In vitro reassociation of phycobiliproteins and membranes to form functional membrane-bound phycobilisomes

A membrane-bound phycobilisome complex has been isolated from the cyanobacterium Fremyella diplosiphon grown in green light, thus containing phycoerythrin in addition to phycocyanin and allophycocyanin. The complex was dissociated by lowering the salt concentration. In the mixture obtained, no energy transfer from phycoerythrin to chlorophyll (Chl) a was observed. Reassociation of the phycobiliproteins and membrane mixture was carried out by a gradual increase of the salt concentration. The complex obtained after reassociation was characterized by polypeptide composition, absorbance and fluorescence emission spectra and electron microscopy. These analyses revealed similar composition and structure for the original and reconstituted membrane-bound phycobilisomes. Fluorescence emission spectra and measurements of Photosystem II activity demonstrated energy transfer from phycoerythrin to Chl a (Photosystem II) in the reconstituted complex. Reassociation of mixtures with varying phycoerythrin / Chl ratio showed that the phycobiliprotein concentration was critical in the reassociation process. Measurements of the amount of phycobilisomes reassociated with the photosynthetic membrane did not show saturation of binding when increasing the phycobiliprotein concentration. The ratio phycoerythrin / Chl a in the native complex was 7:1 (mg / mg). When the phycobiliprotein concentration was increased during the reassociation process, a ratio of 13–15 mg phycoerythrin / mg Chl a could be obtained. Under these conditions, only part of the phycobilisomes attached to the thylakoids was able to transfer energy to Photosystem II.     

Thylakoid polypeptide composition and light-independent phosphorylation of the chlorophyll a,b-protein in Prochloron, a prokaryote exhibiting oxygenic photosynthesis

Thylakoids of the prokaryote Prochloron, present as a symbiont in ascidians isolated from the Red Sea at Eilat (Israel), showed polypeptide electrophoretic patterns comparable to those of thylakoids from eukaryotic oxygen-evolving organisms. Low temperature, fluorescence spectroscopy of Prochloron, having a chlorophyll a/b ratio of 3.8–5, and frozen in situ, demonstrated the presence of Photosystem II chlorophyll-protein complex emitting at 686 and 696 nm, as well as the emission band of Photosystem I at 720 nm which was so far not observed in Prochloron species. The latter emission was absent, if the cells or thylakoids were isolated prior to freezing. Energy transfer from chlorophyll b to chlorophyll a could be demonstrated to occur in vivo. The chlorophyll a,b-protein complex of Photosystem II, isolated by non-denaturing polyacrylamide gel electrophoresis, contained one major polypeptide of 34 kDa. The polypeptide was phosphorylated in vitro by a membrane-bound protein kinase which was not stimulated by light. A light-independent protein kinase activity was also found in isolated thylakoids of another prokaryote, the cyanophyte Fremyella diplosiphon. State I–State II transition could not be demonstrated in Prochloron by measurements of modulated fluorescence intensity in situ. We suggest that the presence of a light-independent thylakoid protein kinase of Prochloron, collected in the Red Sea at not less than 30 m depth, might be the result of an evolutionary process whereby this organism has adapted to an environment in which light, absorbed preferentially by Photosystem II, prevails.     

Changes in the photosynthetic apparatus in the cyanobacterium Synechocystis sp. PCC 6714 following light-to-dark and dark-to-light transitions

The photosynthetic apparatus of Synechocystis sp. PCC 6714 cells grown chemoheterotrophically (dark with glucose as a carbon source) and photoautotrophically (light in a mineral medium) were compared. Dark-grown cells show a decrease in phycocyanin content and an even greater decrease in chlorophyll content with respect to light-grown cells. Analysis of fluorescence emission spectra at 77 K and at 20 °C, of dark- and light-grown cells, and of phycobilisomes isolated from both types of cells, indicated that in darkness the phycobiliproteins were assembled in functional phycobilisomes (PBS). The dark synthesized PBS, however, were unable to transfer their excitation energy to PS II chlorophyll. Upon illumination of dark-grown cells, recovery of photosynthetic activity, pigment content and energy transfer between PBS and PS II was achieved in 24–48 h according to various steps. For O₂ evolution the initial step was independent of protein synthesis, but the later steps needed de novo synthesis. Concerning recovery of PBS to PS II energy transfer, light seems to be necessary, but neither PS II functioning nor de novo protein synthesis were required. Similarly, light, rather than functional PS II, was important for the recovery of an efficient energy transfer in nitrate-starved cells upon readdition of nitrate. In addition, it has been shown that normal phycobilisomes could accumulate in a Synechocystis sp. PCC 6803 mutant deficient in Photosystem II activity.Abbreviations APC allophycocyanin - CAP chloroamphenicol - Chl chlorophyll - DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - CP-47 chlorophyll-binding Photosystem II protein of 47 kDa - EF exoplasmic face - PBS phycobilisome - PC phycocyanin - PS Photosystem     

Functional assembly in vitro of phycobilisomes with isolated photosystem II particles of eukaryotic chloroplasts

Phycobiliproteins obtained by dissociation of phycobilisomes were reassociated in vitro with intact thylakoids or isolated photosystems I and II preparations obtained from cyanophytes (prokaryotes) or green algae (eukaryotes) to form bound phycobilisome complexes. Energy transfer from Fremyella diplosiphon phycobiliproteins to chlorophyll a of reaction centers I and II was measured in: complexes containing intact thylakoids of the cyanophytes F. diplosiphon or Anacystis nidulans and the eukaryotic algae Euglena gracilis and mutants of Chlamydomonas reinhardtii; complexes containing isolated photosystem II particles of A. nidulans or C. reinhardtii; and complexes containing reaction center I of F. diplosiphon or C. reinhardtii. Energy transfer from phycoerythrin to chlorophyll a of photosystem II could be demonstrated in complexes containing phycobilisomes bound to cyanophyte thylakoids or isolated photosystem II particles of A. nidulans or C. reinhardtii. Bound phycobilisomes did not transfer energy to photosystem II within green algae thylakoids containing altered forms of light-harvesting chlorophyll a/b-protein complex (LHC) II antenna, reduced amounts of LHC II, or chlorophyll b, or chlorophyll b-less mutants, nor to chlorophyll a of photosystem I of intact thylakoids or isolated reaction centers. We conclude that phycobilisomes can form a specific and functional association with photosystem II particles of both cyanophytes and eukaryotic thylakoids. This interaction appears to be hindered by the presence of LHC II antenna in the eukaryotic thylakoids.     

Energy transfer in phycobilisomes from phycoerythrin to allophycocyanin

Allophycocyanin appears to be the pigment through which energy trapped by phycobiliproteins is funneled to the chloroplast lamellae. Isolated, intact phycobilisomes from Porphyridium cruentum have a maximum fluorescence emission peak at 675–680 nm when excited at 545 nm. Upon dissociation, when the energy transfer is interrupted the 675–680-nm peak declines. Excitation at 435 nm produced no significant fluorescence at this wavelength.     

Photosystem II characteristics of a constructedSynechocystis 6803 mutant lacking synthesis of the D1 polypeptide

Photosystem II (PSII) composition was studied in a mutant of the cyanobacteriumSynechosystis 6803 in which synthesis of the reaction center polypeptide D1 has been inactivated. The mutant thylakoids had lost also the other reaction center polypeptide D2 and the chlorophylla-binding protein CP47. Cytochromeb559 and the chlorophylla-binding protein CP43 accumulated to almost wild-type amounts in mutant thylakoids. Also the 33 kDa polypeptide involved in water oxidation was present and membrane-bound in mutant thylakoids. The intrinsic 22 kDa polypeptide, so far known only from plants, was detected both in wild-type and mutant thylakoids.     

Selective disruption of energy flow from phycobilisomes to Photosystem I

Efficient production of ATP and NADPH by the light reactions of oxygen-evolving photosynthesis demands continuous adjustment of transfer of absorbed light energy from antenna complexes to Photosystem I (PS I) and II (PS II) reaction center complexes in response to changes in light quality. Treatment of intact cyanobacterial cells with N-ethylmaleimide appears to disrupt energy transfer from phycobilisomes to Photosystem I (PS I). Energy transfer from phycobilisomes to Photosystem II (PS II) is unperturbed. Spectroscopic analysis indicates that the individual complexes (phycobilisomes, PS II, PS I) remain functionally intact under these conditions. The results are consistent with the presence of connections between phycobiliproteins and both PS II and PS I, but they do not support the existence of direct contacts between the two photosystems.Abbreviations Chl chlorophyll - EPR electron paramagnetic resonance - NEM N-ethylmaleimide - PBS phycobilisome - PS photosystem     

Herbicide binding at photosystem II. A new azido-triazinone photoaffinity label

Azido-triazinone (3-dimethylamino-4-methyl-6-(3′-azidophenyl)-1,2,4-triazin-5-one) was found to be an efficient inhibitor of Photosystem II electron transport. This compound has an I₅₀ value of 69 nM (extrapolated to zero chlorophyll concentration), a high-affinity binding constant of 12.6 nM, and a number of binding sites of 1.9 nmol/mg chlorophyll. This corresponds to 550–580 molecules of chlorophyll per bound inhibitor; i.e., one molecule inhibitor per electron transport chain. In isolated spinach thylakoids, [¹⁴C]azido-triazinone upon ultraviolet illumination covalently binds almost exclusively to a 34 kDa protein. Covalent binding is prevented in the presence of other Photosystem II inhibitors. The protein labeled by azido-triazinone is identical to the 34 kDa herbicide-binding protein which is tagged by another photoaffinity label azido-atrazine (2-azido-4-(ethylamino)-6-(isopropylamino)-s-triazine).     

Different degrees of phosphorylation and lateral mobility of two polypeptides belonging to the light-harvesting complex of Photosystem II

Several studies have shown that a subpopulation of the light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHC-II) migrates from the appressed to the stroma-exposed thylakoids upon its phosphorylation. In this study we have analyzed the 27 and 25 kDa apopolypeptides of LHC-II, resolved by two-dimensional electrophoresis, with respect to their relative abundance and phosphorylation in thylakoids and subfractions derived from appressed or stroma-exposed thylakoid regions. The results show that the two polypeptides are heterogeneous with respect to both phosphate incorporation and degree of lateral migration. In intact thylakoids, the specific phosphorylation of the 25 kDa polypeptide exceeded that of the 27 kDa polypeptide by a factor of 3. Following phosphorylation, the 25 kDa polypeptide of the stroma lamellae showed as much as 4–5-times higher specific phosphorylation compared to the 27 kDa polypeptide. Moreover, there was a time-dependent increase in the amount of the 25 kDa polypeptide relative to the 27 kDa polypeptide in the stroma-exposed thylakoids. These results demonstrate a different polypeptide composition of the LHC-II tightly bound to Photosystem II and the free pool of LHC-II able to migrate laterally upon phosphorylation. The mobile pool of LHC-II is estimated to have two 27 kDa polypeptides for every 25 kDa polypeptide, while the ratio in the immobile pool is 4:1.     

Partial disintegration and reconstitution of the photosynthetic oxygen evolution system. Binding of 24 kilodalton and 18 kilodalton polypeptides

Treatment with 1 M NaCl almost totally removed two polypeptides of 24 and 18 kDa from the Photosystem II particles of spinach chloroplasts and reduced the oxygen-evolution activity by about half. Both polypeptides were able to rebind to the NaCl-treated particles in a low-salt medium. The rebinding of the 24 kDa polypeptide showed a saturation curve whose maximum level was close to that naturally occurring in the untreated particles. In parallel with the amount of rebound 24 kDa polypeptide, the oxygen-evolution activity was recovered. The 18 kDa polypeptide bound to the NaCl-treated particles without saturation. When the 18 kDa polypeptide was added to the particles previously treated with NaCl and then supplemented with a saturating amount of 24 kDa polypeptide, there appeared, in addition to the binding without saturation, another binding of the 18 kDa polypeptide with saturation to a maximum level close to that naturally occurring in the untreated particles. The 18 kDa polypeptide did not restore the oxygen-evolution activity. These findings suggest that there are specific binding sites; one for the 24 kDa polypeptide located on the Photosystem II particles, and the other for the 18 kDa polypeptide on the 24 kDa polypeptide.     

Studies on the polypeptide composition of the cyanobacterial oxygen-evolving complex

Various approaches have been used to investigate the polypeptides required for oxygen evolution in cyanobacteria, in particular the thermophile Phormidium laminosum. Antibodies against the extrinsic 33 kDa protein from spinach Photosystem II cross-reacted clearly in immunoblotting experiments with a corresponding polypeptide in isolated thylakoids and Photosystem II particles from P. laminosum and with whole-cell homogenates of three species of cyanobacteria (Phormidium laminosum, Synechococcus leopoliensis and Anabaena variabilis). In contrast, no cyanobacterial proteins reacted with antibodies against the 23 and 16 kDa proteins of spinach Photosystem II. The lack of cross-reactivity and the absence of these polypeptides from highly active Photosystem II particles of Phormidium laminosum strongly suggest that cyanobacteria do not contain polypeptides corresponding to these two chloroplast proteins. Treatment of P. laminosum Photosystem II particles with 0.8 M alkaline Tris, 1 M NaCl, CaCl₂ or MgCl₂ inhibited O₂ evolution, and quantitatively removed a 9 kDa polypeptide from the particles. None of these treatments removed comparable amounts of the 33 kDa polypeptide, and only Tris treatment removed manganese. The release of the 9 kDa polypeptide upon NaCl treatment correlated well with the deactivation at the donor side of Photosystem II. A direct connection between the 33 kDa polypeptide and O₂ evolution was established by the finding that trypsin treatment digested this polypeptide and inhibited O₂ evolution in parallel.     

Energy transfer in the photochemical apparatus of flashed bean leaves

Fluorescence and energy transfer properties of bean leaves greened by brief, repetitive xenon flashes were studied at −196 °C. The bleaching of P-700 has no influence on the yield of fluorescence at any wavelength of emission. The light-induced fluorescence yield changes which are observed in both the 690 and 730 nm emission bands in the low temperature fluorescence spectra are due to changes in the state of the Photosystem II reaction centers. The fluorescence yield changes in the 730 nm band are attributed to energy transfer from Photosystem II to Photosystem I. Such energy transfer was also confirmed by measurements of the rate of photooxidation of P-700 at −196 °C in leaves in which the Photosystem II reaction centers were either all open or all closed. It is concluded that energy transfer from Photosystem II to Photosystem I occurs in the flashed bean leaves which lack the light-harvesting chlorophyll a/b protein.     

N-terminus conservation in the terminal pigment of phycobilisomes from a prokaryotic and eukaryotic alga

High molecular weight polypeptides from phycobilisomes, believed to be involved in facilitating the energy flow from phycobilisomes to thylakoids, are conserved in the prokaryote Nostoc sp. and the eukaryote Porphyridium cruentum. Partial N-terminal sequence analysis of the phycobilisome-polypeptides of Nostoc (94 kilodalton) and Porphyridium (92 kilodalton) revealed 55% identity in the first 20 residues, but no significant homology with sequences of other phycobiliproteins or phycobilisome-linkers. Polypeptides (94 and 92 kilodalton) from Nostoc thylakoids free of phycobilisomes, previously presumed to be involved in the phycobilisome-thylakoid linkage (M Mimuro, CA Lipschultz, E Gantt 1986 Biochim Biophys Acta 852: 126) exhibit the same immunocrossreactivity but are different from the 94 kilodalton-phycobilisome polypeptide by having blocked N-termini and a different amino acid composition.     

Photosynthetic vesicles with bound phycobilisomes from Anabaena variabilis.

Photosynthetically active vesicles with attached phycobilisomes from Anabaena variabilis, were isolated and shown to transfer excitation energy from phycobiliproteins to F696 chlorophyll (Photosystem II). The best results were obtained when cells were disrupted in a sucrose/phosphate/citrate mixture (0.3 : 0.5 : 0.3 M, respectively) containing 1.5% serum albumin. The vesicles showed a phycocyanin/chlorophyll ratio essentially identical to that of whole cells, and oxygen evolution rates of 250 mumol O2/h per mg chlorophyll (with 4 mM ferricyanide added as oxidant), whereas whole cells had rates of up to 450. Excitation of the vesicles by 600 nm light produced fluorescence peaks (-196 degrees C) at 644, 662, 685, 695, and 730 nm. On aging of the vesicles, or upon dilution, the fluorescence yield of the 695 nm emission peak gradually decreased with an accompanying increase and final predominant peak at 685 nm. This shift was accompanied by a decrease in the quantum efficiency of Photosystem II activity from an initial 0.05 to as low as 0.01 mol O2/einstein (605 nm), with a lesser change in the Vmax values. The decrease in the quantum efficiency is mainly attributed to excitation uncoupling between phycobilisomes and Photosystem II. It is concluded that the F685 nm emission peak, often exclusively attributed to Photosystem II chlorophyll, arises from more than one component with phycobilisome emission being a major contributor. Vesicles from which phycobilisomes had been removed, as verified by electron microscopy and spectroscopy, had an almost negligible emission at 685 nm.     

Photosynthetic vesicles with bound phycobilisomes from Anabaena variabilis

Photosynthetically active vesicles with attached phycobilisomes from Anabaena variabilis, were isolated and shown to transfer excitation energy from phycobiliproteins to F696 chlorophyll (Photosystem II). The best results were obtained when cells were disrupted in a sucrose/phosphate/citrate mixture (0.3 : 0.5 : 0.3 M, respectiely) containing 1.5% serum albumin. The vesicles showed a phycocyanin/chlorophyll ratio essentially identical to that of whole cells, and oxygen evolution rates of 250 μmol O₂/h per mg chlorophyll (with 4 mM ferricyanide added as oxidant), whereas whole cells had rates of up to 450. Excitation of the vesicles by 600 nm light produced fluorescence peaks (?196°C) at 644, 662, 685, 695, and 730 nm. On aging of the vesicles, or upon dilution, the fluorescence yield of the 695 nm emission peak gradually decreased with an accompanying increase and final predominant peak at 685 nm. This shift was accompanied by a decrease in the quantum efficiency of Photosystem II activity from an initial 0.05 to as low as 0.01 mol O₂/einstein (605 nm), with a lesser change in the V_max values. The decrease in the quantum efficiency is mainly attributed to excitation uncoupling between phycobilisomes and Photosystem II. It is concluded that the F685 nm emission peak, often exclusively attributed to Photosystem II chlorophyll, arises from more than one component with phycobilisome emission being a major contributor. Vesicles from which phycobilisomes had been removed, as verified by electron microscopy and spectroscopy, had an almost negligible emission at 685 nm.     

Light Energy Transfer Between Phycobiliproteins and Thylakoids of Higher Plants

The excited energy transfer from phycobiliproteins to thylakoids of higher plants was investigated. When incubated with spinach thylakoids, phycobiliproteins isolated from red and blue-green algae have transferred the light energy which they absorbed to spinach PS Ⅱ. The efficiency of energy transfer dependent upon the kind of phycobiliproteins used. If spinach thylakoids were replaced by thylakoids of Brassica chinensis; R-phycoerythrin or C-phycocyanin could transfer the excited energy to PS Ⅱ of B. chinensis only in the presence of allophycocyanin.     

Excitation spectra for Photosystem I and Photosystem II in chloroplasts and the spectral characteristics of the distribution of quanta between the two photosystems

The parameters listed in the title were determined within the context of a model for the photochemical apparatus of photosynthesis.

The fluorescence of variable yield at 750 nm at −196 °C is due to energy transfer from Photosystem II to Photosystem I. Fluorescence excitation spectra were measured at −196 °C at the minimum, F_O, level and the maximum, F_M, level of the emission at 750 nm. The difference spectrum, F_M–F_O, which represents the excitation spectrum for F_V is presented as a pure Photosystem II excitation spectrum. This spectrum shows a maximum at 677 nm, attributable to the antenna chlorophyll a of Photosystem II units, with a shoulder at 670 nm and a smaller maximum at 650 nm, presumably due to chlorophyll a and chlorophyll b of the light-harvesting chlorophyll complex.

Fluorescence at the F_O level at 750 nm can be considered in two parts; one part due to the fraction of absorbed quanta, , which excites Photosystem I more-or-less directly and another part due to energy transfer from Photosystem II to Photosystem I. The latter contribution can be estimated from the ratio of F_O/F_V measured at 692 nm and the extent of F_V at 750 nm. According to this procedure the excitation spectrum of Photosystem I at −196 °C was determined by subtracting 1/3 of the excitation spectrum of F_V at 750 nm from the excitation spectrum of F_O at 750 nm. The spectrum shows a relatively sharp maximum at 681 nm due to the antenna chlorophyll a of Photosystem I units with probably some energy transfer from the light-harvesting chlorophyll complex.

The wavelength dependence of was determined from fluorescence measurements at 692 and 750 nm at −196 °C. is constant to within a few percent from 400 to 680 nm, the maximum deviation being at 515 nm where shows a broad maximum increasing from 0.30 to 0.34. At wavelengths between 680 and 700 nm, increases to unity as Photosystem I becomes the dominant absorber in the photochemical apparatus.     

Excitation energy transfer in aggregates of Photosystem I and Photosystem II of the cyanobacterium Synechocystis sp. PCC 6803: Can assembly of the pigment-protein complexes control the extent of spillover?

The fluorescence profile of Photosystem I/Photosystem II mixtures in different solvent systems shows that both non-hydrophobic and hydrophobic interactions govern their association and control energy transfer from Photosystem II to Photosystem I. The non-hydrophobic interactions lead to a highly efficient excitation energy transfer from Photosystem II to Photosystem I. In view of this, we propose that similar non-hydrophobic interactions, between the Photosystem II and Photosystem I peripheral proteins, also play a significant role in their association in thylakoids that control state transitions in cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Partial reconstitution of the photosynthetic oxygen evolution system by rebinding of the 33-kDa polypeptide

Treatment with 2.6 M urea of the Photosystem II particles depleted of two polypeptides of 24 kDa and 18 kDa completely released a polypeptide of 33 kDa and eliminated the oxygen-evolution activity. The 33-kDa polypeptide rebound to the urea-treated particles and partially reactivated the oxygen evolution. A quantitative analysis of the rebinding suggests tha there is a specific binding site for the 33-kDa polypeptide on the membrane surface.     

A reassessment of the use of herbicide binding to measure photosystem II reaction centres in plant thylakoids

The binding of the herbicide atrazine to thylakoid membranes is often used to quantify Photosystem II reaction centres. Two atrazine binding sites, with high and low affinities, have been observed on the D1 and D2 polypeptides of Photosystem II, respectively (McCarthy S., Jursinic P. and Stemler A. (1988) Plant Physiol. 86S:46). We have observed that the accessibility of the low-affinity binding sites is variable, being limited in freshly isolated thylakoids or in fresh frozen-thawed thylakoids, but increasing during storage of the membranes on ice. In contrast, the accessibility of the high-affinity binding sites, which are titratable at low concentrations (< 500 nM) of herbicide, is much less variable, although the dissociation constant is greatly influenced by ethanol. We conclude that to quantify Photosystem II reaction centres by atrazine binding, it is sufficient and more reliable to assay only the high-affinity binding sites.