Orientation of pigments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. II. Linear dichroism spectra of isolated pigment-protein complexes oriented in polyacrylamide gels at 300 and 100 K

The absorption and linear dichroism (LD) spectra (380–780 nm) of isolated light-harvesting complex (LHC), Photosystem I (PS I), Photosystem II (PS II), as well as intact thylakoids have been determined at 300 and 100 K. The samples were oriented in squeezed polyacrylamide gel. The low-temperature spectra of LHC and PS I present LD signals which are characteristic enough to be recognized in the LD spectrum of thylakoids. Tentative assignments of the various features of the LD spectra to the major photosynthetic pigments are discussed. A shoulder in the low-temperature absorption spectra is observed at about 673 nm in all the systems under investigation. The absence of an associated LD signal suggests that this ubiquitous chlorophyll (Chl) a form is non-dichroic. Furthermore, in the three isolated chlorophyll-protein complexes described in this study the sign of the LD signal indicates that both the Q_y transition of the Chl a and the carotenoid molecules are preferentially oriented parallel to the largest dimension(s) of the particles.     

Steady-state polarized light spectroscopy of isolated Photosystem I complexes

Monomeric and trimeric Photosystem I core complexes from the cyanobacterium Synechocystis PCC 6803 and LHC-I containing Photosystem I (PS I-200) complexes from spinach have been characterized by steady-state, polarized light spectroscopy at 77 K. The absorption spectra of the monomeric and trimeric core complexes from Synechocystis were remarkably similar, except for the amplitude of a spectral component at long wavelength, which was about twice as large in the trimeric complexes. This spectral component did not contribute significantly to the CD-spectrum. The (77 K) steady-state emission spectra showed prominent peaks at 724 nm (for the Synechocystis core complexes) and at 735 nm (for PS I-200). A comparison of the excitation spectra of the main emission band and the absorption spectra suggested that a significant part of the excitations do not pass the red pigments before being trapped by P-700. Polarized fluorescence excitation spectra of the monomeric and trimeric core complexes revealed a remarkably high anisotropy (0.3) above 705 nm. This suggested one or more of the following possibilities: 1) there is one red-most pigment to which all excitations are directed, 2) there are more red-most pigments but with (almost) parallel orientations, 3) there are more red-most pigments, but they are not connected by energy transfer. The high anisotropy above 705 nm of the trimeric complexes indicated that the long-wavelength pigments on different monomers are not connected by energy transfer. In contrary to the Synechocystis core complexes, the anisotropy spectrum of the LHC I containing complexes from spinach was not constant in the region of the long-wavelength pigments, and decreased significantly below 720 nm, the wavelength where the long-wavelength pigments on the core complexes start to absorb. These results suggested that in spinach the long-wavelength pigments on core and LHC-I are connected by energy transfer and have a non-parallel average Q_y(0-0) transitions.Abbreviations PS Photosystem - P Primary donor - Chl chlorophyll - LHC light-harvesting complex - CD circular dichroism - LD linear dichroism - BisTris 2-[bis(2-hydroxyethyl)amino]-2-hydroxy-methylpropane-1,3-diol - RC reaction center     

Photoprotection in higher plants: The putative quenching site is conserved in all outer light-harvesting complexes of Photosystem II

In bright sunlight, the amount of energy harvested by plants exceeds the electron transport capacity of Photosystem II in the chloroplasts. The excess energy can lead to severe damage of the photosynthetic apparatus and to avoid this, part of the energy is thermally dissipated via a mechanism called non-photochemical quenching (NPQ). It has been found that LHCII, the major antenna complex of Photosystem II, is involved in this mechanism and it was proposed that its quenching site is formed by the cluster of strongly interacting pigments: chlorophylls 611 and 612 and lutein 620 [A.V. Ruban, R. Berera, C. Ilioaia, I.H.M. van Stokkum, J.T.M. Kennis, A.A. Pascal, H. van Amerongen, B. Robert, P. Horton and R. van Grondelle, Identification of a mechanism of photoprotective energy dissipation in higher plants, Nature 450 (2007) 575-578.]. In the present work we have investigated the interactions between the pigments in this cluster not only for LHCII, but also for the homologous minor antenna complexes CP24, CP26 and CP29. Use was made of wild-type and mutated reconstituted complexes that were analyzed with (low-temperature) absorption and circular-dichroism spectroscopy as well as by biochemical methods. The pigments show strong interactions that lead to highly specific spectroscopic properties that appear to be identical for LHCII, CP26 and CP29. The interactions are similar but not identical for CP24. It is concluded that if the 611/612/620 domain is responsible for the quenching in LHCII, then all these antenna complexes are prepared to act as a quencher. This can explain the finding that none of the Lhcb complexes seems to be strictly required for NPQ while, in the absence of all of them, NPQ is abolished.     

Orientation of pigments and pigment-protein complexes in the green photosynthetic bacterium Prosthecochloris aestuarii

The orientation of pigments and pigment-protein complexes of the green photosynthetic bacterium Prosthecochloris aestuarii was studied by measurement of linear dichroism spectra at 295 and 100 K. Orientation of intact cells and membrane vesicles (Complex I) was obtained by drying on a glass plate. The photochemically active pigment-protein complexes (photosystem-protein complex and reaction center pigment-protein complex) and the antenna bacteriochlorophyll a protein were oriented by pressing a polyacrylamide gel. The data indicate that the near-infrared transitions (Q_y) of bacteriochlorophyll c and most bacteriochlorophyll a molecules have a relatively parallel orientation to the membrane, whereas the Q_y transitions of the bacteriochlorophyll a in the antenna protein are oriented predominantly perpendicularly to the membrane. Carotenoids and the Q_x transitions (590–620 nm) of bacteriochlorophyll a, not belonging to the bacteriochlorophyll a protein, have a relatively perpendicular orientation to the membrane. The absorption and linear dichroism spectra indicate the existence of different pools of bacteriochlorophyll c in the chlorosomes and of carotenoid and bacteriopheophytin c in the cell membrane. The results suggest that the photosystem-protein and reaction center pigment-protein complexes are oriented with their short axes approximately perpendicular to the plane of the membrane. The symmetry axis of the bacteriochlorophyll a protein has an approximately perpendicular orientation.     

Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts

The lateral distribution of the main chlorophyll-protein complexes between appressed and non-appressed thylakoid membranes has been studied. The reaction centre complexes of Photosystems I and II and the light-harvesting complex have been resolved by an SDS-polyacrylamide gel electrophoretic method which permits most of the chlorophyll to remain protein-bound.

The analyses were applied to subchloroplast fractions shown to be derived from different thylakoid regions. Stroma thylakoids were separated from grana stacks by centrifugation following chloroplast disruption by press treatment or digitonin. Vesicles derived from the grana partitions were isolated by aqueous polymer two-phase partition. A substantial depletion in the amount of Photosystem I chlorophyll-protein complex and an enrichment in the Photosystem II reaction centre complex and the light-harvesting complex occurred in the appressed grana partition region. The high enrichment in this fraction compared to grana stack fractions derived from press or digitonin treatments, suggests that the grana Photosystem I is restricted mainly to the non-appressed grana end membranes and margins, and that the grana partitions possess mainly Photosystem II reaction centre complex and the light-harvesting complex.

In contrast, stroma thylakoids are highly enriched in the Photosystem I reaction centre complex. They possess also some 10–20% of the total Photosystem II reaction centre complex and the light-harvesting complex.

The ratio of light-harvesting complex to Photosystem II reaction centre complex is rather constant in all subchloroplast fractions suggesting a close association between these complexes. This was not so for the ratio of light-harvesting complex and the Photosystem I reaction centre complex.

The lateral heterogeneity in the distribution of the photosystems between appressed and non-appressed membranes must have a profound impact on current understanding of both the distribution of excitation energy and photosynthetic electron transport between the photosystems.     

Evolution and functional properties of photosystem II light harvesting complexes in eukaryotes

Photoautotrophic organisms, the major agent of inorganic carbon fixation into biomass, convert light energy into chemical energy. The first step of photosynthesis consists of the absorption of solar energy by pigments binding protein complexes named photosystems. Within photosystems, a family of proteins called Light Harvesting Complexes (LHC), responsible for light harvesting and energy transfer to reaction centers, has evolved along with eukaryotic organisms. Besides light absorption, these proteins catalyze photoprotective reactions which allowed functioning of oxygenic photosynthetic machinery in the increasingly oxidant environment. In this work we review current knowledge of LHC proteins serving Photosystem II. Balance between light harvesting and photoprotection is critical in Photosystem II, due to the lower quantum efficiency as compared to Photosystem I. In particular, we focus on the role of each antenna complex in light harvesting, energy transfer, scavenging of reactive oxygen species, chlorophyll triplet quenching and thermal dissipation of excess energy. This article is part of a Special Issue entitled: Photosystem II.     

Orientation of pigments and pigment-protein complexes in the diatom Cylindrotheca fusiformis. A linear-dichroism study

The orientation of pigments and pigment-protein complexes of the marine diatom Cylindrotheca fusiformis was studied by linear dichroism at 77 K. The technique of polyacrylamide gel squeezing was used to orient the diatom intact cells, their isolated thylakoid membranes and the three pigment-protein complexes: chlorophyll ac-fucoxanthin, chlorophyll ac and PS I complexes. The data indicate that specific orientation of various pigments exists at all structure levels. Tentative assignments of various features of the linear-dichroism spectra to the major photosynthetic pigments are presented. The orientation of the three pigment-protein complexes with respect to the thylakoid membrane plane and the major axis of the cell is also discussed.     

Orientation of the pigments in Photosystem II: low-temperature linear-dichroism study of a core particle and of its chlorophyll-protein subunits isolated from Synechococcus sp.

The orientation of the pigments in the Photosystem II core particle isolated from the thermophilic cyanobacterium Synechococcus sp. has been investigated by linear dichroism spectroscopy at 10 K of macroscopically oriented samples. The absorbance (A), linear dichroism (LD) and LD/A spectra are remarkably similar to those previously reported for a core complex isolated from Chlamydomonas reinhardtii (Biochim. Biophys. Acta 850 (1986) 156–161). The spectra of the Synechococcus core particle are compared to the corresponding spectra obtained on its two main constituent chlorophyll-protein complexes CP2-b (photochemically active) and CP2-c (photochemically inactive). The various features seen in the spectra of the core particle appear well segregated into the spectra of one or the other of the two subparticles without significant loss of orientation of the pigments. The orientation of the chlorophyll macrocycles, with the Y and X optical axis preferentially parallel and perpendicular to the plane of largest cross-section of the particle, respectively, is very similar in the two subparticles. CP2-b contains mainly the beta-carotene pool absorbing around 505 and 470 nm, which is oriented close to the membrane plane, while CP2-c contains the beta-carotene pool absorbing around 495 and 465 nm and oriented closer to the normal to the membrane plane. A shoulder at 682 nm in the absorbance and linear dichroism spectra of the core complex is fully segregated in the spectra of CP2-c, thus excluding the possibility that this spectral feature could be assigned to the primary donor of PS II. A negative linear dichroism component peaking around 691 nm (LD 691) in the core particle is mainly segregated in CP2-b together with the photoactive pheophytin acceptor molecule responsible for the 544 nm positive linear dichroism signal (LD 544). While the ratio of the amplitudes LD 691/LD 544 is approximately the same for the core particle and for the CP2-b complex, the amplitude of LD 691 is significantly reduced in CP2-b compared to the core particle.     

The influence of carotenoids on the conformation of chlorophyll-protein complexes isolated from the cyanobacterium Plectonema boryanum. Absorption and circular dichroism study

Thylakoid membranes of the cyanobacterium Plectonema boryanum solubilized with Triton X-100 can be resolved into three fractions of pigment-protein complexes (Hladík, J. and Sofrová, D. (1981) Photosynthetica 15, 490–503). Fraction I contained relatively the highest amount of carotenoids as well as monomeric forms of chlorophyll a, Fractions II and III contained chlorophyll-protein complexes with a characteristic exciton-split circular dichroism in the red region. It has been shown that fraction III is an oligomeric form of the chlorophyll-protein complex of fraction II. Circular dichroism spectra indicate that, different from fraction II, fraction III contains specifically oriented and space-fixed molecules of carotenoids. Thermal dissociation of fracion III to fraction II is accompanied by disappearance of the positive circular dichroism effect of carotenoids in the 500–550 nm region, thus causing deorganization of the carotenoids, proceeding in parallel to the geometrical rearrangement of chlorophyll molecules. Extraction of the carotenoids of fraction III with heptane is acompanied by dissociation of fraction III. We assume that the observed effects are due to binding of the two pigments to the protein component of the complex and that carotenoids can mediate a part of the interactions which stabilize the structure of pigment-protein complexes. Thus, besides the light-harvesting and protective functions, carotenoids can also play a structural role.     

Polypeptides of chloroplastic and cytoplastic origin required for development of Photosystem II activity,and chlorophyll-protein complexes,in Euglena gracilis Z chloroplast membranes

The development of photosynthetic activity and synthesis of chloroplast membrane polypeptides was studied during greening of Euglena gracilis Z in alternate light-dark-light cycles. The results show: (a) The development of both Photosystem II and Photosystem I can be dissociated from chlorophyll synthesis. (b) Most of the polypeptides required for development of Photosystem I are already synthesized during the initial light period (10–12 h); the further rise in Photosystem I activity in the dark is not inhibited by cycloheximide nor by chloramphenicol. (c) The development of Photosystem II requires continuous de novo synthesis of polypeptides and is inhibited by chloramphenicol. The water-splitting activity already present at the end of the first light period decays in the presence of chloramphenicol while that of 1,5-diphenylcarbazide oxidation is only partially retained. The activity can be repaired in the absence of chlorophyll synthesis and is correlated with the de novo synthesis of polypeptides of 50 000–60 000 daltons. The synthesis of these polypeptides and associated repair of Photosystem II activity is not inhibited by cycloheximide. (d) The chloroplast membranes can be resolved into about 40 distinct polypeptides, among them several in the molecular weight range 50 000–60 000, 20 000–35 000 and 10 000–15 000, which are major membrane constitutents. (e) The synthesis of two major polypeptides (M_r = 20 000–30 000) required for the formation of chlorophyll-protein complex(es) containing chlorophyll a and traces of chlorophyll b (CPII?) is light-dependent and cycloheximide-inhibited. It is concluded that the synthesis and addition to the growing membrane of chlorophyll and polypeptides required for the formation of Photosystem II and Photosystem I complexes can be dissociated in time. The H₂O-splitting enzyme(s) and possibly other components of Photosystem II complex are of chloroplastic origin and turn over in the dark while at least some of the chlorophyll binding polypeptides are of cytoplastic origin and their synthesis is light-controlled.     

Pigment protein complexes and the concept of the photosynthetic unit: Chlorophyll complexes and phycobilisomes

The photosynthetic unit includes the reaction centers (RC 1 and RC 2) and the light-harvesting complexes which contribute to evolution of one O₂ molecule. The light-harvesting complexes, that greatly expand the absorptance capacity of the reactions, have evolved along three principal lines. First, in green plants distinct chlorophyll (Chl) a/b-binding intrinsic membrane complexes are associated with RC 1 and RC 2. The Chl a/b-binding complexes may add about 200 additional chromophores to RC 2. Second, cyanobacteria and red algae have a significant type of antenna (with RC 2) in the form of phycobilisomes. A phycobilisome, depending on the size and phycobiliprotein composition adds from 700 to 2300 light-absorbing chromophores. Red algae also have a sizable Chl a-binding complex associated with RC 1, contributing an additional 70 chromophores. Third, in chromophytes a variety of carotenoid-Chl-complexes are found. Some are found associated with RC 1 where they may greatly enhance the absorptance capacity. Association of complexes with RC 2 has been more difficult to ascertain, but is also expected in chromophytes. The apoprotein framework of the complexes provides specific chromophore attachment sites, which assures a directional energy transfer whithin complexes and between complexes and reaction centers. The major Chl-binding antenna proteins generally have a size of 16–28 kDa, whether of chlorophytes, chromophytes, or rhodophytes. High sequence homology observed in two of three transmembrane regions, and in putative chlorophyll-binding residues, suggests that the complexes are related and probably did not evolve from widely divergent polyphyletic lines.Abbreviations APC allophycocyanin - B phycoerythrin-large bangiophycean phycoerythrin - Chl chlorophyll - L_CM linker polypeptide in phycobilisome to thylakoid - FCP fucoxanthin Chl a/c complex - LHC(s) Chl-binding light harvesting complex(s) - LHC I Chl-binding complex of Photosystem I - LHC II Chl-binding complex of Photosystem II - PC phycocyanin - PCP peridinin Chl-binding complex - P700 photochemically active Chl a of Photosystem I - PS I Photosystem I - PS II Photosystem II - RC 1 reaction center core of PS I - RC 2 reaction center core of PS II - R phycoerythrin-large rhodophycean phycoerythrin - sPCP soluble peridinin Chl-binding complex     

Pigment-protein complexes are organized into stable microdomains in cyanobacterial thylakoids

Thylakoids are the place of the light-photosynthetic reactions. To gain maximal efficiency, these reactions are conditional to proper pigment-pigment and protein-protein interactions. In higher plants thylakoids, the interactions lead to a lateral asymmetry in localization of protein complexes (i.e. granal/stromal thylakoids) that have been defined as a domain-like structures characteristic by different biochemical composition and function (Albertsson P-Å. 2001,Trends Plant Science 6: 349–354). We explored this complex organization of thylakoid pigment-proteins at single cell level in the cyanobacterium Synechocystis sp. PCC 6803. Our 3D confocal images captured heterogeneous distribution of all main photosynthetic pigment-protein complexes (PPCs), Photosystem I (fluorescently tagged by YFP), Photosystem II and Phycobilisomes. The acquired images depicted cyanobacterial thylakoid membrane as a stable, mosaic-like structure formed by microdomains (MDs). These microcompartments are of sub-micrometer in sizes (~0.5–1.5 μm), typical by particular PPCs ratios and importantly without full segregation of observed complexes. The most prevailing MD is represented by MD with high Photosystem I content which allows also partial separation of Photosystems like in higher plants thylakoids. We assume that MDs stability (in minutes) provides optimal conditions for efficient excitation/electron transfer. The cyanobacterial MDs thus define thylakoid membrane organization as a system controlled by co-localization of three main PPCs leading to formation of thylakoid membrane mosaic. This organization might represent evolutional and functional precursor for the granal/stromal spatial heterogeneity in photosystems that is typical for higher plant thylakoids.     

Pigment compositions,spectral properties,and energy transfer efficiencies between the xanthophylls and chlorophylls in the major and minor pigment-protein complexes of photosystem II

Absorption, fluorescence, and fluorescence excitation spectra have been measured from CP26, CP29, and monomeric and trimeric LHCIIb light-harvesting complexes isolated from Photosystem II subchloroplast particles from spinach. The complexes were purified using a combination of isoelectric focusing and sucrose gradient ultracentrifugation. The chlorophyll (Chl) and xanthophyll pigment compositions were measured using high-performance liquid chromatography (HPLC). Using the pigment compositions from the HPLC analysis as a starting point, the absorption spectral profiles of the complexes have been reconstructed from the individual absorption spectra obtained for each of the pigments. Also, the fluorescence excitation spectra of the complexes have been deconvoluted. The data reveal the energy transfer efficiencies between Chl b and Chl a and between specific xanthophylls and Chl a in the complexes. The spectral analyses reveal the underlying features of the highly congested spectral profiles associated with the complexes and are expected to be beneficial to researchers employing spectroscopic methods to investigate the mechanisms of energy transfer between the pigments bound in these complexes.     

Orientation of plgments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. IV. The 100 K linear dichroism spectra of thylakoids from wild-type and chlorophyll b-less barley thylakoids

Using a polyacrylamide gel squeezing technique, linear dichroism spectra of thylakoids from wild-type and chlorophyll-b less barley have been obtained at 100 K. The calculated difference linear dichroism spectra, based on normalization at 690–695 nm, are identical to those of the light-harvesting complex (LHC) isolated by Triton solubilization. This observation is in agreement with previous conclusions (Tapie, P., Haworth, P., Hervo, G. and Breton, J. (1982) Biochim. Biophys. Acta 682, 339–344) regarding: (i) scattering artifacts are absent in linear dichroism spectra determined using polyacrylamide gels, (ii) the in vivo orientation of LHC pigments is maintained in the isolated complex and (iii) the largest dimension(s) of the isolated LHC is (are), in vivo, parallel to the plane of the photosynthetic membrane.     

Energy transfer for low temperature fluorescence in PS II mutant thylakoids

The Chl-protein complexes of three maize (Zea mays L.) mutants and one barley (Hordeum vulgare L.) mutant were analyzed using low temperature Chl fluorescence emissions spectroscopy and LDS-polyacrylamide gel electrophoresis. The maize mutants hcf-3, hcf-19, and hcf-114 all exhibited a high Chl fluorescence (hcf) phenotype indicating a disruption of the energy transfer within the photosynthetic apparatus. The mutations in each of these maize mutants affects Photosystem II. The barley mutant analyzed was the well characterized Chl b-less mutant chlorina-f2, which did not exhibit the hcf phenotype. Chlorina-f2 was used because no complete Chl b-less mutant of maize is available. Analysis of hcf-3, hcf-19, and hcf-114 revealed that in the absence of CP43, LHC II can still transfer excitation energy to CP47. These results suggest that in mutant membranes LHC II can interact with CP47 as well as CP43. This functional interaction of LHC II with CP47 may only occur in the absence of CP43, however, it is possible that LHC II is positioned in the thylakoid membranes in a manner which allows association with both CP43 and CP47.Abbreviations hcf high chlorophyll fluorescence - LDS lithium dodecyl sulfate - LHC II light-harvesting complex of Photosystem II - LHC I light-harvesting complex of Photosystem I - CPIa chlorophyll-protein complex consisting of LHC I and the PS I core complex - CPI chlorophyll-protein complex consisting of the PS I core complex - CP47 47 kDa chlorophyll-protein of the Photosystem II core - CP43 43 kDa chlorophyll-protein of the Photosystem II core - CP29 29 kDa chlorophyll-protein of Photosystem II - CP26 26 kDa chlorophyll-protein of Photosystem II - CP24 24 kDa chlorophyll-protein of Photosystem II - fp free pigments     

Intersystem exciton transfer in isolated chloroplasts

The following arguments in favor of exciton transfer between the two photosystems are presented:

1. (1) MgCl₂ (1–10 mM range) decreases the intersystem transfer but does not modify the partition of absorbed photons between the photosystems. MgCl₂ addition causes a simultaneous increase of excitation life time (τ) and of fluorescence intensity (F). The same linear relationship is obtained with or without added Mg²⁺.

2. (2) The deactivation of Photosystem II by the Photosystem II to Photosystem I transfer increases with the level of reduced Photosystem II traps. When all Photosystem II traps are closed, half of Photosystem II excitons are deactivated by transfer to Photosystem I.

3. (3) From the relative values of the 685-nm fluorescence yield and System II electron transport rate in limiting light, measured with and without MgCl₂, the values of rate constants of Photosystem II deactivation were calculated.

4. (4) The intersystem transfer determines a 715-nm variable fluorescence, which is lowered by MgCl₂ addition. When this transfer is decreased by MgCl₂ the efficiency of the transfer between Photosystem II-connected units is enhanced, and a more sigmoidal fluorescence rise is obtained.

A double-layer model of the thylakoid membrane where each photosystem is restricted to one leaflet is proposed to explain the decrease of the intersystem transfer after adding cations. It is suggested that MgCl₂ decreases the thickness of the Photosystem I polar region, increasing the distance between the pigments of the two photosystems.     

Reconstitution of electrogenic function in isolated pigment-protein complexes of Anabaena variabilis

Treatment of Anabaena variabilis membranes with lauryldimethylamine N-oxide yielded two fractions of pigment-protein complexes which were separable by gel filtration on Sepharose 6B. A green fraction was characterized which had a maximum of the chlorophyll long-wave absorption band at 678 nm and a small amount of carotenoid. In this fraction, Photosystem I activity was higher than in another (brownish-green) fraction which had a maximum of the chlorophyll absorption band at 673 nm and which was enriched in carotenoids. Similarly to isolated membranes, proteoliposomes containing pigment-protein complexes took up tetraphenylborate anions and tetraphenylphosphonium cations and were found to be capable of light-dependent membrane potential generation, when associated with a planar phospholipid membrane in the presence of reduced phenazine methosulfate upon illumination. The spatial arrangement of the pigment-protein complexes in the native and artificial membranes is discussed.     

The photochemical and fluorescence properties of whole cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum.

The photochemical activities and fluorescence properties of cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum were compared. The photochemical activities were measured in a whole range of wavelengths and expressed as quantum yield spectra (quantum yield vs. wavelength). The following reactions were measured. Photosynthesis (O2 evolution) in whole cells; Hill reaction (O2 evolution) with Fe(CN)63- and NADP as electron acceptors (Photosystem II and photosystem II + Photosystem I reactions); electron transfer from reduced 2,6-dichlorophenolindophenol to diquat (Photosystem I reaction). The fluorescence properties were emission spectra, quantum yield spectra and the induction pattern. On the basis of comparison between the quantum yield spectra and the pigments compositions the relative contribution of each pigment to each photosystem was estimated. In normal cells and spheroplasts it was found that Photosystem I (Photosystem II) contains about 90% (10%) of the chlorophyll a, 90% (10%) of the carotenoids and 15% (85%) of the phycocyanin. In spheroplast particles there is a reorganization of the pigments; they loose a certain fraction (about half) of the phycocyanin but the remaining phycocyanin attaches itself exclusively to Photosystem I (!). This is reflected by the loss of Photosystem II activity, a flat quantum yield vs. wavelength dependence and a loss of the fluorescence induction. The fluorescence quantum yield spectra conform qualitatively to the above conclusion. More quantitative estimation shows that only a fraction (20--40%) of the chlorophyll of Photosystem II is fluorescent. Total emission spectrum and the ratio of variable to constant fluorescence are in agreement with this conclusion. The fluorescence emission spectrum shows characteristic differences between the constant and variable components. The variable fluorescence comes exclusively from chlorophyll a; the constant fluorescence is contributed, in addition to chlorophyll a, by phycocyanine and an unidentified long wavelength component. The variable fluorescence does not change in the transition from whole cells to spheroplasts. However, the constant fluorescence increases considerably. This indicates the release of a small fraction of pigments from the photosynthetic photochemical apparatus which then become fluorescent.     

Evolution of heliobacteria: Implications for photosynthetic reaction center complexes

The evolutionary position of the heliobacteria, a group of green photosynthetic bacteria with a photosynthetic apparatus functionally resembling Photosystem I of plants and cyanobacteria, has been investigated with respect to the evolutionary relationship to Gram-positive bacteria and cyanobacteria. On the basis of 16S rRNA sequence analysis, the heliobacteria appear to be most closely related to Gram-positive bacteria, but also an evolutionary link to cyanobacteria is evident. Interestingly, a 46-residue domain including the putative sixth membrane-spanning region of the heliobacterial reaction center protein shows rather strong similarity (33% identity and 72% similarity) to a region including the sixth membrane-spanning region of the CP47 protein, a chlorophyll-binding core antenna polypeptide of Photosystem II. The N-terminal half of the heliobacterial reaction center polypeptide shows a moderate sequence similarity (22% identity over 232 residues) with the CP47 protein, which is significantly more than the similarity with the Photosystem I core polypeptides in this region. An evolutionary model for photosynthetic reaction center complexes is discussed, in which an ancestral homodimeric reaction center protein (possibly resembling the heliobacterial reaction center protein) with 11 membrane-spanning regions per polypeptide has diverged to give rise to core of Photosystem I, Photosystem II, and of the photosynthetic apparatus in green, purple, and heliobacteria.     

Xanthophyll-induced aggregation of LHCII as a switch between light-harvesting and energy dissipation systems

The xanthophyll cycle pigments, violaxanthin and zeaxanthin, present outside the light-harvesting pigment-protein complexes of Photosystem II (LHCII) considerably enhance specific aggregation of proteins as revealed by analysis of the 77 K chlorophyll a fluorescence emission spectra. Analysis of the infrared absorption spectra in the Amide I region shows that the aggregation is associated with formation of intermolecular hydrogen bonding between the alpha helices of neighboring complexes. The aggregation gives rise to new electronic energy levels, in the Soret region (530 nm) and corresponding to the Q spectral region (691 nm), as revealed by analysis of the resonance light scattering spectra. New electronic energy levels are interpreted in terms of exciton coupling of protein-bound photosynthetic pigments. The energy of the Q excitonic level of chlorophyll is not high enough to drive the light reactions of Photosystem II but better suited to transfer excitation energy to Photosystem I, which creates favourable energetic conditions for the state I-state II transition. The lack of fluorescence emission from this energy level, at physiological temperatures, is indicative of either very high thermal energy conversion rate or efficient excitation quenching by carotenoids. Chlorophyll a fluorescence was quenched up to 61% and 34% in the zeaxanthin- and violaxanthin-containing samples, respectively, as compared to pure LHCII. Enhanced aggregation of LHCII, observed in the presence of the xanthophyll cycle pigments, is discussed in terms of the switch between light-harvesting and energy dissipation systems.