3 different external pH values with and without triphenylmethylphosphonium ion (TPMP') as described in Fig.6. The light intensity was 12.5 mW/cm2 Expt number

External pH

Rate of photophosphorylation control

1

0%.

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with 1 m M TPMP+

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pmol x s-' x mg protein-'

methylphosphonium ion. Non-saturating illumination conditions (12.5 mW/cm2) guaranteed the energyproviding system was limiting the rate of phosphorylation. Table 1 clearly shows that the inhibition of photophosphorylation by triphenylmethylphosphonium ion is strongly pH dependent. The inhibition is 100% at pH 8.5 and only 10-15% at pH 5.0. These results strongly suggest a changing contribution of the membrane potential and the pH difference to the protonmotive force at different external pH values and are consistent with direct measurements of the pH de-

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pendence of the proton-motive force reported in the literature [23,21]. As a consequence of the decreasing contribution of the membrane potential with decreasing pH one might further expect pH-dependent differences in the onset of ATP synthesis upon illumination for the following reason : as an electrical potential difference across a membrane develops much faster than a pH difference a lag phase in ATP synthesis should be observable if the pH gradient is the main driving force. For the H . halobium cell suspension we expect an increasing lag phase of photophosphorylation when the external pH is lowered to pH 5. This is indeed the case (Fig.8). The lag phase at pH 5 can even

R. Hartmann and D. Oesterhelt

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Bacteriorhodopsin-mediated photophosphorylation in Halobacterium halobium.

Eur. J. Biochem. 77, 325 - 335 (1977) Bacteriorhodopsin-Mediated Photophosphorylation in Halobacterium halobium Rainer HARTMANN and Dieter OESTERHELT...
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