The Effect of Permeant Buffers on Initial ATP Synthesis by Chloroplasts Using Rapid Mix-Quench Techniques*

The chemiosmotic hypothesis predicts that buffers which permeate chloroplast membranes should delay the formation of the proton gradient at the onset of illumination. If valinomycin and KC1 are present to collapse the electrical potential as well, this delay should result in a lag in initial ATP synthesis. Using rapid-mix, acid-quench techniques, we have found that in light-driven ATP synthesis the permeant buffer imidazole does not increase the initial lag caused by the valinomycin-KC1 pair. Similar results are obtained under methyl viologen or phenazine methosulfate/ascor-bate-mediated photophosphorylation and are inde- pendent of the internal volume of the chloroplasts. Furthermore, we have observed that chloroplasts can synthesize significant amounts of ATP in darkness following an illumination period as short as 100 ms. This capacity for ATP synthesis in darkness after short pre-illumination periods is decreased in the presence of imidazole, and this may account for the apparent lags reported in earlier studies which have used rapid flash photophosphorylation in the presence of per- meant buffers. The results of the present study argue that in chloroplasts, initial ATP synthesis and post- illumination ATP synthesis are driven by distinct com-ponents of the proton motive potential. The chemiosmotic to organize a great many observations in the field of bioenergetics by its concepts of vectorial proton movements, transmembrane proton and electrical potentials, and proton-translocating ATP synthetases. One set of observations which has been well explained is the effect of weak amine buffers entering a stainless steel tube through which they flowed in darkness before mixing with perchloric acid in a dark mixer.

The chemiosmotic hypothesis predicts that buffers which permeate chloroplast membranes should delay the formation of the proton gradient at the onset of illumination. If valinomycin and KC1 are present to collapse the electrical potential as well, this delay should result in a lag in initial ATP synthesis. Using rapid-mix, acid-quench techniques, we have found that in light-driven ATP synthesis the permeant buffer imidazole does not increase the initial lag caused by the valinomycin-KC1 pair. Similar results are obtained under methyl viologen or phenazine methosulfate/ascorbate-mediated photophosphorylation and are independent of the internal volume of the chloroplasts. Furthermore, we have observed that chloroplasts can synthesize significant amounts of ATP in darkness following an illumination period as short as 100 ms. This capacity for ATP synthesis in darkness after short pre-illumination periods is decreased in the presence of imidazole, and this may account for the apparent lags reported in earlier studies which have used rapid flash photophosphorylation in the presence of permeant buffers. The results of the present study argue that in chloroplasts, initial ATP synthesis and postillumination ATP synthesis are driven by distinct components of the proton motive potential.
The chemiosmotic hypothesis has been able to organize a great many observations in the field of bioenergetics by its concepts of vectorial proton movements, transmembrane proton and electrical potentials, and proton-translocating ATP synthetases. One set of observations which has been well explained is the effect of weak amine buffers on proton movement and phosphorylation in chloroplasts. Work by Nelson et al. (1) and Avron (2) have shown that pyridine, phenylenediamine, aniline, and imidazole not only increased the amount of proton uptake by chloroplasts in the light but also increased ATP synthesis during post-illumination phosphorylation. It was suggested that these buffers permeated the thylakoid membrane and increased the capacity of the inner thylakoid space to store protons. In later work, Ort et al. (3) reasoned, as a corollary, that these buffers should also delay the build-up of the proton gradient at the beginning of illumination. These workers studied the early events of photophosphorylation under conditions where the electrical com-*This work was supported by a grant from the Biochemistry Program of the National Science Foundation. This is Paper 1212 from the Department of Biology, The Johns Hopkins University. The costs of publication of this article were defrayed in part by the Payment of Page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ponent of the electrochemical gradient was abolished by the addition of valinomycin and KCl. An electronic shutter was employed to provide illumination in the form of a short flash which, by generating a burst of protons, drove ATP synthesis. To magnify the signal derived from a short flash, Ort et al. had used a train of short flashes where each single flash was followed by a long dark period (multiple flashes). The duration of the dark period was such as to allow any pH gradient formed by a single flash to be dissipated before a second flash occurred. Using this experimental protocol, Ort et al. demonstrated that although tris(hydroxyamino)methane, orthophosphate, and bicarbonate permeated the inner thylakoid space, these buffers did not delay the onset of photophosphorylation beyond the 50-ms lag caused by valinomycin and KCl. These results were challenged by Vinkler et al. (4) and Davenport and McCarty (5). Using single short flashes, Vinkler et al. observed that pyridine, imidazole, and phosphate did yield an apparent increase in the lag of initial ATP synthesis. Davenport and McCarty found that if samples were illuminated by a single flash, imidazole appeared to increase the lag in initial ATP synthesis; whereas, if samples were illuminated by multiple flashes, imidazole did not appear to increase this lag. Vinkler et al. and Davenport and McCarty postulated that Ort et al. s failure to observe the imidazole-dependent lag in initial ATP synthesis was the result of the experimental design and in specific that multiple flashes caused the progressive build-up of a transmembrane proton gradient. This would lead to a situation where each successive flash would yield progressively more ATP, and the average yield of ATP/ flash would increase as the length of the train of flashes increased, which would result in an underestimation of lags. However, Ort et al. had measured photophosphorylation under single flashes also and had observed essentially the same results as with multiple flashes of equal energy input.
In the above mentioned experiments, the investigators graphed the yield of ATP versus the duration of the light flash and derived curves indicating lags in initial ATP synthesis. It should be noted that the rapid flash technique uses an electronic shutter to begin and end the photophysical events of photophosphorylation with great precision. However, the chemical events cannot be controlled with equal precision because after illumination, the reaction is ended by manual injection of acid. Thus, light-independent chemical transformations are likely to continue for unaccounted periods of time beyond the extinction of the light. Our laboratory has recently used the technique of rapid mixing and quenching to study photophosphorylation (a), and one advantage of this technique over short flash methods is that the interval of darkness between the end of illumination and perchloric acid termination can be precisely controlled. Using this technique, we have obtained evidence that after illumination intervals as short as 100 ms, significant ATP synthesis continues in the dark and that imidazole, as a permeant buffer, inhibits this pre-steady state post-illumination ATP synthesis but not initial light-driven ATP synthesis.

EXPERIMENTAL PROCEDURES
Rapid-mix, acid-quench experiments were carried out using the apparatus described by Froehlich et al. (7). Mixers, illumination tubing, and illumination have been described (8). In the following experiments, which measured initial rates of ATP synthesis, chloroplasts were kept in darkness until the instant of mixing by using a stainless steel tube to carry the sample from the syringe block to the first mixer. Once in the first mixer, the sample path leading to the mixing T-junction was stainless steel tubing set in the plexiglass mixer. Using this arrangement, the chloroplasts were illuminated just as they were mixed with ADP and Pi. Illumination continued as the sample flowed through clear Teflon tubing, and the reaction was ended under illumination as the sample mixed with perchloric acid in a clear plexiglass mixer, Reaction time was controlled by varying the lengths of the clear tubing, and in this protocol the illumination time and the total reaction time were the same. T o simulate a short light flash and the following period of darkness before perchloric acid addition, the clear illumination tubing was connected to stainless steel tubing through which the sample flowed in darkness until the reaction was quenched by mixing with perchloric acid in a dark mixer. In this protocol, illumination time was controlled by the length of clear Teflon tubing, and the time in darkness was controlled by the length of stainless steel tubing. Chloroplasts were isolated from fresh market spinach and were washed in a hypotonic medium to deplete them of residual potassium according to Ort and Dilley (6). Chloroplasts were preincubated in STNM (0.2 M sucrose, 20 mM Tricinel-NaOH, pH 8,lO mM NaCl, 2 mM MgClJ containing permeant buffers for 1 h at 0 "C in darkness. Before rapid mixing, samples were diluted 10-fold in the same medium containing either 1 mM methyl viologen or 50 p~ PMS, 1 mM ascorbate with or without 10 mM KC1 at 18 "C, and valinomycin in ethanol was added to 0.665 p~, 0.5% ethanol. Samples were rapidly mixed with an identical medium which was supplemented with 2 mM ADP and 2 mM Napi, pH 8 (10,000 cpm of 32P, nmol"). Reactions were quenched by mixing with 1 M perchloric acid containing 50 mM NaHsPOr. Formation of labeled ATP and steady state photophosphorylation control rates were determined according to Horner et al. (8). The background was 0.01 nmol mg" of chlorophyll.

Fig. 1 (inset)
shows that in the presence of valinomycin, KC1 causes a lag of between 200 and 300 ms in methyl viologen-mediated photophosphorylation. When 2 mM imidazole was included, no additional lag was observed (Fig. 1). The instability and low rates of methyl viologen-dependent photophosphorylation led us to use PMS/ascorbate-catalyzed cyclic photophosphorylation. In previous studies (4, 5), the use of PMS was avoided due to its possible photoconversion, which might affect the results. This photoconversion of PMS requires between 10 and 30 s to occur under our experimental conditions and is completely inhibited by ascorbate (data not shown). Thus, for our experiments, in which ascorbate was present and ilfumination lasted for at most 300 ms, no PMS photoconversion occurred.
When initial rates of ATP synthesis were determined in the presence of PMS, ascorbate, and valinomycin, we found a small KC1-dependent lag in photophosphorylation (   (12) without potassium depletion, were preincubated in STNM with 10 mM KC1 at 1.0 mg of chlorophyll ml" with or without 2 mM imidazole-HC1, pH 8. The methyl viologen control rate in the absence of imidazole was 0.61 pmol min" mg" of chlorophyll. A, -imidazole; A, +imidazole. Inset, without preincubation, potassium-depleted chloroplasts were suspended at 0.11 mg of chlorophyll ml" in STNM containing 1 mM methyl viologen with or without 10 mM KC1 a t 18 "C prior to rapid mixing, and valinomycin in ethanol was added. The methyl viologen control had a rate of 0.64 pmol min" mg" of chlorophyll. 0, -KCI; a, +KCl. by PMS and ascorbate. Photophosphorylation at pH 8 in the presence of either 10 mM bicarbonate or 2 mM imidazole showed that these two buffers did not extend the valinomycin-KC1 lag (Fig. 3) even a t increased concentrations of valinomycin and KC1 (Fig. 3, inset). Imidazole did decrease the initial rate of ATP synthesis, but because imidazole also decreased the rate of steady state photophosphorylation (Fig.  4), this decrease was not considered as evidence that imidazole delayed initial proton gradient formation but rather that imidazole was an uncoupler of photophosphorylation as has been previously reported (9).
We reasoned that if there were a lag induced by permeant buffers, this lag should be dependent on the internal volume of the thylakoids which is itself dependent on the osmolarity of the medium. If the internal volume were enlarged, the amount of permeant buffers inside would be increased, and any lag in building up the proton gradient would be exaggerated. I t was previously shown by Rottenberg et al. (10) and Ort et al. (3) that the chloroplast thylakoids behave as osmotically sensitive spaces. Fig. 5 shows the results of one of our experiments in which the osmolarities of the 0.2 and 0.01 M sucrose reaction mixtures differed by nearly 10-fold, and yet the initial lags in the presence or absence of imidazole were the same. Moreover, if the 0.01 M sucrose curve was normalized to the 0.2 M curve, the two imidazole curves could be superimposed. Lags in initial rates in the presence or absence of imidazole were not dependent upon osmolarity and hence upon the internal volume of the thylakoids.
To reconcile our results with those of Vinkler et al. (4) and Davenport and McCarty (5), we set up the rapid-mix, acidquench machine to simulate a single short light flash followed by a defined period of darkness before perchloric acid was added to end the reaction. We found that in the presence of valinomycin and KC1, there was significant post-illumination Initial ATP Synthesis by Chloroplasts 11645 ILLUMINATION TIME (rnsec)

FIG. 2.
Photophosphorylation in the presence of PMS/ascorbate, valinomycin, and KCl. Without preincubation, chloroplasts were suspended at 0.11 mg of chlorophyll ml" in STNM containing 50 p~ PMS, 1 mM sodium ascorbate, and with (0) or without (0) 10 mM KC1, and valinomycin in ethanol was added. The PMS/ascorbate control rate was 9.18 pmol min" mg" of chlorophyll.

FIG. 6. The effect of imidazole on illumination and post-illumination ATP synthesis in the presence of valinomycin and KCl.
Chloroplasts were preincubated at 1.00 mg of chlorophyll ml" in 33 mM sucrose, 8 mM Tricine-NaOH, pH 8, 5 mM MgCL, with or without 2 mM imidazole-HC1, pH 8. Afterwards, chloroplasts were diluted 10-fold into 0.2 M sucrose, 20 mM Tricine-NaOH, pH 8, 10 mM KCl, 5 mM MgCI,, 1 mM sodium ascorbate, 50 p~ PMS, with or without 2 mM imidazole-HC1, pH 8, and valinomycin in ethanol was added. a, chloroplasts were mixed with substrates and illuminated for 100 ms before either mixing with perchloric acid or entering a stainless steel tube through which they flowed in darkness for 133 or 183 ms before mixing with perchloric acid in a dark mixer. The PMS/ascorbate control rate without imidazole was 11.4 pmol min" mg" of chlorophyll. A, -imidazole; A, +imidazole. b, chloroplasts were preincubated with 2 mM imidazole-HCI, pH 8 (A), 2 mM piperazine-

N,Nf-bis(2-ethanesulfonic acid) (PIPES)-NaOH, pH 8 (O), or neither (A).
Chloroplasts were mixed with substrates and illuminated for 188 ms before either mixing with perchloric acid or entering a stainless steel tube through which they flowed in darkness before mixing with perchloric acid in a dark mixer.
the amount of ATP made after illumination. When the illumination period was increased to 190 ms (Fig. 6b), the difference caused by imidazole was even more pronounced. Without imidazole, post-illumination synthesis continued for 180 ms, while in the presence of imidazole, post-illumination synthesis stopped after 110 ms. In the absence of imidazole, 190 ms of light yielded approximately 10 nmol of ATP mg" of chlorophyll in post-illumination synthesis, an increase from the 4.5 nmol after 110 ms of illumination. In the presence of imidazole, 110 and 190 ms of light both yielded the same amount of post-illumination synthesis (3 nmol of ATP mg" of chlorophyll).

DISCUSSION
In photophosphorylation catalyzed by either methyl viologen or PMS/ascorbate, the presence of imidazole did not increase the lag in initial ATP synthesis over that caused by valinomycin and KC1 alone (Figs. 1 and 3). Moreover, if the osmolarity of the medium was decreased (which has the effect of increasing the internal volume of the thylakoids), the presence of imidazole did not extend the valinomycin-KC1 lag (Fig. 5). Assuming an internal volume of 30 ~l mg" of chlorophyll (3), equal internal and external concentrations of imidazole, and a proton internalization rate of 2000 kmol mg" of chlorophyll h" (3), 2 mM imidazole should have delayed the initiation of ATP synthesis for 117 ms. This was well within the limits of resolution of our technique. This proton internalization rate is very close to the rate calculated from the steady state rates observed in our controls using the value of 3 H' for every ATP (11).
Studies which have been interpreted to indicate the presence of initial lags in ATP synthesis due to permeant buffers ( 4 5 ) have used short flash photophosphorylation techniques. With the results presented here, we believe that we can now more accurately interpret the results of those earlier studies. In short flash experiments, illumination time is precisely controlled by an electronic shutter, but the termination of the reaction is carried out by manual injection of 1-2 ml of acid from a 3-ml syringe into the 1-ml sample. Although the injection is begun less than 100 ms after the flash (4), the addition is not instantaneous, nor is the mixing of acid with the sample, The time span required to terminate the chemical transformations has never been determined in those experiments. However, even if it takes as little as 100 ms, which is unlikely, significant post-illumination ATP synthesis can take place in that time, as Fig. 6, a and b, has shown. Thus, the ATP synthesized under a short flash experiment is the sum of the ATP made in the light plus the ATP made in darkness before the added perchloric acid can completely quench the reaction. Any buffer, such as imidazole, which inhibits post-illumination ATP synthesis would lower the yield of ATP synthesized under such an experimental protocol, and this could be interpreted as a lag. The rapid mixquench technique does not share this weakness in that the continuous flow of reactants makes it possible to control not only illumination time but also substrate addition and acid termination with great precision. Using this technique, imidazole appears to slow the development of the capacity for presteady state post-illumination ATP synthesis and not to affect initial light-driven ATP synthesis.
Both Ort et al. ( 3 ) and Vinkler et al. (4) have shown that imidazole and pyridine slow the development of the capacity for post-illumination ATP synthesis while increasing the ultimate capacity if illumination is long enough (this concept is illustrated by Fig. 7). This is consistent with the results presented here, although the instability of methyl viologenmediated photophosphorylation and the inhibitory effects of imidazole upon steady state photophosphorylation call for further work to clarify this issue. However, if we assume that the effect of imidazole on the development of the capacity for post-illumination ATP synthesis is due to its action as a permeant buffer, an interesting suggestion can be made. ATP In this model, we will assume that in the presence of valinomycin and KC1, ATP synthesis is driven solely by the transmembrane proton gradient and that a minimum concentration gradient of 2.3 pH units is required for ATP synthesis (13). The permeant buffer imidazole (I) should delay the drop in the internal pH because its pK. is 6.8, between the external pH 8 and internal pH required for ATP synthesis, pH 5.7. The permeant buffer pyridine (P), whose pK, is 5.2, will not delay the initiation of ATP synthesis as much as imidazole because pyridine's maximum buffering range is below the pH 5.7 threshold for ATP synthesis. During steady state phosphorylation, the internal pH will rest around pH 5. 3 (14). When illumination ends, the internal proton reservoir will continue to drive ATP synthesis until the internal pH rises to pH 5.7. Imidazole will extend this post-illumination ATP synthesis, but because its pK, is 6.8, only 5% of its protons can be used before the threshold (pH 5.7) is reached. Pyridine (pK. 5.2) will extend post-illumination ATP synthesis much further because most of its protons can be used before the internal pH reaches 5.7. These effects depend of course on the external pH. For instance, an external pH of 7.0 will imply that the internal pH threshold for ATP synthesis is 4.7, and pyridine will then act to delay initial ATP synthesis as imidazole does at the external pH of 8.0.
synthesis during illumination could be driven by protons conducted from the electron transport system to the CFI. CF, complex through an intramembrane pathway. The common observation that uncouplers increase the rate of electron transport implies that the rate-limiting step in proton utilization is ATP synthesis. At some junction, perhaps at the CFo, those protons which could not be immediately used for ATP synthesis could be shunted to the internal aqueous space. In the absence of a permeant buffer and after, say, 100 ms, the pH of the internal space could drop more than 2.5 units, Le. there would be a pH sufficient to drive post-illumination synthesis, and this pH would increase up to a point as illumination time increases. However, in the presence of a permeant buffer, the excess protons which are shunted into the internal aqueous space would be taken up by the permeant buffer, and the internal pH decrease would be delayed, even though ATP synthesis driven directly by intramembrane protons would not be greatly affected. As illumination proceeds, the buffering capacity of the permeant buffer would be saturated, and the internal pH would drop. The chloroplasts would now become competent for post-illumination synthesis, and the extra protons stored by the permeant buffer would serve to increase the final post-illumination yield of ATP as observed by Avron (2) and Nelson et al. (1). Thus, a permeant buffer could have little effect on initial rates of ATP synthesis during illumination and yet could exert major effects on postillumination yield of ATP.
If every proton must first pass into the inner thylakoid space before being utilized for ATP synthesis by coupling factors, as required by the chemiosmotic coupling hypothesis (Fig. 7), permeant buffers should affect the availability of protons generated either by methyl viologen or by PMS in the same manner. If permeant buffers do cause lags in initial ATP synthesis, the lags should be present in either cyclic or noncyclic photophosphorylation. Since we have found that imidazole does not appear to extend the valinomycin-KC1 lag in initial ATP synthesis, we therefore must concur with Ort et al. (3) that initial ATP synthesis and possible steady state as well may be driven by a pool of protons distinct from the transmembrane gradient which powers post-illumination synthesis. When properly considered, this interpretation emphasizes the element of time in the overall process of proton utilization for ATP synthesis. According to this concept, a given "mobilized" proton does not have to obligatorily migrate into the lumen of the membrane vesicle before it can be utilized by the machinery of the coupling factor. However, there exists a significant probability for a given proton to leave the intramembrane domain and enter the lumen space before it can find its way back to the CF,. CFo complex. The establishment of a transmembrane pH gradient is thus viewed as a consequence of the relative conductance of intramembrane protons through the CF, and to the lumen. Because of the reversibility of the overall process, some of the protons that have accumulated in the thylakoid lumen can and eventually will travel through the CF, to generate ATP.