Detecting localized proton currents in photophosphorylation by procaine inhibition of the transthylakoid pH-gradient

Biochimica et Biophysica Acta, 1140 (1993) 251-261 © 1993 Elsevier Science Publishers B.V. All rights reserved 0005-2728/93/$06.00

251

BBABIO 43731

Detecting localized proton currents in photophosphorylation by procaine inhibition of the transthylakoid pH-gradient Henrik Laasch, Carolin Ihle and Gabriele Giinther Institut fiir 6kologische Pflanzenphysiologie, Heinrich-Heine-Universitiit, Diisseldorf (Germany) (Received 21 February 1992) (Revised manuscript received 15 June 1992)

Key words: Chloroplast; Photophosphorylation; ATPase, thylakoid; Local anesthetic; Localized energy,coupling The relationship between the transthylakoid pH-gradient, ApH, and the velocity of photophosphorylation, Vp, in thylakoid membranes from spinach was investigated using the local anesthetic amine procaine as inhibitor of ApH. When ApH was driven by Photosystem (PS) II + I-dependent electron flow, passing through the cytochrome b6/f complex, inhibition by procaine was accompanied by an increase of ATP formation. It appeared that procaine allowed for values of Vp similar to those in controls (without procaine) at a significantly lower ApH than in the controls. In contrast, when ApH was driven by cyclic electron flow around P S I or by PS-II + I-dependent electron flow via a bypass around the cytochrome b6/f complex, or by PS II alone, procaine simultaneously caused an inhibition of ApH and a decrease of ATP formation. Inhibition of ApH by procaine did not induce an electrical membrane potential gradient that otherwise may have energetically compensated for the observed decline of ApH. The electron flow capacity was unaffected by procaine. However, inhibition of ApH did not significantly relax pH-dependent control of electron flux. Procaine accelerated ATP hydrolysis by pre-activated thylakoid ATPase to rates which were observed in the presence of uncouplers and had no direct effect on the activation state of the ATPase. The shift in the relationship between ApH and llp towards lower ApH persisted in thermodynamic equilibrium between the phosphorylation potential and ApH. The data indicated that the unconventional effect of procaine on photophosphorylation may be related to effects on proton translocation at the cytochrome b6/f complex and that a localized protonic coupling may occur between cytochrome b6/f and thylakoid-ATP-synthase complexes.

Introduction T h e r e is general consensus that a t r a n s m e m b r a n e gradient of proton potential, A/2H+ , serves as the energy link between light-driven redox reactions of electron transport and A T P synthesis at the CFoF 1 A T P a s e [1,2]. Beyond this consensus, however, there is still uncertainty whether d/2H+ is delocalized over the bulk

Correspondence to: H. Laasch, Institut fiir 6kologische Pflanzenphysiologie, Heinrich-Heine-Universit~it, Universit~itsstr. l, D-4000 Diisseldorf 1, Germany. Abbreviations: AA, 9-aminoacridine; CCCP, carbonylcyanide chlorophenylhydrazone; Chl, chlorophyll; cyt, cytochrome; EDTA, ethylenediaminetetraacetic acid; DBMIB, dibromothymoquinone; DTT, dithiothreitol; MV, methylviologen; PAR, photosynthetically active radiation; PBQ, phenyl-p-benzoquinone; Pi, inorganic phosphate; PMS, phenazine methosulfate; PS, photosystem; qE, energydependent quenching of chlorophyll fluorescence; TMPD, tetramethylphenylenediamine;AGp,phosphorylation potential; ApH, pH gradient across the thylakoid membrane; A/2H+, electrochemical gradient of proton potential; Agt, electrical potential gradient; Ve, rate of electron transport; Vh, rate of ATP hydrolysis; Vi, volume of intrathylakoid space; Vp, rate of photophosphorylation;

aqueous phases adjoining the thylakoid m e m b r a n e or is localized to membrane-associated proton domains, or both (for reviews, see Refs. 2-5). A proton domain was generally defined as a space, containing potentially mobile protons which are not in equilibrium with protons in the bulk phases [5]. A suggestion that energy coupling in chloroplasts involves localized proton domains may have implications for the energetics of photophosphorylation, as well as for all processes which are yet believed to be under control of a common lumenal pH. A m o n g these processes are the different sites of p H - d e p e n d e n t electron flow control [6,7] or enzyme activations, e.g., of thylakoid ATPase [8] or violaxanthin deepoxidase [9]. Assuming a delocalized A p H in the absence of an electrical potential gradient, A g t, and a limitation of the rate of photophosphorylation, Vp, only by ApH, a continual decline of Vp with decreasing A p H was expected and experimentally observed. When A p H was varied by light or by the lipophilic uncoupler carbonylcyanide chlorophenylhydrazone (CCCP), a decrease in A p H of about 0.35 units resulted in about 90% inhibition of Vp [10].

252 However, the 'flow-force' relationship of Vp and ApH proved to be influenced by the procedure of ApH adjustment. A selection of experimental data describing the variability between Vp and ApH was summarized in a recent review [4]. A particular line of evidence for a variable relationship of Vp and ApH was derived from effects of uncoupling amines. Low concentrations of ammonium [11-13], imidazole or hexylamine [14] increased Vo up to 30%, while zlpH was unaffected or decreased. These amines did not induce a significant zlqs [11,14]. Their effects on photophosphorylation were assumed .to be incompatible with the existence of a delocalized A~H+[11,14,15]. Giersch [12,13] explained the described effect of amines on energy coupling by assuming that ATP synthesis in chloroplasts in the absence of amines is not limited energetically, i.e., by the strength of A/21a+, but rather kinetically. Sigalat et al. [14] filled in this hypothesis by postulating resistances for the lateral conductance of protons from the proton pumps of electron transport to the ATPase complex, resulting in decreased ATP synthesis. Their model implied that the exchange of protons between the bulk phase and a hypothetical membrane phase is energetically hindered. Only under this condition may lateral resistances be effective on proton translocation. Consequently, localized proton domains which may be neutralized by uncoupling amine effects were assumed. Another explanation of amine ~effects was given by Pick and Weiss [15]. They suggested that amine effects on ATP formation are primarily due to effects on electron transport, resulting in a localization of coupling rather than in a delocalization. Dilley and coworkers [5,16] finally reexamined amine effects using their criteria for a demonstration of localized coupling. These were the delayed onset of ATP formation under flashing light and the inefficacy of increased proton uptake in continuous light on photophosphorylation in the presence of permeable buffers [16]. In contrast to Sigalat et al. [14], they concluded that low concentrations of amine had no significant effect on localization of energy coupling. In this work, we present a detailed study of effects of the local anesthetic amine 4-aminobenzoic acid-(2dimethylaminoethyl) ester, procaine, on energy coupling in chloroplasts. The results are related to effects of 'classical' amine-type uncouplers and are discussed as to support the hypothesis that localized proton currents are involved in photophosphorylation. A possible mechanism of action of local anesthetic amines in energy coupling is presented.

Materials and Methods

Preparation of chloroplasts. Chloroplasts from Spinacia oleracea L. cv. Monatol were isolated with the

procedure described in Ref. [17] and osmotically ruptured immediately after the isolation procedure. The shocked chloroplasts were stored in the dark on ice in a medium containing 300 mM sorbitol, 25 mM hydroxyethyl-l-piperazineethanesulfonic acid, Hepes, adjusted to pH 8 by KOH, 40 mM KC1, 1 mM MgCI 2, 1 mM MnC12 and 2 mM EDTA. The Chl concentration was photometrically determined. All experiments were conducted using a medium containing 300 mM sorbitol, 15 mM KC1, 2 mM MgC12, 0.5 mM EDTA, 0.5 mM MnC12 and 15 mM Hepes/KOH (pH 8). Rates of electron transport, Ve. PS II + I-dependent electron transport from H2 ° to dioxygen, catalyzed by 25 /zM methylviologen, MV, was polarographically measured at 20°C under phosphorylating conditions. A by-pass of electrons around the cytochrome (cyt) b6/f complex during linear electron flow was enforced by an addition of 100/zM TMPD, in the presence of 2 /~M DBMIB [18]. PS-II-dependent electron flow proceeded from H 2 0 via phenyl-p-benzoquinone, PBQ, to K3Fe(CN) 6, as well as in the presence of DBMIB [19]. The chloroplasts were illuminated with red light of wavelengths > 620 nm (RG 630 filter, Schott, and heat reflecting mirror, Oriel 3-57400, Lot). The indicated light flux densities represent photosynthetically active radiation, PAR. The Chl concentrations varied from t5 to 25 mg/1. Rates of photophosphorylation, Vp. Photophosphorylation was either driven by cyclic electron flow via PS I and mediated by 50 p~M phenazine methosulfate, PMS, or driven by linear PS-II + I-dependent electron flow from H 2 0 to 25 txM MV. Linear electron transport was energized by red light as described above. Cyclic electron transport was driven by far-red light of wavelengths between 685 and 730 nm (RG 695 filter, Schott, and Oriel 3-57400, Lot). Vp was assayed at room temperature or, when required, at 20°C together with ApH and V~. 10 ~M diadenosine pentaphosphate was added to 1 mM ADP and 4 mM K2HPO 4 for inhibition of the adenylate kinase (EC 2.7.4.3). The ATP synthesized was determined with the luciferin/ luciferase (EC 1.14.14.3) test [20] when cyclic electron flow was involved, or enzymatically with glucose-6phosphate dehydrogenase (EC 1.1.1.49) and hexokinase (EC 2.7.1.1) when ATP was synthesized during linear electron transport. Rates of A T P hydrolysis, Vh. The CFoF l ATPase of the thylakoid membrane was activated for 2 min in the presence of 5 mM dithiothreitol, DT-I', in the light. If not otherwise stated, the activation ApH was adjusted by the flux density of red light of wavelengths > 620 nm. ATP hydrolysis was allowed after enzyme activation in the presence of ATP concentrations stated below. The amount of ATP hydrolyzed was determined enzymatically with lactate dehydrogenase (EC 1.1.1.27) and pyruvate dehydrogenase (EC 2.7.1.40). 10 ~M

253 diadenosine-pentaphosphate was added for inhibition of the adenylate kinase.

Energy-dependent quenching of Chl fluorescence, qe. Osmotically-shocked chloroplasts were suspended in standard medium with 4 mM MgC12, at a Chl concentration of 25 mg/l. Electron flow was mediated by 25 ~M MV. Photophosphorylation was allowed by additions of 0.5 mM ADP and 4 mM K2HPO 4. 5 /zM diadenosine-pentaphosphate was added for a suppression of adenylate kinase activity. The Chl fluorescence was measured with a pulse amplitude modulated fluorometer (PAM, Walz) [21]. After a determination of basic fluorescence with the measuring light pulses only, actinic light of 500 ~ E m -2 s -1, PAR, was switched on. After 2 rain in the light, 20/zM DCMU was added to the rapidly stirred assay to block electron transport. From the biphasic rise of variable Chl fluorescence, qE was calculated according to Ref. 22. Aliqots of the assay were used for a determination of ATP synthesized. Determination of ApH and proton uptake. The ApH was calculated either from quenching of 9-aminoacridine (AA) fluorescence or from the uptake of [14C]methylamine into the intrathylakoid compartment in the light. From AA fluorescence quenching the ApH was calculated following the equation ApH = Iog[AF.F-I-50"C -1]

(1)

(/iF, quenching of fluorescence; F, fluorescence remaining). The empirical expression '50. C -1' (C, Chl concentration expressed in g/l) was chosen instead of the originally used ratio of external and internal volumes [23], since quenching of AA fluorescence was found to be independent of internal volume [24]. The fluorescence was excited and detected at peak wavelengths of 400 and 465 nm, respectively. The final concentration of AA was 5/~M. The Chl concentration was 10 to 25 mg/1. The uptake of [14C]methylamine was assayed using the technique of silicon oil centrifugation, as described in Ref. 25. A mixture of silicon oils AP 150 and AR 20 (Wacker, Miinchen) was used for the separation of chloroplasts from the aqueous supernatant. 1.5 MBq of [~4C]methylamine, corresponding to a concentration of 0.8 /.~M, was added to chloroplasts equivalent to 100 mg Chl/l. The chloroplasts were illuminated with white light of 2500/~E m -2 s -1, PAR, which was filtered by heat reflecting mirrors. The ApH was calculated according to the equation ApH =

Iog(AI'Aol'Ve'Vi -I)

(2)

(A I, A o, amounts of amine bound to thylakoids or free in the medium; Ve, Vi, volumes of the external and the intrathylakoid spaces). Vi was determined by the

method described below. Proton uptake by illuminated thylakoid vesicles was measured with a glass electrode at pH 8 in the absence of ADP and Pi in a medium composed as the standard medium, but with only 0.4 mM Hepes-KOH present. The concentration of Chl was 30 rag/1. Volumes of intrathylakoid space, V1. The values of Vi were determined by means of a silicon-oil centrifugation technique [25]. 0.2 ml of a suspension of osmotically-shocked chloroplasts, equivalent to 100 mg Chl per 1, was incubated with 17 MBq [3H]HzO and 2.4 MBq [14C]sorbitol. After 2 rain of incubation in the dark or light, the chloroplasts were centrifuged through a layer of silicon oil, composed of 39% AP 150 and 61% AR 20 (both from Wacker, Munich), into a compartment containing 3 M HC104. From the contents of 3H- and of 14C isotopes in the HC104-containing compartment, the volume of the sorbitol impermeable space was calculated.

Determination

of the membrane potential,

A~.

Chloroplasts were osmotically shocked in standard medium which instead of KCI contained 40 mM KNO 3. After sedimentation by centrifugation, the membranes were washed twice and finally stored in the medium described. Chloroplast membranes at a concentration of 200 mg Chl/l were illuminated for 2 min and then sedimented through a layer of silicon oil into HC104, as described in the previous paragraph. The contents of CI- in the supernatant and in the sediment were determined by coulometric titration with Ag +, which was generated from a silver electrode by current pulses. The contents of C1- evaluated for the HC104-containing compartment was corrected for the amounts of CIimported by suspension medium, attached to the chloroplast membranes. Results

Effects of procaine on electron transport, ApH and ATP formation during linear electron flow across PS-II and I The effects of procaine on the rates of linear electron transport, V~, photophosphorylation, Vp, and on the light-induced ApH are pictured in Fig. 1. ApH was increasingly inhibited by rising concentrations of procaine. Electron transport was slightly accelerated but, despite of a strong inhibition of ApH, remained quite remote from its maximum capacity. The maximum capacity was observed after rigorous uncoupling with 50 nM gramicidin and 1 mM NH4CI in the absence of procaine [26]. The rate of electron transport measured in the presence of these uncouplers was not influenced by addition of 2 mM procaine. This indicated that procaine was no inhibitor of electron flow. Apparently, electron flow control, which was thought to depend on the pH in the thylakoid lumen [7], was maintained, even in the presence of 2 mM procaine. Comparable

254 results on electron flow control have been reported previously [27]. Despite a continuous inhibition of ApH by increasing concentrations of procaine, Vp increased up to procaine concentrations _< 200 /~M, before it finally decreased at higher concentrations. Thus, similar Vp could be related with two quite different values of ApH. With regard to the dependence of Vp on ApH, procaine effects resemble those of other amine-type uncouplers, e.g., hexylamine or ammonium [11,14]. It was shown previously that the ratio of ATP synthesized per two electrons transported, A T P / 2 e ratio, was almost unaffected by the amine-type uncoupler methylamine [11]. A similar observation was made when procaine was used for inhibition of ApH, in the range of procaine concentration where Vp was increasing. The data required for a calculation of the ratio is available from Fig. 1. A value of 0.85 was calculated for controls. After the addition of 0.3 mM procaine, the ratio was still equal to 0.81, despite of a severe ApH decline. Obviously, related procaine effects resemble those of methylamine. When a 'classical' uncoupler (e.g., CCCP) is used for inhibition of ApH, the ratio rapidly decreased with increasing inhibition of ApH, since Vp is inhibited and Ve accelerated (not shown). An alternative method for a determination of the light-driven ApH was used here by measuring the uptake of []4C]methylamine [28]. The ApH calculated from this approach was smaller than that obtained from AA fluorescence (Fig. 2). The degree of ApH inhibition by procaine, however, was comparable to that derived from AA fluorescence (Figs. 1, 4). The volume of the intrathylakoid space, V~, used for this determination, was unaffected by procaine concentra-

150 I.

o

1.5 2

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E 200

1.0 <::]

o_ 0.0

0.5

1,0

1.5

0.5

Procoine (raM) Fig. 2. Inhibition of light-induced proton uptake by thylakoid vesicles (©) and zlpH calculated from [~4C]methylamine uptake (zx) by thylakoid vesicles with increasing concentrations of procaine. The strength of zlpH was calculated from methylamine uptake using the separately determined intrathylakoid volume of 11 p.l/mg Chl. The Chl concentration was 30 m g / l .

tions up to 2 mM in the standard medium used here. Table I displays the values of Vi in the absence and presence of procaine, in light and dark and under condition of different electron transport sequences operative. The decrease of ApH with increasing procaine concentrations was correlated with a diminuition of proton uptake into the thylakoid vesicles (Fig. 2). This decline in proton uptake with increasing procaine concentrations also indicated an uncoupler-like effect of the amine. We considered that protonation/deprotonation of procaine may overlap the pH changes driven by electron transport. However, a probing of light-induced procaine binding to thylakoid membranes showed that possible effects of deprotonation/protonation are significantly smaller than the total decrease of proton uptake observed. As a further test of a ApH decline in the presence of procaine, we studied the dependence of the rates of photophosphorylation on energy-dependent quenching

3.4

0 E 0 (J

2.0

400 1

TABLE I

I00

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>?

2.4 o.0

1.0

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(raM)

Fig. 1. Effects of procaine on the light-induced ApH ( • ) , the rates of photophosphorylation, Vp (z x) and electron transport, Ve (©). ApH was calculated from A A fluorescence quenching. Photophosphorylation was allowed for 2 min in red light of wavelengths > 620 nm. ATP formation was driven by linear electron transport from H 2 0 to methylviologen. 100% of Vp and Ve correspond to 228/.~mol A T P / m g Chl per h and 1540 /.~mol e - / m g Chl per h, respectively. Ve and ApH were determined simultaneously with ATP formation. 100% of electron transport rate was observed in the presence of 2 mM procaine, 50 nM gramicidin and 1 mM NH4CI. The Chl concentration was 15 mg/1.

Effect of procaine on the colume of the intrathylakoid space, Vi Osmotically shocked chloroplasts, suspended in standard reaction medium, were kept in the dark or under white light of 2500 i x E m -2 s - l , PAR, for 2 min, Electron flow in controls (without procaine) and in the presence of procaine was either linear, from H 2 0 via MV to dioxygen, or cyclic, as mediated by PMS. 1300 U of catalase (EC 1.11.1.6) was added to each sample for a recovery of dioxygen. The values of Vi were determined from the differences of [14C]sorbitol and [3H]H20 permeable spaces. Mean values of Vi from n >__20 measurements with four chloroplast preparations are shown. Electron flow

Vi ( # l / m g CtlI) controls

linear, H z O ~ MV cyclic, PMS

+ 2 mM procaine

dark

light

dark

light

12.6+2.4 12.1_+2.3

12.9±2.3 8.7_+2.2

10.5±1.3 11.3_+1.9

8.3_+2.3 8.0_+2.9

255

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200

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15o

o'~

lO0

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E

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0.0

0.1

0.2

0.3

0.4

qe Fig. 3. Dependence of the rate of photophosphorylation, Vo, on the magnitude of energy-dependent quenching of Chl fluorescence, qEVo and qE were influenced by procaine (zx) and nigericin (©). Osmotically-shocked chloroplasts were illuminated with continuous actinic light of wavelengths > 620 nm. Aliquots of the samples were used for a determination of ATP synthesized. The chloroplasts were dark-adapted prior to the measurements for at least 1 h. The Chl concentration was 25 mg/l.

of Chl fluorescence, qz" The parameter qz is known to reflect a mechanism of thermal PS-II inactivation. This inactivation is dependent on the intrathylakoid pH [6] and, thus, on ApH. Fig. 3 compares the influences of nigericin and procaine on the relationship between Vp and ApH. While Vp declined in line with qz when nigericin was used for inhibition of ApH, an increase of Vp with decreasing qz was observed in the presence of procaine. It is apparent that the relationship between the two parameters in the presence of procaine compares to that of lip and ApH shown in Figs. 1 or 4. Regarding qE as an intrinsic measure for the magnitude of ApH. these results confirm the increase of Vp, despite decreased ApH.

The transmembrane electrical potential gradient As outlined above, an electrical potential gradient, AT may contribute to AI2H+ also in chloroplasts [29].

In osmotically-ruptured spinach chloroplasts, a concentration of 0.2 IzM gramicidin was sufficient for a reliable inhibition of light-induced AT [30]. In contrast, ApH became impaired only at higher concentrations of the protonophor. We employed gramicidin in the presence of procaine to probe for the existence of an amine-induced AT. Fig. 4 describes the 'flow-force' relationship between ATP formation and ApH under a light-flux density saturating for ATP synthesis. ApH and Vp were varied by additions of gramicidin (assay I), procaine (assay II) and gramicidin plus a constant concentration of procaine (assay III), respectively. Assay I revealed a linear dependence of Vp and ApH. A linear relationship of these parameters was usually observed when the influence of uncouplers was tested. In assay 1I (Fig. 4), a non-linear 'flow-force' relationship was found, as suggested by Fig. 1. A decline of ApH of about 0.6 units did not decrease Vp below the level of the controls (without amine added). A AT of about - 35 mV (inside positive, see Eqn. 4) would have been required to compensate for this decrease of ApH. In assay III, finally, a linear 'flow-force' relationship appeared, as was observed for assay I. Undoubtedly, gramicidin concentrations of 7/zM were sufficient to wipe out any AT. Assuming that AT contributed to A/2r~÷ in the presence of procaine, the graphs of assay I and III would have converged already at low concentrations of gramicidin due to the inhibition of AT. This was not the case. The magnitude of AT in the presence of procaine and under continuous illumination was determined from the distribution of C1- between the thylakoid lumen and the suspension medium (Table II). AT was calculated from Eqn. 3, wherein [C1-] i and [C1-] o denote the concentration of C1- in the thylakoid lumen and in the medium, respectively. Aq t = [ - R . T / z .

F]. 2.303 log([Cl- ] i / [ C I - ]o )

(3)

2sl TABLE II

:>~ 2.0 o

1.5 2.7

3.2 3.7 A pH Fig. 4. Relationship between Vp and ApH after an inhibition of ApH by increasing concentrations of gramicidin (o), procaine (Ix) and gramicidin in the presence of 250 p,M procaine (0), respectively. ATP synthesis was allowed for 2 min under saturating red light ( > 620 nm). Gramicidin and procaine were added prior to 'light on'. Gramicidin concentrations up to 7/z M were used. Electron transport proceeded from H 2 0 to MV. The ApH was calculated from AA fluorescence. The Chl concentration was 20 mg/l. Vp is plotted in a logarithmic scale.

Electrical potential gradient (inside positive) across the thylakoid membrane, A~P, in the presence o f procaine During 2 min of illumination with white light of 2500 ~E m -2 s - 1 PAR, electron flow proceeded from H 2 0 via MV to dioxygen. For a recovery of dioxygen, 6500 U ml- 1 of catalase and 100 U ml- l of superoxide dismutase (EC 1.15.1.1) were added to the samples. A~ was calibrated by assuming that it was equal to zero in the dark and presence of 1 IzM gramicidin D. Mean values of A ~ ± s t a n d a r d deviation (S.D.) and the number of samples evaluated (n) are shown. Procaine (mM)

Aqt ± S.D. (mV)

n

0 0.2 0.4 0.7 1.0

-17.9±6.9 -27.4±7.9 -26.4±8.2 -25.3±4.5 -26.5±5.9

19 17 17 6 12

256 [C1-] i was calculated using the values of Vi displayed in Table I. Up to 1 m M of procaine present, Aq~ increased by at most 8 mV. A~ZH+ = A ~ -

2.303.(R.T/F).ApH

(4)

According to Eqn. 4, this increase of A ~ may energetically compensate a A p H decrease of only 0.15 units. As a consequence, Vp as shown in Figs. 1 and 4, was apparently increased or maintained despite a factual decrease of A/2H+.

Effect of procaine on ATP hydrolysis Procaine did not inhibit A T P hydrolysis by preactivated thylakoid ATPase (Fig. 5). The velocity of A T P hydrolysis, Vh, was low when hydrolysis was coupled to the build-up of ApH. Procaine induced a five-fold increase of Vh. This pointed to an uncoupler-like activity of procaine. In the presence of high amine concentrations, A T P hydrolysis decreased again. A plot of the logarithm of Vh vs. A p H revealed a linear relationship of the two p a r a m e t e r s up to concentrations of 2.5 mM procaine (Fig. 5, inset). 'Classical' uncouplers like nigericin or CCCP, also accelerate A T P hydrolysis by dissipating ApH. The decrease of Vh observed at high concentrations of procaine or 'classical' uncouplers is probably due to an inactivation of the ATPase at low ApH [8]. The optimum values of Vh achieved with procaine were comparable to those obtained with nigericin or ammonium. The activation of the ATPase is dependent on the A p H existing during activation procedure; an increase of A p H yielded an increase of enzyme activity (Fig. 6). When the A p H was varied by varying light flux density, a decrease by 1 unit of A p H revealed about 75% loss of ATPase activity. A similar result was obtained when A p H was diminished by procaine instead of light. We regarded this as further evidence for inhibition of ApH

300

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o

c-

200

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100 2.8

O

E

o

z~.

~-

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Z~pH o

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Procaine (mM)

IO

> Fig. 5. Acceleration of the rate of ATP hydrolysis, Vh, by procaine (0). The ATPase was preactivated for 2 min in the light in the presence of 5 mM DTT. After the light was turned off, procaine was added and 30 s later 1 mM ATP was added. ATP hydrolysis proceeded for 60 s in the dark. The inset represents the dependence of the logarithm of Vh on the ApH adjusted by procaine during ATP hydrolysis in the dark. The Chl concentration was 25 mg/l.

co

10o

2

-.~ cO ~ ©

75 5O

~

25 c-

>

o

2.5

3.0

3.5

A pH during a c t i v a t i o n Fig. 6. Effect of the A p H adjusted during ATPase activation on the hydrolytic activity of the ATPase. A p H was varied either by changes of the light flux density ( z~ ), or by addition of procaine at a maxium light flux density of 1500/.~E m -2 s -1 (©). A p H was determined by the A A method. Immediately after 'light off' 5 m M NH4CI (zx) or procaine to a final concentration of 3 m M (©) was added. 30 s later, A T P hydrolysis was initiated by addition of 1 m M A T P and proceeded for 60 s in the dark. The Chl concentration was 25 m g / I .

by procaine. Beyond this, the experiment suggested that procaine acted on A T P hydrolysis/formation only by an interference with A/2H+, not by direct inhibition or activation of ATPase activity.

Thermodynamic equilibrium in the presence of procaine Previous studies of unconventional amine effects on photophosphorylation argued that the maintenance of Vp despite decreased ApH may be related to a 'removal of kinetic limitations in the energy-converting process' by means of amines [11,13]. We tried to exclude this tentative mechanism by studying procaine effects on A T P synthesis and hydrolysis under conditions of thermodynamic equilibrium. The method applied here was adopted from Ref. 31. When the free energy of the phosphorylation potential, A G p , is in balance with that of A/2H+, A T P formation and hydrolysis are in equilibrium. U n d e r this condition, Agt could be omitted because of addition of valinomycin. A determination of ApH, hence, revealed the mangnitude of A/2H+. In Fig. 7, the rates of net A T P synthesis and hydrolysis were monitored after imposition of a defined AGp in the presence of the indicated ApH. When AGp was adjusted by addition of ATP, A D P and Pi, the a p H deviated from its initial strength; net A T P hydrolysis caused an increase and net A T P synthesis a decrease of A p H (not shown, see Ref. 31). The A p H values shown in Fig. 7a were adjusted by a reduction of the light flux prior to the imposition of AGp. The energetic equilibrium in the presence of an [ A T P ] . [ A D P ] - 1 . [Pi] - t ratio of 103, indicated by zero A T P formation, was observed at A p H approx. 3.5. In Fig. 7b, the initial A p H was adjusted by the uncoupler CCCP or by procaine, starting from a lightflux density which was sufficient for a maximum A p H in the absence of said inhibitors. Using CCCP for the

257 inhibition of ApH, an equilibrium of ATP formation and hydrolysis was obtained at ApH approx. 3.5, as in the case of varied light-flux density. A decrease of ApH below 3.5, however, allowed for higher rates of ATP hydrolysis than observed under conditions chosen for Fig. 7a. This was related to the fact that only under the conditions in Fig. 7a ADP formation was thermodynamically hindered by the ApH build-up in the course of ATP hydrolysis. When procaine was used for the reduction of ApH, the equilibrium condition was found at ApH approx. 3.0. This discovery suggested that the anomalous relationship of Vp and ApH in the presence of procaine persisted under equilibrium conditions. At ApH approx. 3.3, still allowing for Vp approx. 100 ~mol A T P / m g Chl per h in the presence of procaine, a similar rate of hydrolysis occurred in the presence of CCCP. A possible contribution of Aqz to A#H+ in the presence of procaine could also be excluded by this approach, since the build-up of A~ under equilibrium conditions is unlikely.

Localization of procaine effects on photophosphorylation In the course of seeking to localize procaine effects on energy coupling, linear electron transport was excited with different light qualities. A predominant excitation of PS II or I was obtained with light of maximum intensities either at wavelengths around 650 nm or > 715 nm. The influence of procaine on the relationship of ApH and Vp during linear electron flow, excited by different light qualities, is monitored in Fig. 8. An increase of Vp despite decreased ApH did not occur when P S I was predominantly excited. A prevalent excitation of P S I obviously hindered a 'stimula-

150

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o-50 3

>~

A pH

4

A pH

Fig. 7. Determination of equilibrium ApH for ATP synthesis and hydrolysis. ATP hydrolysis is indicated by negative values of Vo. Activation of the ATPase took place in the presence of 5 mM DTT and 2 min of saturating red light of wavelengths > 620 nm. Subsequently, the indicated ApH was adjusted by: (a), variation of the light-flux density (o) or (b), by additions of CCCP (ix) and procaine (xz), respectively, in the presence of saturating red light. After further 45 s, 0.2 mM ATP, 0.2 mM ADP and 1 mM K2HPO 4 were added. ATP synthesis/hydrolysis then proceeded for 60 s in the light. Rates are plotted vs. the ApH adjusted prior to additions of ATP, ADP and Pi. The Chl concentration was 25 m g / l 0.3 /xM valinomycin was added to all samples.

A

I

150 f-

f(D

T E o

' I00

l

i

g

~ / ~

~ o -~-~ 'light 1 ~

~ 50

E >

i::1_

2.8

3.0

3.2

3.4

ApH

Fig. 8. Dependence of the rate of photophosphorylation, Vp, on the ApH obtained under different light regimes. Electron transport proceeded from H 2 0 to MV. ATP formation was allowed for 1 min. ApH was calculated from AA fluorescence. Thylakoids were illuminated by red light of maximum wavelengths around 650 nm (light 2) or > 715 nm (light 1). The variation of ApH was induced by additions of procaine. The Chl concentration was 20 mg/I.

tion' of ATP formation by procaine. This observation suggested that the procaine effects are linked to PS II or to the electron transport chain between PS II and I. Measurements of steady-state Chl fluorescence at room temperature, as described in Ref. 32, indicated that the primary quinone-type acceptor of PS II, QA, w a s far more reduced under short-wavelength than under long-wavelength red light. The degree of QA reduction was estimated from photochemical Chl fluorescence quenching after 60 s of illumination in the presence of ADP and P~ (data not shown). For a study of PS-I-dependent cyclic photophosphorylation, we illuminated thylakoid membranes with red light of A > 685 nm. Only a low PS II activity, necessary to compensate for the oxidation of PMS by dioxygen, was maintained. Under this condition, the 'flowforce' relationship in Fig. 9a appeared. Any decrease of ApH caused by procaine resulted in a decrease of Vo. The influence of procaine on ApH and Vp resembled that of CCCP. Apparently, procaine "stimulated' ATP formation only when PS II or the intersystem electron-transport chain was involved in light-induced proton translocation. In a further approach, electron transport from H 2 0 to MV and dioxygen was allowed in the presence of DBMIB and TMPD. Under this condition, the cyt b6/f complex was by-passed in the electron transport sequence [18]. Fig. 9b demonstrates that uncoupling with procaine yields a similar relationship between flow (Vp) and force (ApH) to the uncoupler CCCP. Apparently the involvement of the cyt b6/f complex is a prerequisite for 'non-classical' amine effects on photophosphorylation. When ammonium was used as an inhibitor of ApH in the type of experiments shown in Fig. 9a,b, a similar result was obtained (not shown). The effects of procaine and ammonium were comparable.

258

IO0[A

>

2.8

o.~

3.2 A pH

3.6

Fig. 9. Inhibition of ApH and Vo by procaine (o) and CCCP (zx). ApH was driven by cyclic electron flow mediated by 50 ~M PMS (A) or by linear electron flow from HzO via MV to dioxygen, bypassing the cyt b 6 / f complex via TMPD (B). Cyclic electron flow was energized with light of wavelengths > 685 nm for minimization of PS II activity (A). Linear flow was driven by wavelengths > 620 nm (B). Photophosphorylation was allowed for 1 rain. The Chl concentrations were (A) 10 and (B) 20 mg/1, respectively. Maximum concentrations of inhibitors were 2.5 mM procaine and 5 /~M CCCP. 100% of Vp corresponds to (A), 558 (©) and 553 (z~) and (B), 256 (©) and 286 (z~)/~mol ATP/mg Chl per h.

We tested the relationship of ApH and Vp under a condition where only PS-II-dependent electron flow from H / O via PBQ to K3Fe(CN) 6 was operative. 2 /zM DBMIB was added to prevent a contribution of the cyt b6//f complex to membrane energization. Under this condition, photophosphorylation declined in line with ApH, irrespective whether ApH was inhibited by CCCP or by procaine (data not shown). A similar dependence as in Fig. 9a was found. It was concluded that neither PS-I- nor PS-II-dependent electron flow

TABLE III

Velocities of electron transport, mediated by artificial electron donors and acceptors PS-II+I-dependent electron flow proceeded either from H 2 0 to MV and dioxygen, or from H 2 0 via TMPD to MV and 0 2. Since DBMIB was present in the latter case, the cyt b6 / f complex was by-passed. PS-II-dependent electron flow from H 2 0 to Fe(CN) 3was mediated by PBQ in the presence of DBMIB. The maximum of electron transport, Vem, was assumed to occur in the presence of 0.5 /xM nigericin. The Chl concentration was 15 mg/l. Mean values from n > 8 measurements, carried out with 3 chloroplast preparations are shown. Sequence of electron flow

H 2 0 ~ MV H 2 0 ~ TMPD ~ MV (+ 2 # M DBMIB) H 2 0 ~ PBQ Fe(CN) 3 (+ 2 # M DBMIB)

Rates of electron flow (/xmol O 2 / m g Chl per h)

Vem, after addition of 0.5/z M nigericin

Ve, after addition of 2 mM procaine

392_+ 17

225 + 36

465_+29

443 + 38

180_+11

186_+28

alone was able to elicit the amine effect described in Figs. 1, 3 and 4. Finally, we studied the PHi-dependent flux-control of electron transport in the presence of the mediators used in the experiments on photophosphorylation, above. Table III shows the capacities of electron flow, Vem, and the values of Ve obtained in the presence of 2 mM procaine. This amine concentration largely reduced ApH, irrespective of the electron mediators involved. PS-II + I-dependent flow passing through the cyt be/f complex was accelerated only to about 60% of the related Vem. This is in agreement with the data from Fig. 1. In contrast, both, electron flow via PS II + I, by-passing the cyt b 6 / f complex, and PS-II-dependent flow were accelerated to their maximum velocities by procaine. It appeared that a maintenance of flow control in the presence of procaine originated from an amine effect on the cyt b6/f region of electron transport. Discussion

Amines with local anesthetic properties in nerve-cell membranes [33] inhibit the apparent transmembrane ApH and photophosphorylation of thylakoid vesicles [34]. In contrast to the effects of 'classical' uncouplers, however, they do not relax the control of linear electron flow from H 2 0 to MV. This feed-back control is usually thought to be dependent on the acidification of the thylakoid lumen [6,7]. With other amines of local anesthetic activity, procaine was classified within the group of 'selective' uncouplers [34]. Uncoupling by local anesthetic effects was termed 'selective', since ApH and photophosphorylation, but not pH-dependent electron flow control, were inhibited [34,35]. This study was centered on the relationship between the activities of ATP formation and hydrolysis by CFoF I ATPsynthase and the assumed intermediate of energy coupling, A/2H+, in the presence of a 'selective' uncoupler. The experiments shown in Figs. 1 and 4 indicate that the effects of procaine on the relationship of Vo and ApH during PS-II + I-dependent electron transport resemble those of hexylamine, ammonium or imidazole [12,14]: ATP formation was increased despite a lowered ApH. The inhibition of ApH by procaine was indicated by the methods of AA fluorescence quenching (Figs. 1 and 4), by [14C]methylamine uptake and, more indirectly, by proton uptake (Fig. 2). A comparison of the effects of light and procaine on the activation of the CFoF1 ATPase (Fig. 6) and the acceleration of ATP hydrolysis by procaine (Fig. 5) also indicated the inhibition of ApH by procaine. Energy-dependent quenching of Chl fluorescence, qe, was furthermore used as an 'intrinsic' indicator of ApH [6,21]. The apparent correspondence of the relationships between Vp and ApH (Fig. 4) on the one

259 hand and V~ on the other (Fig. 3), strongly confirmed that photophosphorylation may increase despite a decreased ApH. At least four independent observations pointed out that the decrease of ApH in the presence of procaine was not counterbalanced by an increase of A~. First, a relative increase of Vp by means of procaine was not reversed by gramicidin concentrations up to 7 ~M (Fig. 4). This concentration of the ionophore would erase any Aqt in thylakoid vesicles [30]. Second, the anomalous relationship of Vp and ApH only appeared when the cyt b6/f complex was involved in electron transport. Since an accumulation of positively-charged amine in the thylakoid lumen was thought to be in charge of amine-induced A~, it is unreasonable to assume that PS-I-dependent proton translocation or intrathylakoid acidification originating only from water splitting, may not cause a A~ (Fig. 9). Third, the activation of CFoF ~ ATPase showed a similar dependence on ApH when the latter was influenced by light or procaine (Fig. 6). CFoF 1 ATPase may be activated either by ApH or by A~ [36,37]. Assuming that a significant A~ was induced by procaine, the ATPase would have remained active, despite an inhibition of ApH. Finally, a direct measurement of A~ indicated that the amine-induced increase of Aqz was < 8 mV and, thus, by far too small to compensate for the related decrease of ApH. Another essential continuation of previous reports on amine effects in photophosphorylation was to study equilibrium between AGp and ApH in the presence of procaine. In thermodynamic equilibrium, the phosphorylation potential may be described by the equation AGp = AG ° + RT" In(tATP]" [ADP] - " [Pi 1 - ' )

(5)

(AG °, standard free energy change; R, gas constant and T, absolute temperature; [ATP], [ADP] and [Pi], concentrations of these eompounds). AGp was adjusted only by the concentrations of ATP, ADP and Pi under our conditions. It is related to A/2H + by the equation AGp = n.A/2rt+

(6)

(n, mol protons translocated per mol ATP synthesized or hydrolyzed). Assuming that A~ is equal to zero under equilibrium conditions in the presence of valinomycin, A/2H+ in the latter equation may be substituted by ApH. Hence, AGp is a function of ApH when the coefficient n is assumed to be constant. In fact, there is no reason to assume different values of n for ATP formation driven by PS II + I or PS I alone [31,38]. Strotmann and Lohse [31] demonstrated that a ApH, initially adjusted by light flux, was unaffected by additions of ATP, ADP and Pi, when there was no net ATP synthesis and hydrolysis. Following this approach for determination of thermodynamic equilibrium, a de-

fined AGp was imposed on all samples [31]. The ApH was influenced by varying the light flux density or by additions of CCCP or procaine (Fig. 7). A thermodynamic equilibrium in the presence of procaine was obtained at a lower ApH than in the presence of CCCP or variable light flux densities. Evidently, the unconventional procaine effects on ATP formation are not due to a removal of kinetic limitations as suggested in Refs. 12 and 13 and persisted under equilibrium conditions. The evidence presented in this work strongly implies the new concept that in the presence of 'selective' uncouplers, electron flow through the cyt b6/f complex is in charge of a driving force of ATP formation which was not based on a Aqt and was not amenable to conventional methods of ApH determination. This driving force, however, was still related to a proton potential gradient, since 'classical' uncouplers were able to inhibit ATP formation in the presence of procaine (Fig. 4). We hypothesize that the procaine effects disclosed a mechanism of localized energy coupling in the cyt b6/f region of the thylakoid membrane. Localized energy coupling may be an alternative pathway of proton conductance from the cyt b6/f complex to the CFoF 1 ATP synthase and may be active side by side with the delocalized pathway of coupling. The 'amineeffect' on photophosphorylation exerted by hydrophilic amines like NH4C1 is also dependent on the involvement of the cyt b6/f complex (not shown). The methods of ApH determination applied here were those most frequently used in studies on the energetics of photophosphorylation [26,38,39]. However, it must be noticed that these methods appear to be unsuitable for a detection of the postulated localized gradients of proton potential. Even AA fluorescence quenching, which, different from methylamine uptake [28], can be produced by AA binding to energized membranes [39] rather than by accumulation in the thylakoid lumen [23], failed to indicate a ApH matching the observed values of Vo. Like AA fluorescence or methylamine uptake, the value of Vh as an intrinsic indicator of A/2H+ failed to be influenced by the postulated localized gradient. Furthermore, the present investigation again justified a delimination of 'selective' uncouplers [34] and decouplers, as described by Rottenberg [4]. While decouplers inhibited ATP formation without affecting the ApH [4], the 'selective' uncoupler procaine in comparison stimulated ATP formation associated with an obvious inhibitory effect on ApH. Several hypotheses have been advanced to explain the effects of local anesthetics on membrane excitation. An interference of these compounds with electrical charges on the membrane surfaces [40] and their binding to specific receptor protein complexes, such as ion channels [41], has been assumed. Previously, we

260 s h o w e d a d i s p l a c e m e n t of m a g n e s i u m ions f r o m thyla k o i d m e m b r a n e s by a local a n e s t h e t i c amine, dibuc a i n e [42]. This effect m a y tie o u r results to r e c e n t r e p o r t s on a c a l c i u m - g a t e d switch o f p r o t o n flux b e t w e e n d e l o c a l i z e d a n d l o c a l i z e d p r o t o n g r a d i e n t s . Dilley arid c o - w o r k e r s [5,43] s u g g e s t e d t h a t t h e d i s p l a c e m e n t o f a specific c a l c i u m ion f r o m t h e t h y l a k o i d m e m b r a n e o p e n s a gate, allowing an e q u i l i b r a t i o n of a l o c a l i z e d d o m a i n with l u m e n a l p r o t o n s . A n intriguing p r o p e r t y o f a m i n e - t y p e u n c o u p l e r s a n d also o f p r o c a i n e is t h e m a i n t e n a n c e o f a r a t h e r c o n s t a n t A T P / 2 e r a t i o over a w i d e r a n g e of A p H d e c r e a s e (Ref. 11, Fig. 1). G i e r s c h [12] was a b l e to show t h a t t h e initial velocity o f p r o t o n efflux f r o m t h y l a k o i d vesicles a f t e r a l i g h t - d a r k t r a n s i t i o n was inc r e a s e d as c o m p a r e d to controls. This o b s e r v a t i o n was also m a d e with p r o c a i n e ( n o t shown). It m a y b e conc l u d e d from a c c e l e r a t e d p r o t o n efflux t h a t t h e a m i n e s u s e d i n c r e a s e d t h e a p p a r e n t p e r m e a b i l i t y of t h y l a k o i d m e m b r a n e s for .protons. A l t h o u g h m o r e p r o t o n s left t h e t h y l a k o i d l u m e n t h e r m o d y n a m i c a l l y inefficient, t h e ratio o f A T P / 2 e was c o n s t a n t . This p h e n o m e n o n m a y b e e x p l a i n e d by two d i f f e r e n t m e c h a n i s m s . First, in t h e p r e s e n c e o f amines, less t h a n 3 p r o t o n s m a y b e r e q u i r e d for t h e synthesis of o n e A T P . This a l t e r n a t i v e w o u l d r e q u i r e t h a t p r o t o n s passing t h r o u g h t h e A T P synthase p r o v i d e m o r e free energy for A T P synthesis t h a n in t h e a b s e n c e of amines. Such m e c h a n i s m w o u l d also r e q u i r e a flexibility in t h e H ÷ / A T P r a t i o at t h e level o f t h e A T P synthase. W e c o n s i d e r e d this m e c h a n i s m as r a t h e r unlikely. S e c o n d , m o r e t h a n two p r o t o n s m a y b e t r a n s l o c a t e d across t h e m e m b r a n e p e r e l e c t r o n t r a n s f e r r e d , in the p r e s e n c e o f amine. Since a m i n e effects w e r e r e l a t e d to e l e c t r o n p a s s a g e t h r o u g h the cyt b6/f c o m p l e x , it was t e m p t i n g to a s s u m e t h a t a Q-cycle m e c h a n i s m o f p r o t o n t r a n s l o c a t i o n [44] m a y b e in c h a r g e o f a d d i t i o n a l p r o t o n u p t a k e in t h e p r e s e n c e of amines. T h e activity of t h e Q-cycle was shown to b e d e p e n d e n t on t h e r e d o x s t a t e of cyt f , as well as o f t h e p l a s t o q u i n o n e p o o l [45]. P r o c a i n e e x e r t e d an u n u s u a l effect on p H d e p e n d e n t e l e c t r o n flow c o n t r o l at t h e level o f t h e cyt b6/f c o m p l e x ( T a b l e III); c o n t r o l was m a i n t a i n e d despite a d r a s t i c d e c r e a s e of A p H . It m a y b e this p r o p erty of p r o c a i n e w h i c h causes a c o n d i t i o n for i n c r e a s e d activity of t h e Q-cycle, since effects on flux c o n t r o l m a y well i n f l u e n c e t h e r e d o x - s t a t e s o f cyt f a n d the p l a s t o q u i n o n e pool.

Acknowledgements T h e a u t h o r s a r e i n d e b t e d to M a r g a r e t e W a r m u s for h e r skillful a s s i s t a n c e a n d e n d l e s s p a t i e n c e in t h e realization of e x p e r i m e n t s . T h e y also t h a n k T i m S k e l l e t t for his advice c o n c e r n i n g m a t t e r s o f English l a n g u a g e .

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