Reconstitution of Photosynthetic Energy Conservation. II. Photophosphorylation in Liposomes Containing Photosystem-I Reaction Center and Chloroplast Coupling-Factor Complex (original) (raw)
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Journal of Bioenergetics and Biomembranes, 2012
Photosynthetic reaction center (RC) is the minimal nanoscopic photoconverter in the photosynthetic membrane that catalyzes the conversion of solar light to energy readily usable for the metabolism of the living organisms. After electronic excitation the energy of light is converted into chemical potential by the generation of a charge separated state accompanied by intraprotein and ultimately transmembrane proton movements. We designed a system which fulfills the minimum structural and functional requirements to investigate the physico/chemical conditions of the processes: RCs were reconstituted in closed lipid vesicles made of selected lipids entrapping a pH sensitive indicator, and electron donors (cytochrome c 2 and K 4 [Fe(CN) 6 ]) and acceptors (decylubiquinone) were added to sustain the photocycle. Thanks to the low proton permeability of our preparations, we could show the formation of a transmembrane proton gradient under illumination and low buffering conditions directly by measuring proton-related signals simultaneously inside and outside the vesicles. The effect of selected ionophores such as gramicidin, nigericin and valinomycin was used to gain more information on the transmembrane proton gradient driven by the RC photochemistry.
Stimulation of ATP Synthesis by a Membrane Potential in Chloroplasts
European Journal of Biochemistry, 1973
Chloroplasts preloaded with protons by light-induced proton uptake do not synthesize ATP in a following dark period if the proton concentration gradient (ApH) is lower than 2.5-3.0 pH units. At such suboptimal ApH values synthesis of ATP can be obtained by imposing a diffusion potential across the chloroplast membrane. This potential was realized by providing a high external concentration of either KC1 in the presence of valinomycin or of NaCl in the presence of monactin. Diffusion-potential-induced stimulation of ATP synthesis was observed both in chloroplasts and in bacterial chromatophores.
Functional reconstitution of photosynthetic cyclic electron transfer in liposomes
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1989
The interaction of solubilized reaction centers from the phototrophic bacterium, Rhodobacter sphaeroides, and the solubilized ubiquinol-cytochrome c oxidoreductase of Rhodobacter capsulatus was studied in solution and after coreconstitution in liposomes prepared from Escherichia coli phospholipids. Under both conditions, the ubiquinol-cytochrome c oxidoreductase increased the light-induced cyclic electron transfer, induced by reaction centers with 2,3-dimethoxy-5-methyl-6-(prenyl)z-l,4-benzoqumone and cytochrome c as redox mediators. This effect was more pronounced at acid pH values. The light-induced cyclic electron transfer in these liposomes resulted in the generation of a protonmotive force. Under conditions where the protonmotive force was composed of a membrane potential only, the highest membrane potential (approx.-200 mV) was generated when 2,3-dimethoxy-5-methyl-6-(prenyl)lo-l,4-benzoquinone was used as redox mediator and when both electron transfer proteins were co-reconstituted in a 2:1 molar ratio. At acid pH non-transitent membrane potentials could be generated only in liposomes containing both reaction centers and the ubiquinol-cytochrome c oxidoreductase. These observations show that the pH-dependent direct oxidation of cytochrome c by ubiquinol in the liposomes was indeed catalyzed by the ubiquinol-cytochrome c oxidoreductase and that this oxidoreductase participates in proton pumping. This could also be concluded from the stimulating effect of 2,3-dimethoxy-5-methyl-6-(prenyl)t0-1,4-benzoquinone on the membrane-potential-generating capacities in liposomes containing both electron transfer complexes. Such a stimulation was not observed in liposomes containing only reaction centers. The presence of cytochrome c in the co-reconstituted system was found to be essential for proton pumping.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1972
Chlamydomonas reinhardi y-I cells are rendered permeable to substrates and cofactors for photosynthetic electron transfer and photophosphorylation by applying low shearing forces in a hypertonic viscous medium. The chloroplast integrity is well preserved in such open-cell preparations which exhibit high and stable photophosphorylation rates for both photosystems and proton pump activity. These activities were measured during greening of dark-grown y-I cells using open-cell preparations. It was found that photophosphorylation and light-dependent proton uptake are absent in preparations obtained from cells grown in the dark for 5-6 generations which still contained 1-2 /zg chlorophyll per lO T cells. The activities became measurable following exposure of the cells to the light and increased rapidly reaching a maximal rate per chlorophyll unit 3-4 times higher than that exhibited by light-grown cells after 2-3 h of illumination when the chlorophyll content of the cells has increased only slightly. As chlorophyll continues to accumulate the activity per chlorophyll unit declined and became equal to that usually found in light grown cells. Photosynthetic ATP formation and pH rise do not develop in cells greening in the presence of chloramphenicol which specifically inhibits the synthesis of chloroplast made proteins. Chloroplast membranes formed in the presence of chloramphenicol which are enriched in proteins of cytoplasmic origin resume the light-dependent proton pump activity and photophosphorylation by both photosystems if the cells are further incubated in absence of chloramphenicol. The repair of the inactive membranes requires synthesis of proteins within the chloroplast and does not require light and additional synthesis of chlorophyll or proteins of cytoplasmic origin. * Light-dependent pH rise measured in whole cells is considered to be an indication ot photosynthetic CO 2 uptake 1°, n rather than proton uptake as reported earlier ~.
Proceedings of the National Academy of Sciences of the United States of America, 1984
The ATP-synthase in chloroplasts is built from two blocks, CF0, which is integral to the thylakoid membrane and which serves as a proton channel, and CF1, attached to CF0, which is catalytically active. This study is aimed at understanding proton conduction through CF0. By a mild procedure we extracted <10% of total CF1, predominantly the four-subunit CF1 without the delta subunit. Extracted chloroplasts were excited with short flashes of light and the time course of the transmembrane potential and of the pH changes in both phases was measured spectrophotometrically. Mild extraction of CF1 caused two effects. (i) Up to 50% of the protons rapidly released from water oxidation transiently escaped detection in the thylakoid interior. (ii) The initial extent of the transmembrane potential was decreased by some 10% (20-mus resolution). Protons that were not detected inside appeared in the external phase after having passed the thylakoid membrane. pH titrations of the transient loss of...
The Journal of Cell Biology, 1985
Chlorophyll a/b light-harvesting complexes (chl a/b LHC) and photosystem II (PSII) cores were isolated from an octyl glucoside-containing sucrose gradient after solubilization of barley thylakoid membranes with Triton X-100 and octyl glucoside. No cation precipitation step was necessary to collect the chl a/b LHC. PAGE under mildly denaturing and fully denaturing conditions showed that the chl a/b LHC fraction contained chlorophyll-protein complexes CP27, CP29, and CP64. The PSII core material contained CP43 and CP47, and little contamination by other nonpigmented polypeptides. Freeze-fracture electron microscopy of the chl a/b LHC after reconstitution into digalactosyldiglyceride (DG) or phosphatidylcholine (PC) vesicles showed that the protein particles (approximately 7.5 +/- 1.6 nm) were approximately 99 and 90% randomly dispersed, respectively, in the liposomes. Addition of Mg++ produced particle aggregation and membrane adhesion in chl a/b LHC-DG liposomes in a manner analogous...