Coassembly of Photosystem II and ATPase as Artificial Chloroplast for Light-Driven ATP Synthesis.

Adenosine triphosphate (ATP) is one of the most important energy sources in living cells, which can drive serial key biochemical processes. However, generation of a proton gradient for ATP production in an artificial way poses a great challenge. In nature, photophosphorylation occurring in chloroplasts is an ideal prototype of ATP production. In this paper we imitate the light-to-ATP conversion process occurring in the thylakoid membrane by construction of FoF1-ATPase proteoliposome-coated PSII-based microspheres with well-defined core@shell structures using molecular assembly. Under light illumination, PSII can split water into protons, oxygen, and electrons and can generate a proton gradient for ATPase to produce ATP. Thus, an artificially designed chloroplast for PSII-driven ATP synthesis is realized. This biomimetic system will help to understand the photophosphorylation process and may facilitate the development of ATP-driven devices by remote light control.

Effect of cerium on photosynthetic pigments and photochemical reaction activity in soybean seedling under ultraviolet-B radiation stress.

Effects of cerium (Ce) on photosynthetic pigments and photochemical reaction activity in soybean (Glycine max L.) under ultraviolet-B (UV-B) radiation stress were studied under laboratory conditions. UV-B radiation caused the decrease in chlorophyll content, net photosynthetic rate, Hill reaction activity, photophosphorylation rate and Mg(2+)-ATPase activity. Ce (III) (20 mg L(-1)) could alleviate UV-B-induced inhibition to these photosynthetic parameters because values of these photosynthetic parameters in Ce (III) + UV-B treatment were obviously higher than those with UV-B treatment alone. Dynamic changes of the above photosynthetic parameters show that Ce (III) could slow down the decrease rate of these photosynthetic parameters during a 5-day UV-B radiation and quicken the restoration during recovery period. The final restoration degree of five parameters mentioned above in leaves exposed to low level of UV-B radiation (0.15 W m(2)) was higher than that exposed to high level (0.45 W m(2)). Correlating net photosynthetic rate with other four parameters, we found that the regulating mechanisms Ce (ΠΙ) on photosynthesis under various level of UV-B radiation were not the same. The protective effects of Ce (III) on photosynthesis in plants were influenced by the intensity of UV-B radiation.

Cyclic, pseudocyclic and noncyclic photophosphorylation: new links in the chain.

Photosynthetic electron transport is coupled to ATP synthesis. This process - photosynthetic phosphorylation - proceeds by several alternative electron-transport pathways in isolated chloroplasts. The question: 'Which of these works in real life?' has long occupied students of photosynthesis. Recent results from structural biology and genomics suggest that the answer is 'All of them'. The interplay between the pathways might explain the flexibility of photosynthesis in meeting different metabolic demands for ATP.

The Z-scheme--down hill all the way.

The Z-scheme is considered in the context of personal recollections of events during the time that it was conceived and an evaluation of its enduring importance.

Guard-cell chloroplasts provide ATP required for H(+) pumping in the plasma membrane and stomatal opening.

To elucidate the role of guard-cell chloroplasts (GCCs) in stomatal movement, we investigated the effects of oligomycin, an inhibitor of oxidative phosphorylation, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II, on fusicoccin (FC)-induced H(+) pumping and stomatal opening. FC was found to induce H(+ )pumping in guard-cell protoplasts (GCPs) from Vicia faba and stomatal opening in the epidermis of Commelina benghalensis; and, red light (RL) slightly stimulated these responses. Oligomycin strongly inhibited the pumping and stomatal opening in the dark. RL partially reversed the inhibitions, and DCMU decreased the effect of RL. FC activated the plasma membrane H(+)-ATPase (EC 3.6.1.35) in GCPs similarly irrespective of these treatments, indicating that the H(+)-ATPase activity was not the limiting step in H(+) pumping. Oligomycin significantly decreased the ATP content in GCPs in the dark. RL partially reversed this effect, and DCMU eliminated the effect of RL. A significant part of the ATP produced by photophosphorylation to H(+) pumping was indicated under RL. These results suggest that GCCs supply ATP to the cytosol under RL, and that the ATP is utilized by the plasma membrane H(+)-ATPase for H(+) pumping.

Adenilate pool and thylakoid ATP-synthase content in pea leaves under clinorotation.

It is known that plant resistance to stress factors is connected with energy metabolism. The energy stored in the process of photophosphorylation in the form of ATP is used then to support respiration, transpiration, organic compound synthesis, growth and development as well as to restore cell structure after its damage under extremal environmental factors. Transformation of light energy into chemical energy of ATP is catalyzed by thylakoid membrane enzymatic complex of ATPsynthase-CF1CF0. Its activity and amount in the thylakoid membrane depends on plant growth conditions. The aim of this work was investigation of clinorotation effect on light-induced dynamics of adenyl nucleotides (AMP, ADP and ATP) and estimation of CF1CF0 content in thylakoids of pea leaves grown under slow clinorotation and vertical control.

Light-induced Mg2+ ATPase activity of coupling factor in intact chloroplasts.

Intense illumination isolated, intact, spinach chloroplasts triggers the well known proton-pumping Mg2+ ATPase activity of coupling factor, which can be assayed in subsequently lysed chloroplasts by monitoring ATP-driven quenching of 9-aminoacridine fluorescence. The light-triggered ATPase activity decays slowing in the dark and is inhibited by N,N'-dicyclohexylcarbodiimide. After osmotic lysis and washing of the chloroplasts, preillumination no longer triggers maximal proton-pumping ATPase until methylviologen and dithiothreitol are added to the medium. It is suggested that intact organelles contain soluble or loosely bound cofactors necessary for light-triggering of coupling factor ATPase. On osmotic lysis, these endogenous cofactors are diluted or inactivated and must be replaced by addition of a dithiol reagent and an electron acceptor.

Effects of light intensity on membrane differentiation in Rhodopseudomonas capsulata.

Cells of Rhodopseudomonas capsulata, strain 37b4, leu-, precultivated anaerobically under low light intensity, were exposed to high light intensity (2000 W.m-2). The cells grew with a mass doubling time of 3 h. The synthesis of bacteriochlorophyll (BChl) began after two doublings of cell mass. Reaction center and light-harvesting BChl I (B-875) were the main constituents of the photosynthetic apparatus incorporated into the membrane. The size of the photosynthetic unit (total BChl/reaction center) decreased and light-harvesting BChl I became the dominating BChl species. Concomitant with the appearance of the different spectral forms of BChl the respective proteins were incorporated into the membrane, i.e. the three reaction center polypeptides, the polypeptide associated with light-harvesting BChl I, the two polypeptides associated with BChl II. A polypeptide of an apparent molecular weight of 45 000 was also incorporated. A lowering of the light intensity to 7 W.m-2 resulted in a lag phase of growth for 6 h. Afterwards, the time for doubling of cell mass was 11 h. The concentration of all three BChl complexes (reaction center, light-harvesting BChl I and II complexes)/cell and per membrane protein increased immediately. Also the size of the photosynthetic unit and the amount of intracytoplasmic membranes/cell increased. The activities of photophosphorylation, succinate dehydrogenase, NADH dehydrogenase and NADH oxidation (respiratory chain)/membrane protein are higher in membrane preparations isolated from cells grown at high light intensities than in such preparations from cells grown at low light intensities.

The initial stages of photophosphorylation. Studies using excitation by saturating, short flashes of light.

1. Photophosphorylation was studied in spinach chloroplasts on illumination, from the dark state, with saturating short ("single turnover") flashes of light. 2. At rapid flash rates (100 Hz), phosphorylation began within the first five flashes. The ATPase inhibitor protein appeared to be displaced from its inhibitory site on the ATPase also within five flashes, as deduced from the flash-induced ATPase activity. 3. At slower flash rates, or if the rate of electron transfer were reduced with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), phosphorylation began only after a larger number (50--60) of flashes. The displacement of the ATPase inhibitior protein was similarly delayed. 4. Partial displacement of the inhibitor protein from its inhibitory site on the ATPase (by pretreatment with dithioerythritol) allowed phosphorylation to proceed without a perceptible lag, even in the presence of DCMU. It was concluded that the ATPase inhibitor protein must be displaced on the ATPase before phosphorylation can begin, and that this process is energy dependent. 5. During the flash regime used, release of inhibitor from its inhibitory site seemed to be governed largely by the membrane potential. The light-induced pH gradient seemed to have little effect under these conditions. Our results are not compatible with a direct conformational interaction between the electron transfer chain and the ATPase causing displacement of the inhibitor. 6. The maximal rate of photophosphorylation induced by less than 200 flashes was 0.12--0.15 mol ATP made/mol ATPase per flash. This rate seemed to be limited not be the supply of energy to the ATPase molecules, nor by the maximal turnover capacity of the ATP synthesising system, but by the number of ATPase molecules which were active in synthesis, i.e., which lacked the inhibitor protein. 7. The bound nucleotides of the coupling ATPase exchanged with added nucleotides during single turnover flashes. At high flash rates, exchange began within 5 flashes. The average amount of nucleotide exchanged per flash over 100 flashes was about one tenth the amount of ATP synthesised in each flash. 8. It is concluded that, during phosphorylation, a steady state level of active coupling ATPases is set up. The energy-dependent displacement of the inhibitor protein, and its (energy-independent) relaxation back on to the inhibitory site are the two opposing factors involved in this steady state.

Photophosphorylation as a function of illumination time. I. Effects of permeant cations and permeant anions.

(1) Very brief periods of illumination do not initiate photophosphorylation in isolated chloroplast lamellae. The time of illumination required before any phosphorylation can be detected is inversely proportional to the light intensity. At very high intensities, phosphorylation is initiated after illumination for about 4 ms. (2) There is no similar delay in the initiation of electron transport. The rate of electron transport is very high at first but declines at about the time the capacity for ATP synthesis develops. When the chloroplasts are uncoupled with gramicidin the high initial rate persists. (3) Various ions which permeate the thylakoid membrane (K+ or Rb+ in the presence of valinomycin, SCN-, I-, or C1O4-) markedly increase the time of illumination required to initiate phosphorylation. Potassium ions in the presence of valinomycin increase the delay to a maximum of about 50 ms whereas thiocyanate ions increase the delay to a maximum of about 25 ms. The effects of K+ with valinomycin and the effect of SCN- are not additive. Permeant ions and combinations of permeant ions have little or no effect on phosphorylation during continuous illumination. (4) The reason for the threshold in the light requirement and the reason for the effect of permeant ions thereon are both obscure. However, it could be argued that the energy for phosphorylation initially resides in an electric potential gradient which is abolished by migration of ions in the field, leaving a more slowly developing proton concentration gradient as the main driving force for phosphorylation during continuous illumination. If so, the threshold in the presence of permeant ions should depend on internal hydrogen ion buffering.

Photophosphorylation as a function of illumination time. II. Effects of permeant buffers.

(1) The amounts of orthophosphate, bicarbonate and tris (hydroxymethyl)-aminomethane found inside the thylakoid are almost exactly the amounts predicted by assuming that the buffers equilibrate across the membrane. Since imidazole and pyridine delay the development of post-illumination ATP formation while increasing the maximum amount of ATP formed, it follows that such relatively permeant buffers must also enter the inner aqueous space of the thylakoid. (2) Photophosphorylation begins abruptly at full steady-state efficiency and full steady-state rate as soon as the illumination time exceeds about 5 ms when permeant ions are absent or as soon as the time exceeds about 50 ms if valinomycin and KC1 are present. In either case, permeant buffers have little or no effect on the time of illumination required to initiate phosphorylation. A concentration of bicarbonate which would delay acidification of the bulk of the inner aqueous phase for at least 350 ms has no effect at all on the time of initiation of phosphorylation. In somewhat swollen chloroplasts, the combined buffering by the tris(hydroxymethyl) aminomethane and orthophosphate inside would delay acidification of the inside by 1500 ms but, even in the presence of valinomycin and KC1, the total delay in the initiation of phosphorylation is then only 65 ms. Similar discrepancies occur with all of the other buffers mentioned. (3) Since these discrepancies between internal acidification and phosphorylation are found in the presence of saturating amounts of valinomycin and KC1, it seems that photophosphorylation can occur when there are no proton concentration gradients and no electrical potential differences across the membranes which separate the medium from the greater part of the internal aqueous phase. (4) We suggest that the protons produced by electron transport may be used directly for phosphorylation without even entering the bulk of the inner aqueous phase of the lamellar system. If so, phosphorylation could proceed long before the internal pH reflected the proton activity gradients within the membrane.

[Light-induced redox conversions of cytochrome f in pea leaves]. / Svetoindutsirovannye okislitel'no-vosstanovitel'nye prevrashcheniia tsitokhroma f v list'iakh gorokha

Redox conversions of cytochrome f were studied in intact pea leaves by double wavelength difference spectrophotometry. Using the inhibition of the photosystem II activity by far red light (719 nm) or diurone, it was found that cytochrome f is located between two photosystems on the oxidative side of photosystem I. The inhibitors of phosphorylation, , e.g. antimycin A and phloridzine, as well as the uncoupler, methylamine, strongly decreased electron transport through the carrier. It is concluded that cytochrome f is functioning in the non-cyclic phosphorylation. It is suggested that in vivo cytochrome f is not coupled with cyclic electron transfer.

Sulphydryl groups in photosynthetic energy conservation. I. Light-dependent inhibition of photophosphorylation by the sulphydryl reagent 2-2'dithio bis-(5-nitropyridine).

1. The sulphydryl reagent 2-2'dithio bis-(5-nitropyridine) (DTNP) inhibited photophosphorylation when the chloroplasts were preincubated with the reagent in the light. A maximum inhibition of about 50% was obtained in the presence of pyocyanine and MgCl 2 at 0.3 mumol DTNP per mg chlorophyll and was completed in about 40 s of preillumination. 2. Dithioerythritol, ADP plus Pi (or arsenate) and uncouplers prevented the inhibition when present during the preillumination while phloridzin, Dio-9 and discarine B were ineffective. Low concentrations of ADP or ATP afforded partial protection but other nucleotides had no effect. 3. DTNP inhibited the coupled electron transport rate to the basal level and had no effect on the uncoupled electron transport. The stimulation of proton uptake and inhibition of electron transport by ATP was prevented by DTNP. 4. The trypsin-activated but not the light- and dithioerythritol-triggered ATPase was inhibited by light preincubation of chloroplasts with DTNP. 5. Reversal of DTNP inhibition of photophosphorylation was obtained by a second preillumination in the presence of thiol groups. 6. More DTNP reacted with chloroplasts in the light than in the dark. Two mol of thione were formed in the light per mol of DTNP disappeared. 7. The results suggested that DTNP inhibition is related to the oxidation by DTNP of chloroplast vicinal dithiols probably exposed by a light-induced conformational change.