Properties of ATP-driven reverse electron flow in chloroplasts

1. The reverse reactions induced by coupled ATP hydrolysis were studied in spinach chloroplasts by measurements of the ATP-induced increase in chlorophyll fluorescence reflecting reverse electron flow, and of the ATP-induced decrease in 9-aminoacridine fluorescence, representing formation of the transthylakoidal proton gradient (ΔpH). ATP-driven reverse electron flow was kinetically analysed into three phases, of which only the second and third one were paralleled by corresponding phases in ΔpH formation. The rapid first phase and formation of a ΔpH occur also in the absence of the electron transfer mediator phenazine methosulfate.2. The rate and extent of the reverse reactions were measured at temperatures in the range from 0 to 30°C. The rate of formation of ΔpH and of reverse electron flow were faster at high temperatures, but the maximal extent of ΔpH and chlorophyll fluorescence increase were observed at the lowest temperature. Considering rate and extent of the ATP-stimulated reactions, a temperature optimum around 15°C was found. Light activation of the ATPase occurred throughout the range studied. At 0°C and in the presence of inorganic phosphate the activated state for ATPase was maintained for more then 10 min.3. The ATP-induced rise in chlorophyll fluorescence yield was found to be of similar magnitude as the rise induced by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), when both were measured with an extremely weak measuring beam. It is concluded, that both effects, although derived via distinctly different pathways, are limited by the same electron donating or electron accepting pool.     

Comparison of ATP-induced and DCMU-induced increases of chlorophyll fluorescence

A comparative study of the ATP-induced and the DCMU-induced increases of dark chlorophyll fluorescence after activation of the latent ATPase gave the following results: (1) The ATP-induced fluorescence rise exceeds the DCMU-induced rise by an amount equivalent to the rapid component of the biphasic ATP-induced change. There is complementarity between the slow component and any preceding DCMU-induced fluorescence rise. (2) Up to 10^?4 M DCMU (3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea)), with the slow component being completely suppressed, the rapid ATP-induced phase is unaffected. It becomes eliminated, though, with an I₅₀ of about 3 · 10^?4 M. (3) No binary oscillations in dependence of the number of preilluminating flashes are observed for the rapid ATP-induced fluorescence increase. Under identical conditions such oscillations are found upon DCMU-addition. (4) The amplitude of the rapid ATP-induced fluorescence rise is unaffected by closure of Photosystem II reaction centers in presence of DCMU and NH₂OH by a single saturating flash (removal of about 50% of total quenching). With further flashes and gradual complete removal of quenching, the rapid ATP-induced change is eliminated with a two-step dependency. It is concluded that the rapid phase of the ATP-induced increase in fluorescence reflects reverse electron flow at non-B-type reaction centers, while the slow phase is linked to reverse electron flow at B type centers. On the basis of these results a model is proposed for heterogeneous interactions between the ATPase and B-type and non-B-type electron-transport chains. ‘Direct coupling’ appears to be possible between CF₀-CF₁ and those electron-transport chains which are located in the stroma-exposed margin region of the grana stacks (PS IIβ units with non-B-type properties).     

Properties of ATP-driven reverse electron flow in chloroplasts.

1. The reverse reactions induced by coupled ATP hydrolysis were studied in spinach chloroplasts by measurements of the ATP-induced increase in chlorophyll fluorescence reflecting reverse electron flow, and of the ATP-induced decrease in 9-aminoacridine fluorescence, representing formation of the transthylakoidal proton gradient (deltapH). ATP-induced reverse electron flow was kinetically analysed into three phases, of which only the second and third one were paralleled by corresponding phases in deltapH formation. The rapid first phase and formation of a deltapH occur also in the absence of the electron transfer mediator phenazine methosulfate. 2. The rate and extent of the reverse reactions were measured at temperatures in the range from 0 to 30 degrees C. The rate of formation of delta pH and of reverse electron flow were faster at high temperatures, but the maximal extent of delta pH and chlorophyll fluorescence increase were observed at the lowest temperature. Considering rate and extent of the ATP-stimulated reactions, a temperature optimum around 15 degrees C was found. Light activation of the ATPase occurred throughout the range studied. At 0 degrees C and in the presence of inorganic phosphate the activated state for ATPase was maintained for more than 10 min. 3. The ATP-induced rise in chlorophyll fluorescence yield was found to be of similar magnitude as the rise induced by 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea (DCMU), when both were measured with an extremely weak measuring beam. It is concluded, that both effects, although derived via distinctly different pathways, are limited by the same electron donating or electron accepting pool.     

ATP-induced increase in chlorophyll fluorescence. Characterization of rapid and slow induction phases

The biphasic rise of chlorophyll fluorescence induced in the dark (following activation of the latent ATP-ase) upon ATP-hydrolysis was investigated in detail, yielding the following main results: (1) The rapid phase is independent of artificial reductants or redox mediators. On the contrary, the slow phase requires such additions. (2) The slow phase is selectively eliminated by substances which collapse the transmembrane proton gradient, while the rapid phase may even be stimulated. (3) The ratio of rapid-to-slow phase is favored by a high degree of chloroplast integrity. The same factors which favor the rapid phase appear to be essential for a pronounced ‘slow electrogenic reaction’ in the flash-induced P 515 absorbance change. (4) For the rapid phase of the ATP-induced fluorescence increase, neither a ΔpH nor a Δψ are obligatory intermediates. (5) Hydroxylamine at about 5 · 10^?3 M causes a preferential stimulation of the rapid phase by about a factor 2. (6) There is selective inhibition of the slow phase by DBMIB, dinitrophenylether of iodonitrothymol, Bathocuproine and HQNO (2-heptyl-4-hydroxy quinoline-N-oxide) which are known to block at the level of the Cyt $\text{b}\text{f}$ FeS-complex. (7) The rapid phase is not affected by presence of 5 mM ferricyanide; however, there is substantial suppression if in addition a lipophilic redox mediator, like diamino-durene, is present. It is concluded that the two components of the reverse coupling reactions, reflected by the biphasic ATP-induced fluorescence rise, involve different coupling intermediates and different types of reverse electron flow. The rapid component appears to reflect close interaction between the coupling factor and a redox component in the vicinity of Photosystem II.     

Adenosine triphosphate-generated transmembrane electric potential in chloroplasts

Utilizing oxonol VI as a transmembrane electric potential indicating dye, chloroplasts are shown to develop rapid transient light-induced and ATP-induced potentials. Following the large transient signal smaller steady-state potentials are maintained with either driving system. The ATP-induced potential in the dark depends upon preactivation of the light-triggered ATPase of the chloroplasts, and is inhibited by uncouplers, ionophores such as valinomycin, and energy-transfer inhibitors such as tentoxin, Dio-9 or DCCD. Nigericin increased the signal of both the light- and the ATP-induced reactions. The fact that relatively large transient membrane potentials are induced by either a dark-to-light transition or ATP in the dark provides an explanation for previously observed phenomena such as early kinetics of photophosphorylation and the ATP-induced luminescence.     

The Respiratory Chain of Plant Mitochondria: XII. Some Aspects of the Energy-linked Reverse Electron Transport from the Cytochromes c to the Cytochromes b in Mung Bean Mitochondria

The cytochromes c of mung bean (Phaseolus aureus) mitochondria become reduced when sulfide, a cytochrome oxidase inhibitor free from uncoupling side effects, is added to the aerobic mitochondrial suspension in the absence of added substrate. The cytochromes b remain largely oxidized. Subsequent addition of ATP results in partial oxidation of the cytochromes c and partial reduction of the cytochromes b due to ATP-driven reverse electron transport through the second site of energy conservation, or coupling site, of the respiratory chain. Cytochrome a is also oxidized under these conditions, but there is no concomitant reduction of the flavoprotein components, of ubiquinone, or of endogenous pyridine nucleotide. The reaction is abolished by oligomycin. The reducing equivalents transported from the cytochromes c and a in ATP-driven reverse electron transport are about 2-fold greater than those which appear in the cytochromes b. It is suggested that the equivalents not accounted for are present in a coupling site enzyme at the second site of energy conservation which interacts with the respiratory chain carriers by means of the dithiol-disulfide couple; this couple would not show absorbance changes with redox state over the wavelength range examined. With succinate present, reverse electron transport can be demonstrated at both coupling sites in both the aerobic steady state and in anaerobiosis. ATP-driven reverse electron transport in anaerobiosis maintains cytochrome a 30% oxidized while endogenous pyridine nucleotide is 50% reduced.     

ATP-driven transhydrogenase provides an example of delocalized chemiosmotic coupling in reconstituted vesicles and in submitochondrial particles

The mechanism of coupling between mitochondrial ATPase (EC 3.6.1.3) and nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) was studied in reconstituted liposomes containing both purified enzymes and compared with their behavior in submitochondrial particles. In order to investigate the mode of coupling between the transhydrogenase and the ATPase by the double-inhibitor and inhibitor-uncoupler methods, suitable inhibitors of transhydrogenase and ATPase were selected. Phenylarsine oxide and A3'-O-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)-NAD+ were used as transhydrogenase inhibitors, whereas of the various ATPase inhibitors tested aurovertin was found to be the most convenient. The inhibition of the ATP-driven transhydrogenase activity was proportional to the inhibition of both the ATPase and the transhydrogenase. Inhibitor-uncoupler titrations showed an increased sensitivity of the coupled reaction towards carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)--an uncoupler that preferentially uncouples localized interactions, according to Herweijer et al. (Biochim. Biophys. Acta 849 (1986) 276-287)--when the primary pump was partially inhibited. However, when the secondary pump was partially inhibited the sensitivity towards FCCP remained unchanged. Similar results were obtained with submitochondrial particles. These results are in contrast to those obtained previously with the ATP-driven reverse electron flow. In addition, the amount of uncoupler required for uncoupling of the ATP-driven transhydrogenase was found to be similar to that required for the stimulation of the ATPase activity, both in reconstituted vesicles and in submitochondrial particles. Uncoupling of reversed electron flow to NAD+ required much less uncoupler. On the basis of these results, it is proposed that, in agreement with the chemiosmotic model, the interaction between ATPase and transhydrogenase in reconstituted vesicles as well as in submitochondrial particles occurs through the delta mu H+. In contrast, the energy transfer between ATPase and NADH-ubiquinone oxidoreductase appears to occur via a more direct interaction, according to the above-mentioned results by Herweijer et al.     

Relative lack of ATP-driven H+ translocase activity in isolated parotid secretory granules

The possible presence of ATP-driven H+ translocase activity in isolated rat parotid secretory granules has been examined by several approaches. First the transmembrane pH difference measured by either [14C] methylamine or [3H]acetate distribution is not substantially affected by ATP in the presence of membrane-permeating anions. Second, despite a low intrinsic H+ permeability of parotid granule membranes, only a small variably detectable inside-positive transmembrane potential is observed (by altered distribution of radioactive ions) when ATP is added in the absence of permeant anions. Third, ATP-induced lysis of parotid granules is minor and appears to be independent of ATP hydrolysis. Finally, ATP-hydrolase activity of the parotid granule fraction is not stimulated by an H+ ionophore, nor is it susceptible to inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole at a concentration which decreases the measured ATPase of purified chromaffin granule membranes by more than 80%. These findings suggest that this exocrine secretory granule type, which is characterized by storage of a heterogeneous mixture of secretory proteins, exhibits H+ pump activity which is at most a small fraction of that observed in biogenic amine storage granules of neural and endocrine tissues.     

Uncoupler-inhibitor titrations of ATP-driven reverse electron transfer in isolated rat-liver mitochondria

Uncoupler-inhibitor titrations of ATP-driven reverse electron transfer across the first site of the respiratory chain were performed in isolated rat-liver mitochondria, and the experimental results were compared with the predictions of a simple delocalized chemiosmotic mechanism. The rates of ATP hydrolysis (Jp) and reverse electron transfer (-J0) were measured at different uncoupler (S-13) concentrations, either in the absence or in the presence of rotenone. When the rates -J0 and Jp measured at different uncoupler concentrations were expressed as percentages of the activity at zero uncoupler concentration, it was found that the efficiency of S-13 to uncouple the reverse electron transfer and to stimulate ATP hydrolysis was not significantly changed upon partial inhibition with rotenone. These results are in contrast with data from a study of uncoupler-inhibitor titrations in submitochondrial particles published previously, in which a higher effectiveness of several uncouplers to inhibit ATP-driven reverse electron transfer was observed in the presence of rotenone.     

The defective proton-ATPase of uncA mutants of Escherichia coli: ATP-binding and ATP-induced conformational change in mutant alpha-subunits

Mutations in the uncA gene of Escherichia coli cause loss of both oxidative phosphorylation and ATP-driven generation of the transmembrane proton gradient. The uncA gene encodes the alpha-subunit of the F1-sector of the E. coli membrane proton-ATPase. F1-alpha-subunit from normal (unc+) E. coli binds ATP tightly (KD = 0.1 microM) and undergoes a large ATP-induced conformational change, but the functional role of the ATP-binding site is currently unknown. There is disagreement in the literature as to whether the ATP-binding site is present or lacking in F1-alpha-subunit from uncA mutant strains. One obstacle in studying this question is the difficulty of purifying mutant alpha-subunits in native form. In order to circumvent this difficulty we have studied ATP binding and ATP-induced conformational changes in mixtures of F1 subunits obtained by dissociating uncA mutant F1. Anti-alpha antibody was used in conjunction with immunoblotting to identify the alpha-subunits in the mixtures. Retention of native conformation by the alpha-subunits was demonstrated by the fact that the dissociated alpha-subunits were fully competent to repolymerize with other F1 subunits to yield intact F1 aggregate. The results show that, contrary to previous reports, alpha-subunits from three catalytically defective uncA mutants do indeed bind ATP and do undergo an ATP-induced conformational change. The binding affinity of alpha-subunit for ATP was lower than normal in each of the three mutants, but this is not likely to be a significant factor under physiological conditions.     

The effect of delta mu H+ on the interaction of rotenone with complex I of submitochondrial particles.

The inhibition by rotenone of the forward (NADH-oxidase) and reverse (delta mu H(+)-dependent succinate-NAD+ reductase activities of submitochondrial vesicles was measured. The inhibition of NADH-oxidase, measured in the presence of uncoupler, followed a monophasic inhibition curve with Ki < or = 2 nM. The reverse electron flow was only partially (40%) inhibited at these rotenone concentrations. The rest of the activity was less sensitive to the inhibitor (Ki approximately 30 nM). The lower affinity for the inhibitor of the reverse electron flow is a consequence of enhanced rate of rotenone dissociation caused by the high delta mu H+ value required for this reaction. The analysis of the results indicates that the AS-SMP preparation consists of two subpopulations: one with a relatively low degree of coupling, which exhibits high sensitivity to rotenone and the other which is highly coupled with lower affinity to the inhibitor.     

Evidence for a Block between Plastoquinone and Cytochrome f in a Photosynthetic Mutant of Lemna with Abnormal Flowering Behavior

Mutant strain 1073 of Lemna perpusilla is concluded to be blocked between plastoquinone and cytochrome f in the photosynthetic electron transport system. The location of the block is based on the following observations of activities in chloroplasts isolated from the mutant and wild-type plants. (a) Relative to wild type, electron flow rates from water to ferricyanide, 2,6-dichlorophenol indophenol or NADP were very low in the mutant, but rates of photosystem I-dependent electron flow and cyclic phosphorylation were high. (b) Chlorophyll a fluorescence induction curves for mutant and wild type were similar. (c) Silicomolybdate and lipophilic acceptors in the mutant were photoreduced at rates comparable to wild type. (d) Cytochrome f of the mutant chloroplasts was not reduced by red light, but was oxidized by red or far red light. (e) Reduction of the primary electron acceptor of photosystem II (Q) by ATP-driven reverse electron flow was not observed in the mutant.     

Reverse electron flow in chloroplasts

Energy dependent reverse electron flow reactions in isolated thylakoids provide a unique tool to study, in the dark, the coupling between the ATP synthase, proton transport and the electron transfer system. Appropriate experimental conditions have been established to follow experimentally the following reactions:

1. ATP driven proton uptake into the inner-thylakoid space, which requires preactivation of the ATP synthase.
2. ATP driven reverse electron transport, which involves proton transport as an intermediate, and results in the reduction of Q_A by an externally added electron donor.
3. ATP driven luminescence, which requires the presence of an oxidized partner on the water side of photosystem II, and involves electron transport from Q_B to Q_A.
4. ΔpH driven reverse electron flow, which does not require the participation of the ATP synthase, and uses reduced intermediates between the two photosystems as electron donors for the reduction of Q_A.
5. ΔpH driven luminescence which again uses reduced intermdiates between the two photosystems as electron donors for Q_A reduction, and requires the presence of an oxidized partner on the water side of photosystem II.

Several of these reactions have been shown to occur in intact chloroplasts and may provide an important regulatory mechanism in vivo.     

Extracellular ATP itself elicits superoxide generation in guinea pig peritoneal macrophages.

Extracellular ATP itself elicited the generation of superoxide (O2-) in guinea pig peritoneal macrophages associated with an increase in cytosolic calcium ([Ca2+]i). The ATP-induced O2- generation was completely inhibited by pretreatment with pertussis toxin (PT) accompanied by the suppression of [Ca2+]i mobilization. Pre-exposure to a small amount of phorbol myristate acetate (PMA) primed the ATP-induced generation of O2- without a change of [Ca2+]i. The results suggest that ATP-induced O2- generation is mediated by [Ca2+]i mobilization and by PT-sensitive G protein.     

Conformational change induced by ATP binding in the multidrug ATP-binding cassette transporter BmrA

ATP-binding cassette (ABC) transporters are involved in the transport of a wide variety of substrates, and ATP-driven dimerization of their nucleotide binding domains (NBDs) has been suggested to be one of the most energetic steps of their catalytic cycle. Taking advantage of the propensity of BmrA, a bacterial multidrug resistance ABC transporter, to form stable, highly ordered ring-shaped structures [Chami et al. (2002) J. Mol. Biol. 315, 1075-1085], we show here that addition of ATP in the presence of Mg2+ prevented ring formation or destroyed the previously formed rings. To pinpoint the catalytic step responsible for such an effect, two classes of hydrolysis-deficient mutants were further studied. In contrast to hydrolytically inactive glutamate mutants that behaved essentially as the wild-type, lysine Walker A mutants formed ring-shaped structures even in the presence of ATP-Mg. Although the latter mutants still bound ATP-Mg, and even slowly hydrolyzed it for the K380R mutant, they were most likely unable to undergo a proper NBD dimerization upon ATP-Mg addition. The ATP-driven dimerization step, which was still permitted in glutamate mutants and led to a stable conformation suitable to monitor the growth of 2D crystals, appeared therefore responsible for destabilization of the BmrA ring structures. Our results provide direct visual evidence that the ATP-induced NBD dimerization triggers a conformational change large enough in BmrA to destabilize the rings, which is consistent with the assumption that this step might constitute the "power stroke" for ABC transporters.     

Diurnal variations in the kinetics of delayed luminescence from Scenedesmus obtusiusculus after exposure to various light and CO2 regimes

Delayed luminescence was measured from samples of a synchronously growing cell culture of the unicellular green alga, Scenedesmus obtusiusculus Chod., every second hour during the 24 h cell cycle under a 15/9 h lighi/dark regime. Both high (air + 2.5% CO₂) and low (0.03% CO₂) CO₂ conditions were used. Under high CO₂ conditions, while light excitation induces formation of a late (maximum reached after 10–60 s) transient peak in delayed luminescence in cells sampled after 10–16 h in the cell cycle. During most of the cell cycle low CO₂ conditions stimulate a late transient peak formation. Excitation with 700 nm light, but not with 680 nm light, induces a late transient peak in delayed luminescence under high CO₂ conditions. The transient peak is more or less pronounced depending on the cell stage. The variations might be due to a changing capacity for light-induced state I/stale II transitions during the cell cycle. It is assumed that the formation of a late transient peak in delayed luminescence is due to ATP hydrolyzation and is thus favoured by a high ATP/NADPH ratio. Hydrolyzation of ATP affects the transthylakoidal ΔpH, which regulates the reverse electron flow to the plastoquinone-pool and Q_A/Q_B, thus affecting the decay kinetics of the delayed luminescence.     

Identification of a novel post-hydrolytic state in CFTR gating

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, ubiquitous proteins found in all kingdoms of life, catalyze substrates translocation across biological membranes using the free energy of ATP hydrolysis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of this superfamily in that it functions as an ATP-gated chloride channel. Despite difference in function, recent studies suggest that the CFTR chloride channel and the exporter members of the ABC protein family may share an evolutionary origin. Although ABC exporters harness the free energy of ATP hydrolysis to fuel a transport cycle, for CFTR, ATP-induced dimerization of its nucleotide-binding domains (NBDs) and subsequent hydrolysis-triggered dimer separation are proposed to be coupled, respectively, to the opening and closing of the gate in its transmembrane domains. In this study, by using nonhydrolyzable ATP analogues, such as pyrophosphate or adenylyl-imidodiphosphate as baits, we captured a short-lived state (state X), which distinguishes itself from the previously identified long-lived C2 closed state by its fast response to these nonhydrolyzable ligands. As state X is caught during the decay phase of channel closing upon washout of the ligand ATP but before the channel sojourns to the C2 closed state, it likely emerges after the bound ATP in the catalysis-competent site has been hydrolyzed and the hydrolytic products have been released. Thus, this newly identified post-hydrolytic state may share a similar conformation of NBDs as the C2 closed state (i.e., a partially separated NBD and a vacated ATP-binding pocket). The significance of this novel state in understanding the structural basis of CFTR gating is discussed.     

Simultaneous stimulation of uric acid synthesis and gluconeogenesis in chicken hepatocytes by alpha-adrenergic action of epinephrine and calcium

Formation of a transmembrane electric potential coupled to ATP hydrolysis is demonstrated in chloroplast ATPase complex containing proteoliposomes. The ATP-induced signals were detected through absorbance changes of the membrane potential-responding dye oxonol VI. They were inhibited by the specific energy-transfer inhibitor, tentoxin and the ionophore valinomycin while stimulated by nigericin. Calibration of the transmembrane potential signal was possible by the application of a proton diffusion potential. The ATP-induced transmembrane potential was estimated to be 40–50 mV.     

Limited reversibility of transmembrane proton transfer assisting transmembrane electron transfer in a dihaem-containing succinate:quinone oxidoreductase

Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential (Δp), either by supporting transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by supporting transmembrane proton transfer. The first mechanism has been unequivocally demonstrated to be operational for Δp-dependent catalysis of succinate oxidation by quinone in the case of the dihaem-containing succinate:menaquinone reductase (SQR) from the Gram-positive bacterium Bacillus licheniformis. This is physiologically relevant in that it allows the transmembrane potential Δp to drive the endergonic oxidation of succinate by menaquinone by the dihaem-containing SQR of Gram-positive bacteria. In the case of a related but different respiratory membrane protein complex, the dihaem-containing quinol:fumarate reductase (QFR) of the ?-proteobacterium Wolinella succinogenes, evidence has been obtained that both mechanisms are combined, so as to facilitate transmembrane electron transfer by proton transfer via a both novel and essential compensatory transmembrane proton transfer pathway (“E-pathway”). Although the reduction of fumarate by menaquinol is exergonic, it is obviously not exergonic enough to support the generation of a Δp. This compensatory “E-pathway” appears to be required by all dihaem-containing QFR enzymes and results in the overall reaction being electroneutral. However, here we show that the reverse reaction, the oxidation of succinate by quinone, as catalysed by W. succinogenes QFR, is not electroneutral. The implications for transmembrane proton transfer via the E-pathway are discussed.     

Anomalous uncoupling of photophosphorylation by palmitic acid and by gramicidin D

Palmitic acid and gramicidin D at low concentrations uncouple photophosphorylation in a mechanism that is inconsistent with classical uncoupling in the following properties: (1) delta pH, H+ uptake, or the transmembrane electric potential is not inhibited. (2) O2 evolution is stimulated under nonphosphorylating conditions but slightly inhibited in the presence of adenosine 5'-diphosphate + inorganic phosphate (Pi). (3) Light-triggered adenosine 5'-triphosphate (ATP)-Pi exchange is hardly affected, and ATPase activity is only slightly stimulated. (4) ATP-induced delta pH formation is selectively inhibited. This characteristic uncoupling is observed only when the native coupling sites of the electron transport system are used for energization such as for methylviologen-coupled phosphorylation. With pyocyanine, which creates an artificial coupling site, 1000-fold higher gramicidin D and higher palmitic acid concentrations are required for inhibition, and the inhibition is accompanied by a decrease in delta pH. Moreover, comparison between photosystem 1 and photosystem 2 electron transport and the effects of membrane unstacking suggest that low gramicidin D preferentially inhibits photosystem 2, while palmitic acid inhibits more effectively photosystem 1 coupling sites. The inhibitory capacity of fatty acids significantly drops when the chain length is reduced below 16 hydrocarbons or upon introduction of a single double bond in the hydrocarbon chain. It is suggested that palmitic acid and gramicidin D interfere with a direct H+ transfer between specific electron transport and the ATP synthase complexes, which provides an alternative coupling mechanism in parallel with bulk to bulk delta microH+. The sites of inhibition seem to be located in chloroplast ATP synthase, photosystem 2, and the cytochrome b6f complexes.