Evidence for chemiosmotic coupling of electron transport to ATP synthesis in spinach chloroplasts

In spinach chloroplasts it has been shown that (1) the size of the proton gradient under phosphorylating conditions is smaller than under non-phosphorylating conditions; (2) ADP, ATP or Dio-9, added under non-phosphorylating conditions, decrease the rate of electron transport but increase the size of the proton gradient; (3) ADP, ATP or Dio-9 inhibit not only electron transport but also the rate of decay of the proton gradient; (4) the H⁺/e⁻ ratio under non-phosphorylating conditions is 1.0. It is not affected by ADP, ATP or Dio-9.

These results show that protons pass out of the thylakoids at the site of ATP synthesis and that this leakage is inhibited by ADP, ATP or Dio-9, compounds that interact with the site of ATP synthesis. As these compounds do not alter the H⁺/e⁻ ratio the formation of the proton gradient must be an intermediate between electron transport and ATP synthesis. These data are in support of the chemiosmotic theory of coupling of electron transport to ATP synthesis.     

Effect of chronic ethanol consumption on energy-linked processes associated with oxidative phosphorylation: Proton translocation and ATP-Pi exchange

Male rats were administered an ethanol-containing diet for 31 days during which time they demonstrated fatty liver. Mitochondria and submitochondrial particles were prepared from their livers (ethanol mitochondria, ethanol submitochondrial particles) and from their pair-fed partners (control mitochondria, control submitochondrial particles). The H⁺/coupling site ratio was not significantly different in ethanol and control mitochondria with succinate as electron donor. A 13% decrease in the H⁺/coupling site ratio was observed in ethanol mitochondria, however, when β-hydroxybutyrate was used as substrate. The rate of ATP-P_i exchange was decreased significantly in both ethanol mitochondria and submitochondrial particles as compared to control preparations. These observations demonstrate ethanol-elicited decreases in energy conservation in the site I region of the electron transport chain and in the activity of the ATP synthetase complex.     

Lack of site-specificity of spinach chloroplast coupling factor 1

The irreversible inhibition of chloroplast phosphorylation by either sulfate anions, or N-ethylmaleimide, is energy dependent. Chloroplasts must first be illuminated in the presence of the inhibitors and a mediator of electron flow, for the subsequent phosphorylation to show any inhibition. Both inhibitors affect the chloroplast coupling factor 1.Electron transport only through Photosystem I can be used to activate either of these inhibitions. The subsequent inhibition in a second light reaction is the same whether ATP synthesis is supported by Photosystem I, or by Photosystem II electron transport. The reverse experiment, activating inhibition by electron transport only through Photosystem II, is possible in the case of sulfate. Again, the inhibition is expressed whether Photosystem II or Photosystem I electron flow supports ATP synthesis. We conclude that the two electron transport regions probably generate the same high energy state which is able to activate all members of a functionally uniform coupling factor population. These enzyme molecules must catalyze phosphorylation coupled to electron transport through either region of the chain. The results tend to discredit models requiring a separate group of coupling factor molecules unique to each part of the chain.     

Effect of phloridzin on photosynthetic electron transport

Phloridzin (2',4',6',4-tetraoxyhydrochalcon-2'-glucoside) was used to study the localization of synthesis of ATP in the electrontransporting chain of photosynthesis. It was shown that phloridzin inhibits the rate of photoreduction of NADP+ by isolated pea chloroplasts by 40%, electron transport via cytochrome f by 100% and via plastocyanin--by 50%. The "crossover" experiments demonstrated that phloridzin inhibits ADP-induced photoreduction of cytochrome f, having no effect on plastocyanin under identical conditions. It is assumed that the site of ATP synthesis is localized on the reduced site of cytochrome f, while the carrier itself is located in the electron transporting chain coupled to phosphorylation. It is possible that only part of the plastocyanin molecules are located in the phosphorylating pathway of electron transport.     

Oxidative phosphorylation. The relation between the specific binding of trimethlytin and triethyltin to mitochondria and their effects on various mitochondrial functions

1. A binding site (site 1) is present in mitochondria with affinity for trimethyltin and triethyltin adequate for a site to which they could be attached when the processes of energy conservation are inhibited. 2. The quantitative relationships between the binding of trimethyltin and triethyltin to site 1 and their effects on various mitochondrial functions have been examined. 3. ATP synthesis linked to the oxidation of pyruvate, succinate and intramitochondrial substrate, ATP synthesis and oxygen uptake (succinate or pyruvate as substrate) stimulated by uncoupling agents are all inhibited by trimethyltin and triethyltin; when inhibition is less than 50% the ratio (percentage inhibition)/(percentage of binding site 1 complexed) is approx. 10:1. 4. ATP synthesis linked to the oxidation of reduced cytochrome c (ascorbate+NNN'N'-tetramethyl-p-phenylenediamine), ATP hydrolysis and oxygen uptake in the presence of low concentrations of trimethyltin and triethyltin approach zero activity as the proportion of binding site 1 complexed approaches 100%. 5. Possible interpretations of these findings are discussed with reference to published arrangements for coupling of electron transport to ATP synthesis and also to our present knowledge of the chemical and biological specificity of trialkyltin compounds.     

ATP generation during reduced inorganic sulfur compound oxidation by<Emphasis Type="Italic"> Acidithiobacillus caldus</Emphasis> is exclusively due to electron transport phosphorylation

The synthesis of adenosine 5-triphosphate (ATP) (increase in phosphorylation potential) during the oxidation of reduced inorganic sulfur compounds was studied in the moderately thermophilic acidophileAcidithiobacillus caldus (strain KU) (formerly Thiohacillus caldus). The phosphorylation potential increased during the oxidation of all reduced inorganic sulfur compounds tested compared with resting cells. The generation of ATP in whole cells was inhibited by the F0F1 ATPase inhibitor oligomycin, electron transport chain inhibitors, valinomycin and potassium ions. There was no increase in the phosphorylation potential, nor synthesis of ATP. in the absence of electron transport. An apparent lack of substrate-level phosphorylation was indicated by the lack of adenosine 5-phosphosulfate reductase in tetrathionate-grown At. caldus. Studies were also performed on the synthesis of ATP by membrane vesicles of At. caldus when presented with an artificial proton gradient. Complete inhibition of ATP synthesis in these vesicles occurred when they were loaded with N,N-dicyclohexylcarbodiimide (DCCD), but not when they were loaded with oligomycin, vanadate or electron transport chain inhibitors. The data presented here suggest that during the oxidation of reduced inorganic sulfur compounds by At. caldus, all ATP is synthesized by oxidative phosphorylation via a membrane-bound F0F1 ATPase driven by a proton gradient.     

A novel aerobic respiratory chain-linked NADH oxidase system in Zymomonas mobilis.

Membrane vesicles prepared from Zymomonas mobilis oxidized NADH exclusively, whereas deamino-NADH was little oxidized. In addition, the respiratory chain-linked NADH oxidase system exhibited only a single apparent Km value of approximately 66 microM for NADH. The NADH oxidase was highly sensitive to the respiratory chain inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide. However, the NADH:quinone oxidoreductase was not sensitive to 2-heptyl-4-hydroxyquinoline-N-oxide and was highly resistant to another respiratory chain inhibitor, rotenone. Electron transfer from NADH to oxygen generated a proton electrochemical gradient (inside positive) in inside-out membrane vesicles. In contrast, electron transfer from NADH to ubiquinone-1 generated no electrochemical gradient. These findings indicate that Z. mobilis possesses only NADH:quinone oxidoreductase lacking the energy coupling site.     

Investigation of the apparent inefficiency of the coupling between Photosystem II electron transfer and ATP formation

The maximum phosphorylation efficiency achieved with synchronous turnovers of Photosystem II (PS II) in spinach chloroplast lamellae is 0.3 molecules of ATP per pair of electrons transferred. This is the same as the efficiency observed for PS II operating alone in continuous light and would seem to indicate less than 50% coupling efficiency. Flash-induced ATP synthesis associated with both photosystems acting in unison closely approaches twice the flash-induced ATP synthesis associated with the Photosystem-I-dependent oxidation of duroquinol (itself 0.6) and comes close to equalling the highest efficiency observed in steady-state PS I + PS II electron transport. The anomalously low coupling efficiency seen when PS II is operating alone can be overcome by a ΔpH of two units imposed before flash illumination, or by a prior flash series involving the entire electron transfer chain. In contrast, prior electron transport through PS II alone is only slightly effective in enhancing the coupling efficiency of subsequent PS II turnovers. (It should be noted that in all cases where supplementary energy was provided, either by a proton gradient or by prior illumination, this supplementary energy was always below the energetic threshold for phosphorylation. Furthermore, the enhancement of PS II coupling efficiency by supplementary energy persisted even after a large number of subsequent PS II-inducing flashes). The efficiency of flash-induced ATP synthesis associated with whole-chain electron transfer or with PS-I-dependent duroquinol oxidation is also enhanced by the supplementary energy, but only during the first few inefficient flashes, suggesting that in this case the supplementary energy may simply be contributing to the initial build-up of an energetic threshold for ATP synthesis. This cannot be the case when the same supplementary energy contributes to the efficiency of the PS II reaction, since the enhancement then persists for a long time and contributes to an essentially constant flash yield of ATP. Our results imply that during electron transfer involving both photosystems, PS II participates in generating about half of the total ATP, whereas it operates inefficiently only when operating alone. Since hydrogen ions produced by PS I are able to raise the efficiency of subsequent PS-II-dependent phosphorylation, at least some cooperation between the two photosystems takes place and this suggests some donation of protons from PS I to PS II. However, the inability of PS II alone to achieve high efficiency, even with prolonged pre-illumination, would seem to indicate some functional distinction of protons from the two photosystems.     

Metabolism and energy generation in homoacetogenic clostridia

Abstract Clostridium thermoautotrophicum and C. thermoaceticum contain an anaerobic electron transport chain. It involves hydrogen and carbon monoxide as electron donors and, presumably methylenetetrahydrofolate as physiological electron acceptor. Cytochrome b ₅₅₄, cytochrome b ₅₅₉, menaquinone, a flavoprotein, ferredoxin and rubredoxin are parts of the electron transport chain. The electron transport results in the generation of a proton motive force which drives the synthesis of ATP or the uptake of amino acids.     

Inhibition of mitochondrial ATP-synthetase by redox-agents]

Effects of phenol and phenothiazine on ATP synthesis and electron transport in submitochondrial particles were studied. Nitrophenols and phenothiazines inhibited ATP synthesis without notable effect on electron transport. On the contrary chlorphenols equally decreased the velocities of electron transport and ATP synthesis. The inhibitors studied showed the properties of electron acceptors in relation to the radicals, their acceptor properties corresponding to their ability to inhibit ATP synthesis.     

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.     

The reconstitution of oxidative phosphorylation in mitochondria isolated from a ubiquinone-deficient mutant ofSaccharomyces cerevisiae

Mitochondria, isolated from the ubiquinone-deficient nuclear mutant ofSaccharomyces cerevisiae E3-24, are practically unable to oxidize exogenous substrates. Respiratory activity, coupled to ATP synthesis, can, however, be reconstituted by the simple addition of ethanolic solutions of ubiquinones. A minimal length of the isoprenoid side chain (3) was required for the restoration. Saturation of the reconstitution required a large amount of exogeneous ubiquinone, in excess over the normal content present in the mitochondria of the wild type strain. A similar pattern of reconstituted activities could be also obtained using sonicated inverted particles. Mitochondria and sonicated particles are also able to carry out a dye-mediated electron flow coupled to ATP synthesis in the absence of added ubiquinone, using ascorbate or succinate as electron donor. This demonstrates that the energy conserving mechanism at the third coupling site of the respiratory chain is fully independent of the presence of the large mobile pool of ubiquinone in the membrane.     

The phosphorylation site associated with the oxidation of exogenous donors of electrons to photosystem I.

1. The Photosystem I-mediated transfer of electrons from diaminodurene, diaminotolune and reduced 2,6-dichlorophenolindophenol to methylviologen is optimal at pH 8-8.5, where phosphorylation is also maximal. In the presence of superoxide dismutase, the efficiency of phosphorylation rises from smaller than or equal to 0.1 at pH 6.5 to 0.6-0.7 at pH 8-8.5, regardless of the exogenous electron donor used. 2. The apparent Km (at pH 8.1) for diaminodurene is 6-10-minus 4 M and for diaminotoluene is 1.2- 10- minus 3 M. The concentrations of diaminodurene and diaminotoluene required to saturate the electron transport processes are greater than 2 mM and greater than 5 mM, respectively. At these higher electron donor concentrations the rates of electron transport are markedly increased by phosphorylation (1.5-fold) or by uncoupling conditions (2-fold). 3. Kinetic analysis of the transfer of electrons from reduced 2,6-dichlorophenolindophenol (DCIPH2) to methylviogen indicates that two reactions with very different apparent Km values for DCIPH2 are involved. The rates of electron flux through both pathways are increased by phosphorylation or uncoupling conditions although only one of the pathways is coupled to ATP formation. No similar complications are observed when diaminodurene or diaminotoluene serves as the electron donor. 4. In the diaminodurene yields methylviologen reaction, ATP formation and that part of the electron transport dependent upon ATP formation are partially inhibited by the energy transfer inhibitor HgC12. This partial inhibition of ATP formation rises to about 50 percent at less than 1 atom of mercury per 20 molecules of chlorophyll, then does not further increase until very much higher levels of mercury are added. 5. It is suggested that exogenous electron donors such as diaminodurene, diaminotoluene and DCIPH2 can substitute for an endogenous electron carrier in donating electrons to cytochrome f via the mercury-sensitive coupling site (Site I) located on the main electron-transporting chain. If this is so, there would seem to be no reason for postulating yet another coupling site on a side branch of the electron transport chain in order to account for cyclic photophosphorylation.     

Dependence of yeast mitochondrial unselective channel activity on the respiratory chain

The dependence of yeast mitochondrial unselective channel activity on the respiratory chain was investigated. Modulation of the respiratory chain with different substrates and inhibitors showed that channel activity was dependent on the electron flow rate through the chain and that external NADH only could provide a sufficient rate to activate the channel. These results support the hypothesis that the yeast mitochondrial unselective channel may be involved in the oxidation of cytosolic NADH without coupling to ATP synthesis.     

Mitochondrial respiratory activity of the trematode Fasciola hepatica

Polarographic studies have been made on the respiratory activity of isolated mitochondria of the trematode F. hepatica. Respiratory chain transferring electrons to oxygen and which is sensitive to cyanide was found in the mitochondria. Certain coupling between respiration and phosphorylation was observed. Intact mitochondria of the trematode exhibit the respiratory control although its level is significantly lower than that in the mitochondria from rat liver. The existence of an alternative respiratory chain was demonstrated in which electron transport is not associated with ATP synthesis.     

Photosynthesis of ATP-electrons,proton pumps,rotors, and poise

Light-driven electron transport is coupled to ATP synthesis in chloroplasts. While the nature of the coupling and the structures of key components are now known, there has long been disagreement over pathways of electron transport. Recent results now put an old idea back on the agenda-cyclic electron transport around photosystem I.     

Flexibility of coupling and stoichiometry of ATP formation in intact chloroplasts.

Since coupling between phosphorylation and electron transport cannot be measured directly in intact chloroplasts capable of high rates of photosynthesis, attempts were made to determine ATP/2 e ratios from the quamdum requirements of glycerate and phosphoglycerate reduction and from the extent of oxidation of added NADH via the malate shuttle during reduction of phosphoglycerate in light. These different approaches gave similar results. The quantum requirement of glycerate reduction, which needs 2 molecules of ATP per molecule of NADPH oxidized was found to be pH-dependent. 9-11 quanta were required at pH 7.6, and only about 6 at pH 7.0. The quantum requirement of phosphoglycerate reduction, which consumes ATP and NADPH in a 1/1 ratio, was about 4 both at pH 7.6 ant at 7.0. ATP/2 e ratios calculated from the quantum requirements and the extent of phosphoglycerate accumulation during glycerate reduction were usually between 1.2 and 1.4, occasionally higher, but they never approached 2. Although the chloroplast envelope is impermeable to pyridine nucleotides, illuminated chlrooplasts reduced added NAD via the malate shuttle in the absence of electron acceptors and also during the reduction of glycerate or CO2. When phosphoglycerate was added as the substrate, reduction of pyridine-nucleotides was replaced by oxidation and hydrogen was shuttled into the chloroplasts to be used for phosphoglycerate reduction even under light which was rate-limiting for reduction. This indicated formation of more ATP than NADPH by the electron transport chain. From the rates of oxidation of external NADH and of phosphoglycerate reduction at very low light intensities ATP/2e ratios were calculated to be between 1.1 and 1.4. Fully coupled chloroplasts reduced oxaloacetate in the light at rates reaching 80 and in some instances 130 mumoles times mg-1 chlorophyll times h-1 even though ATP is not consumed in this reaction. The energy transfer inhibitor phlorizin did not significantly suppress this reduction at concentrations which completely inhibited photosynthesis. Uncouplers stimulated oxaloacetate reduction by factors ranging from 1.5 to more than 10. Chloroplasts showing little uncoupler-induced stimulation of oxaloacetate reduction were highly active in photoreducing CO2. Measurements of light intensity dependence of quantum requirements for oxaloacetate reduction gave no indication for the existence of uncoupled or basal electron flow in intact chloroplasts. Rather reduction is brought about by loosely coupled electron transport. It is concluded that coupling of phosphorylation to electron transport in intact chloroplasts is flexible, not tight. Calculated ATP/2e ratios were obtained under con a decreENG     

Effects of Water Stress on Energy Transduction in Chloroplasts

Water stress inhibited the photosynthetic O2 evolution rate of wheat leaves. It was shown that water stress decreased the electron transport rate, the activities of photophosphorylation and, coupling factor, and, the synthesis of ATP in chloroplasts. PS Ⅱ electron transport was more senstitive to water stress than PS Ⅰ. The reduction in photophosphorylation activity might be the results of reduction in electron transport rate and coupling factor activity, as well as the uncoupling effect of water stress on chloroplasts. The uncoupling effect could be due to the inhibition of light induced proton translocation in chloroplasts.     

Inhibition of chloroplast electron transport reactions by trifluralin and diallate

The herbicides trifluralin (alpha,alpha,alpha-trifluoro-2,6-dinitro-N, N-dipropyl-p-toluidine) and diallate (S-[2,3-dichloroallyl] diisopropylthiocarbamate) inhibit electron transport, ATP synthesis, and cytochrome f reduction by isolated spinach (Spinacia oleracea L.) chloroplasts. Both compounds inhibit noncyclic electron transport from H(2)O to ferricyanide more than 90% in coupled chloroplasts at concentrations less than 50 mum. Neither herbicide inhibits electron transport in assays utilizing only photosystem I activity, and the photosystem II reaction elicited by addition of oxidized p-phenylenediamine or 2,5-dimethylquinone is only partially inhibited by herbicide concentrations which block electron flow from H(2)O to ferricyanide. Inhibition of ATP synthesis parallels inhibition of electron flow in all noncyclic assay systems, and cyclic ATP synthesis catalyzed by either diaminodurene or phenazine metho-sulfate is susceptible to inhibition by both herbicides. These results indicate that trifluralin and diallate both inhibit electron flow in isolated chloroplasts at a point in the electron transport chain between the two photosystems.     

The phosphorylation site associated with the oxidation of exogenous donors of electrons to Photosystem I

1. The Photosystem I-mediated transfer of electrons from diaminodurene, diaminotoluene and reduced 2,6-dichlorophenolindophenol to methylviologen is optimal at pH 8–8.5, where phosphorylation is also maximal. In the presence of superoxide dismutase, the efficiency of phosphorylation rises from ? 0.1 at pH 6.5 to 0.6–0.7 at pH 8–8.5, regardless of the exogenous electron donor used.2. The apparent K_m (at pH 8.1) for diaminodurene is 6·10^?4 M and for diaminotoluene is 1.2·10^?3 M. The concentrations of diaminodurene and diaminotoluene required to saturate the electron transport processes are > 2 mM and > 5 mM, respectively. At these higher electron donor concentrations the rates of electron transport are markedly increased by phosphorylation (1.5-fold) or by uncoupling conditions (2-fold).3. Kinetic analysis of the transfer of electrons from reduced 2,6-dichlorophenolindophenol (DCIPH₂) to methylviologen indicates that two reactions with very different apparent K_m values for DCIPH₂ are involved. The rates of electron flux through both pathways are increased by phosphorylation or uncoupling conditions although only one of the pathways is coupled to ATP formation. No similar complications are observed when diaminodurene or diaminotoluene serves as the electron donor.4. In the diaminodurene → methylviologen reaction, ATP formation and that part of the electron transport dependent upon ATP formation are partially inhibited by the energy transfer inhibitor HgCl₂. This partial inhibition of ATP formation rises to about 50% at less than 1 atom of mercury per 20 molecules of chlorophyll, then does not further increase until very much higher levels of mercury are added.5. It is suggested that exogenous electron donors such as diaminodurene, diaminotoluene and DCIPH₂ can substitute for an endogenous electron carrier in donating electrons to cytochrome f via the mercury-sensitive coupling site (Site I) located on the main electron-transporting chain. If this is so, there would seem to be no reason for postulating yet another coupling site on a side branch of the electron transport chain in order to account for cyclic photophosphorylation.