Involvement of Photosystem Two in Non-Oxygen Evolving Non-Cyclic, and in Cyclic Electron Flow Processes in Chloroplasts

Eur. J. Biochem. 15 (1970) 155-160 Involvement of Photosystem Two in Non-Oxygen Evolving Non-Cyclic, and in Cyclic Electron Flow Processes in Chloroplasts Gozal BEN-HAYYIM and Mordhay AVRON Biochemistry Department, Weizmann Institute of Science, Rehovot (Received January 25/March 26, 1970) 1. Ascorbate is concluded to serve as an electron donor at a site which is prior to photosystem 2. This conclusion is supported by the following observations : Addition of ascorbate doubled the apparent rate of oxygen uptake in the presence of diquat. NADPf photoreduction was not affected by addition of ascorbate, while the concomitant oxygen evolution was markedly inhibited. Both of these reactions were as sensitive to 3-(3,4-dichloropheno1)-1,l-dimethylurea in the presence of ascorbate as a normal Hill reaction. Both reactions shawed the red-drop phenomenon in the presence of ascorbate. 2. Ascorbate did not serve as an electron donor prior to photosystem two either in heat-or in Tris-treated chloroplast preparations. However, ascorbate and hydroquinone did. 3. Tris-treated chloroplast preparations catalyse a hydroquinone dependent cyclic photophosphorylation in which photosystem two is involved. Illuminated chloroplast preparations, as well as model systems were shown to oxidize ascorbic acid [1,2]. It was found that this oxidation consisted of two phases, a slow phase in which oxidation of ascorbate to dehydroascorbate occurs, and a fast phase in which further oxidation of dehydroascorbate to oxalic and threonic acids takes place. In one case [l] this reaction was shown to be mediated by far-red light and was partially inhibited by 3-(3,4-dichlorophenol)-1,l-dimethylurea. However, it was also found [2] that the oxidation of ascorbate exhibited a drop in quantum yield at the far-red light region, and that it was sensitive to dichlorophenol-dimethylurea to the same extent as Hill reaction. Katoh and San-Pietro [3] showed that ascorbate could act as an electron donor in heat-treated Euglena chloroplast preparations. Under these conditions, oxygen evolution was fully inhibited, and ascorbate was as active as a reduced dye in restoring NADPf photoreduction. However, the photoreduction restored with ascorbate was found to be sensitive to dichlorophenol-dimethylurea while that restored with the reduced dye was not. It was therefore proposed that ascorbate interacted with the electron transport chain at a site prior to photosystem 2. Chloroplast preparation from higher plants when subjected to a similar treatment [4] behaved differently. The evolution of oxygen was fully inhibited, but ascorbate only partially restored NADP+ photo- Unusual Abbreviatione. Dichlorophenol-dimethylma, 3- (3,4-dichlorophenol)- 1,l-dimethylurea ; diquat, 1,l -ethylene-2,y-dipyridylium dibromide. reduction, and the latter was mostly insensitive to dichlorophenol-dimethylurea. Only by coupling the aacorbate with dyes one can obtain high activity of NADP+ photoreduction mediated by photosystem 1 [5]. Inhibition of oxygen evolution was also observed after treating chloroplast preparations with high concentration of Tris buffer [6]. Ascorbate by itself served as a rather poor electron donor for restoration of NADP+ photoreduction. Only when coupled to reduced dyes, such as p-phenylenediamine or hydroguinone the reaction proceeded at a high rate. Such chloroplast preparations were also shown to have dichlorophenol-dimethylurea-sensitive photophosphorylation activity provided both electron donor and electron acceptor were supplied [7]. In this article we present evidence that ascorbate can serve as a good electron donor in untreated chloroplast preparations and that the electron transport path from it, is as sensitive to dichlorophenol-dimethylurea as the ferricyanide Hill reaction. Its site of donation of electrons precedes photosystem 2, and this site is either destroyed or changed by heat or Tris treatment of the chloroplast preparation. Some properties of the ATP formation by Tristreated chloroplasts are also described. METHODS Chloroplasts were isolated from lettuce leaves as previously described [S]. Chlorophyll waa sssrtyed after Arnon [9]. Oxygen uptake and evolution were

166 Involvement of Photosystem Two in Electron Flow Processes in Chloroplasts Eur. J. Biochem. measured with a Yellow Springs Instruments Clark type oxygen electrode. NADP+ photoreduction was measured in a Cary 14 recording spectrophotometer equipped with a scattering attachment. The photomultiplier was protected from the actinic light by a saturated solution of CuSO, and a Corning C. 8.7-60 filter. ATP formation was assayed as previously described [8]. Illumination was provided by a 500 watt projector and was filtered through Baird Atomic sharp cut off interference filters (all blocked to infinity) peaking at 640 nm (25 nm half band width) and 715 nm (16 nm half band width). When monochromatic light was not required a corning C. S. 2-58 was used. The intensity of the actinic light was measured by a Yellow Springs Instrument Radiometer model 65 and was varied by using calibrated metal screens. Absorption data were calculated from a curve previously published [lo]. Tris-treated chloroplasts were obtained following the procedure of Yamashita and Butler [6] except that 0.05 M Tris-C1 buffer ph 9.1 replaced the 0.8 M Tris-C1 buffer ph 7.8. The required incubation time for elimination of oxygen evolution was 17-18 min at 0". Heat-treated chloroplasts were obtained by heating the chloroplast suspension (about 1 mg chlorophyll/ml) for 5 min at 65". A 0 9" on Ascwbate (20prnole) I KIP (0.1 kmole) Off Fig. 1. The effect of ascorbate on oxygen uptake in the presence of diquat. Reaction mixture contained : 45 pnoles Tricine, ph 7.8, 60 ymoles NaCI, 0.1 ymole diquat, and 1.5 pmoles Na-azide, in a total volume of 3.0 ml. Chloroplasts contained 35 pg chlorophyll/ml. Numbers in parentheses are the initial rates of oxygen uptake expressed as ymoles 0, x mg chlorophyll-' x hour-'. DCMU = dichlorophenol-dimethylurea ; DCIP = dichlorophenolindophenol 15/, F i '-DCMU P' DCMU I RESULTS Effect of Ascorbate on Electron Transport Fig. 1 A shows the effect of addition of ascorbate on the rate of oxygen uptake in the presence of diquat. As can be seen, the apparent rate of oxygen uptake was about doubled on addition of the ascorbate, and the latter was fully inhibited by dichlorophenol-dimethylurea. Similar results were observed also in the presence of an uncoupler (10 mill methylamine). For comparison, Fig. 1 B represents the same experiment but with 2,6-dichlorophenolindophenol added. The addition of the dye rendered the system rather intensitive to dichlorophenol-dimethylurea. By following a concentration curve of the reduced dye (Fig. 2) one can show a continuous transformation from a reaction which exhibits absolute sensitivity to dichlorophenol-dimethylurea (ascorbate only) to another which is rather insensitive to dichlorophenoldimethylurea. The degree of sensitivity can be used, therefore, to indicate the relative rates of the two reactions operating simultaneously. In our hands, p-phenylenediamine, at somewhat higher concentrations, gave identical results to dichlorophenolindophenol. A possible interpretation of these results is illustrated in Eqn. (1)-(6). Pig. 2. Dichlorophenol-cliithylurea semsitivity of oxygen uptake with diquat in the presence of aswrbate or aswrbate + dichlorophenolindophenol a8 donor systems. Reaction mixture as in Fig. 1, with the addition of 20 ymoles Na-ascorbate. Each sample was tested for its activity in three stages. First, prior to the addition of the dye, second, after addition of the various concentrations of dichlorophenolindophenol, and third, after addition of 0.01 ymole of dichlorophenoldimethylurea (DCMU). 0-0, rate of the second stage and 0-0, rate of the third stage H,O + diquat -+ light (diquat - H,) + 1/2 0, (I) (diquat * H,) + 0, -+ H,Oz + diquat (2) light+ H,O + 1/2 0, ~ H,O, (3) ascorbate + diquat *+ (diquat * H,) + dehydroascorbate (4) (diquat - H,) + 0, + H,Oz + diquat ascorbate + 0, 2% dehydroascorbate (2) + HZO,. (5)

Vol. 15, No. 1, 1970 G. BEN-HAYYIM and 31. AVRON 157 Table 1. Effect of aswrbate on oxygen evolution and NADPIphotoreduction Reaction mixture contained 45 pnoles tricine ph 7.8, 60 pmoles NaCl, 1 pmole NADP+, saturating amount of ferredoxin and chloroplasts containing 128 pg chlorophyll in total volume of 3.0ml. The experiment was carried on as follows: The chloroplasts were illuminated for 1 min prior to the addition of ascorbate and 1 min in presence of various concentrations of ascorbate. The light was turned off and 2.0 ml of the chloroplast suspension were assayed for NADPH [11J btea of oxygen evolution were calculated from the second minute in the light. chl = chloroplasts Ascorbate Oxygen evolution NADPC reduction mm pmoles x mg chl- x hour- None 22.7 48.5 2 11.7 44 6.6 8.2 44 13 3.4 44 With diquat as an electron acceptor reactions 1 and 2 take place, resulting [Eqn. (3)] in the uptake of 1/2 0, per 2 electrons traversing the chain. If ascorbate replaces water as an electron donor reaction 2 and 4 will take place resulting in one 0, taken up per 2 electrons traversing the chain, and so in the doubling of the rate of oxygen uptake observed. In order to ascertain that ascorbate was not acting at the acceptor site (reaction2) we measured the effect of ascorbate on the electron transport from water to NADP+, where it was possible to measure the donor and acceptor sites independently. As can be seen in Table 1, NADP+ photoreduction was not affected by addition of ascorbate, while the concomitant oxygen evolution was markedly inhibited. The inhibition required 2-3 min of preincubation in the dark or much shorter time in the light. Ferredoxin and NADP+ could be omitted during the preincubation time. The Site of Action of Ascorbate The dichlorophenol-dimethylurea sensitivity of the ascorbate-dependent NADP+ photoreduction, or oxygen uptake in presence of diquat, indicates that its site of donation of electrons is close to photosystem 2. However, this is not sufficient to decide whether this site is in the electron donating or the electron accepting side of photosystem 2. Since it is well established that reactions which involve photosystem 2 exhibit a Red Drop in their quantum efficiencies, we measured ratios of quantum requirements for the light dependent oxygen uptake in presence of diquat, or NADP+ reduction, with three donor systems : (a) water, (b) dichlorophenolindophenol and ascorbate in the presence of dichlorophenol-dimethylurea, and (c) ascorbate. The results are presented in Table 2. Reaction (a) served as a control reaction which is known to utilize both photosystems, and reaction (b) for a reaction utilizing Table 2. Quantum requirement for electron transport in presence and absence of ascorbate Reaction mixture contained 45 pmoles tricine ph 7.8, 6Opmoles NaCl, either lpmole NADP+, 18pmoles CH3NH3Cl and saturating amount of ferredoxin; or 0.1 pmole diquat and 1.5 voles Na-azide. Chloroplasts Contained 39 and 111 pg chlorophyll for NADP+ and diquat, respectively, in total volume of 3.0ml. Electron transfer to NADP+ was measured by the change in absorbance at 350 nm in a Cary 14 equipped with a scatter attachment. Electron transfer to diquat was described under Methods, assuming the uptake of one oxygen atom to be equivalent to the transport of two electrons when water served as an electron donor, and of one electron when ascorbate or ascorbate + dichlorophenolindophenol served as electron donors. Where mentioned, 20 pmoles of Na-ascorbate and 0.1 pmole of dichlorophenolindophenol were added. 0.01 pmole dichlorophenoldimethylurea was present in the ascorbete-dichlorophenolindophenol system. All quantum requirement data represent values extrapolated to zero intensity (see [lo]) Electron Wavelength of light Ratio Of Electron donor 715 acceptor 716nm 640nm 640 H20 quanta/electron Diquat - - 4.5 NADP+ 12.6 5.1 2.5 Ascorbate + di- Diquat - - 0.65 chlorophenol- NADP+ 1.5 2.7 0.54 indophenol Ascorbate Diquat - - 2.7 NADP+ 12.5 5.0 2.5 photosystem 1 alone. As can be seen, we indeed observed a Red Drop when water was the electron donor, and a Red Rise in the ascorbate dichlorophenolindophenol reaction where photosystem 2 was blocked by dichlorophenol-dimethylurea. The reaction where ascorbate served as an electron donor had a clear Red Drop (though in the case of diquat to a smaller degree with ascorbate than that with water). This result indicates that the site of donation of electrons by ascorbate must be localized somewhere on the electron donating side of photosystem 2. Chloroplast preparations treated with either heat [4] or high concentration of Tris buffer [6] do not evolve oxygen when supplied with any of the known electron acceptors. On the other hand, addition of some donor systems could restore the photoreduction of NADP+. As can be seen in Table 3, ascorbate could only poorly restore NADPf photoreduction with either heat or Tris-treated chloroplast preparations. Also, the dichlorophenol-dimethylurea sensitivity of this restored activity was markedly diminished in the heat-treated chloroplasts, and somewhat in the Tristreated ones. The poor activity of the ascorbate in these systems suggests that its site of action is either changed or destroyed, by the treatment employed. As can be seen in Table 3, the best dichlorophenoldimethylurea-sensitive restoration was obtained in the presence of hydroquinone.

158 Involvement of Photosystem Two in Electron Flow Processes in Chloroplasts Eur. J. Biochem. Table 3. Photoreduction of NADP+ by normal, Tris- and heat-treated chloroplast preparations The reaction mixture contained 45 pmoles tricine ph 7.8, 60 pmoles NaCl, 0.5 pmole NADP, saturating amount of ferredoxin and chloroplasts containing 60 pg chlorophyll in 3.0 ml. Other additions were as follows: 20 ymoles Naascorbate, 0.1 pmole p-phenylenediamine, 0.6 *ole hydroquinone, 0.1 pmole dichlorophenolindophenol and 0.01 pmole dichlorophenol-dimethylurea (DCMU). NADP+ reduction was measured as described under Methods and the activity of normal chloroplasts, taken aa 100 /,,, was 150 pmoles x mg chlorophyll-' x hour-1 Normal Heat-treated Tris-treated Electron donor chloroplasts chloroplasts ohloroplasts Addition + + + None DClU 'One DCMU DCMU of control H*O (loct) 0 0 0 7 0 Ascorbate 108 3 18 15 21 6 Ascorbate + p-phenylened i a m i n e 111 8 27 18 55 13 Ascorbate + hydroquinone 101 5 21 13 75 7 Ascorbate + dichlorophenolindophenol 79 38 64 64 58 52 Table 4. Requirements for photoreduction of NADPt and photophosphorylation in Tris-treated chloroplasts The reaction mixture contained 45 pmoles tricine ph 8.0, 60 pmoles NaCl, 12 pmoles MgCl,, 12 pmoles phosphate ph 8.0 (containing 6 x lo6 counts YaP/min), 4 ymoles ADP and Tris-treated chloroplasts containing 53 yg chlorophyll in a total volume of 3.0ml. Where indicated, the concentrations of ascorbate, hydroquinone, dichlorophenol-dimethylurea (DCMU) and NADP+ (with saturating amount of ferredoxin) were 6.6, 0.2, 0.003 and 0.3 mm respectively. NADP+ photoreduction was followed as described under Methods and ATP formation was measured after 2 min of illumination in the same set-up NADPt Additions ATP formation NADP+ reduction *moles x mg chlorophyll-' x hour-' 0 7.8 NADP+ + ascorbate 30 33 NADP+ + hydroquinone 193 14 NADP+ + hydroquinone + ascorbate 257 83 NADP+ + hydroquinone + ascorbate + DCMU 8 0 Ascorbate 26 - Hydroquinone 228 - Ascorbate + hydroquinone 222 - Ascorbate + hydroquinone + DCMU 0 - A list of inhibitors including o-phenanthroline, ioxynil, 2-n-hepty1-4-hydroxyquinoline-N-oxide, 4,5,6,7 - tetrabromo - 2 - trifhoromethylbenziazole, n-butyl-3,5-diiodo-4-hydroxybenzoate and dichlorophenol-dimethylurea were tested with normal chloroplast preparations in the absence and presence of ascorbate. No change in sensitivity towards all these inhibitors was observed as a result of the addition of ascorbate. It seems, therefore, that the site of interaction of ascorbate with the electron transport chain precedes the site of inhibition by these inhibitors. Cyclic Photophosphorylation in Tris-Treated Chloroplast Preparations Tris-treated chloroplast preparations were found to catalyze ATP formation with ascorbate and hydroquinone in the absence of any electron acceptor. The requirements for ATP formation and NADP+ photoreduction are summarized in Table 4. Both these reactions were sensitive to dichlorophenol-dimethylurea. The ascorbate-hydroquinone catalyzed photophosphorylation seems independent of the presence of an added electron acceptor. Moreover, under these conditions no net oxygen uptake could be detected indicating the absence of a pseudocyclio electron flow. Thus, it seems that we are dealing with a new type of cyclic photophosphorylation which is dichlorophenol-dimethylurea sensitive. In contrast to Table 5. Quantum requirement for NADP+ photoreduction and photophaspho ylation by Tris-treated chloroplasts The reaction mixture contained 45 pmoles tricine ph 8.0, 60 pmoles NaCl, 12 pmoles MgCl,, 12 pmoles phosphate ph 8.0 (containing lo' counts "P/min), 4 ymoles ADP and Tris-treated chloroplasts containing 62 pg chlorophyll in 3.0 ml. For NADP+ reduction, 1 pmole of NADP+ and a saturating amount of ferredoxin were added into the reaction mixture. The concentration of ascorbate, hydroquinone, dichlorophenolindophenol (DCIP) and dichlorophenol-dimethylurea (DCMU) were 6.6, 0.2, 0.03 and 0.003 mm, respectively. NADP+ photoreduction ww measured as described in Table 2 and ATP formation was measured after 2-min illumination. Quantum requirement data represent values extrapolated to zero intensity (see [lo]) Wavelength Ratio Reaction 715 840nm 715nm 640 quanta/atp ATP formation Ascorbate + hydroquinone 6.0 13.4 2.2 Ascorbate + DCIP + DCMU 9.4 3.6 0.38 quanta/eleetron NADP+ photoreduction Ascorbate + hydroquinone 4.8 12.3 2.6 Ascorbate + DCIP + DCW 4.9 2.0 0.41 the ascorbate + dichlorophenolindophenol or phenazine methosulphate-induced cyclic photophosphorylation, the data in Table 5 present evidence that in the hydroquinone-induced cyclic ATP formation

Vol.15, No.l,1070 G. BEN-HAYYIM. and M. AYRON 159 photosystem 2 is involved. Whether or not photosystem 1 is also active in this process cannot be determined from these quantum requirement measurements. Untreated chloroplast preparations showed only a slight stimulation of ATP formation induced by either ascorbate or ascorbate-hydroquinone over the endogeneous rate measured in absence of added electron donor or electron acceptor. Slight stimulation of ATP formation in the presence of ascorbate and in the absence of electron acceptor other than oxygen was also shown by Jacobi [12]. DISCUSSION The increase of apparent rate of oxygen uptake mediated by diquat in presence of ascorbate could be interpreted by either of two ways, (a) real increase in the rate of electron flow, or (b) a change in stoichiometry between the number of electrons moved through the electron transport chain per one molecule of oxygen taken up. The data obtained from the experiments on NADP+ photoreduction, strongly suggests alternative (b) (Table l), since in this case it was obvious that while the net rate of electron flow was unaffected ascorbate largely replaced water as the electron donor. It seems therefore, that ascorbate is a good competitor for water as an electron donor, and that both may be operating at the same time. Since it was impossible to fully eliminate the reaction where water served as an electron donor even at very high concentrations of ascorbate (Table 1), the calculated rates for the diquat-dependent oxygen uptake in the presence of ascorbate, assuming one molecule of oxygen to be equivalent to two electrons, are somewhat inaccurate. The same mechanism was already proposed by Trebst et al. [13]. They tried different quinones as electron acceptors and found that for those which had negative redox potentials oxygen uptake was stimulated by the addition of ascorbate. Ascorbate has a redox potential of about zero volts and was shown to interact with a few components of the electron transport chain which can serve as possible donation sites. Both cytochromes f and b-669, localized between the two photosystems were shown to be ascorbate reducible [14-1661. However, in the presence of both ascorbate and of dichlorophenolindophenol, the major site of electron donation seems to by-pass both cytochromes and is closer to photosystem 1 [14]. The quantum requirement measurements presented in this article indicate that ascorbate s major site of electron donation is at the electron donating side of photosystem 2, between the oxygen evolution step and the photoact. In the case of NADP+ photoreduction the Red Drop measured in the presence of ascorbate was the same as that of the water to NADP+ reaction. Somewhat Merent values for the drop in quantum requirement were obtained for the diquat system. However, as was shown before [lo] the distribution of light, at a certain wavelength, between the two photosystems may be varied with changing electron acceptors, such as diquat and NADP+. Following the same hypothesis we can assume that addition of ascorbate (i. e., changing of electron donor) may also result in a change of distribution of the light between the photosystems. The results obtained with Tris-treated chloroplast preparations restricted even more the site of action of ascorbate. It was already shown [S] that these preparations have active photosystem 2, and therefore the failure of ascorbate to restore NADP+ photoreduction in these preparations indicates that the region of its donation site must be closer to the oxygen evolution step than the point of inhibition obtained by this treatment. Thus, we propose the following scheme : Tris - treatmenl /--pho!osystem 2 h ydroquinone, or p - phenylenediamine t + Uichlorophenol - dichloropheno( - dimethylurea 7photosyitem 1- ascorbate indophenol NAUP The ascorbate-hydroquinone dependent ATP formation by Tris-treated chloroplast preparations presented in this paper differs from that already described by Yamashita and Butler [7]. They measured ATP formation in presence of ascorbate + p-phenylenediamine under anaerobic conditions and showed an absolute requirement for an electron acceptor. It seems to us that this discrepancy may be due to the different measuring conditions, employed, i. e. anaerobic versus aerobic systems. Though we found no significant oxygen uptake during ATP formation its presence may be important for maintaining the required redox state of the system. It was previously shown that the cyclic photophosphorylation in the presence of phenazine methosulphate [17], or dichlorophenolindophenol-ascorbate [ls] were strongly dependent on the redox state of the medium. Thus, the role of the electron acceptor in the anaerobic system could be solely the keeping of a suitable redox potential for ATP formation, as was previously shown in the ascorbate + dichlorophenolindophenol case [181. After completion of this work, similar conclusions were arrived at by Bohme and Trebst [19]. Ascorbate was photooxidized in presence of electron acceptors for photosystem 1 and its site of donation of electrons was indicated to be prior to photosystem 2. In contrast to our work no such reaction took place in presence of NADPf.

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