Peripheral Antenna Research Articles

Temporally and spectrally resolved subpicosecond energy transfer within the peripheral antenna complex (LH2) and from LH2 to the core antenna complex in photosynthetic purple bacteria.

We report studies of energy transfer from the 800-nm absorbing pigment (B800) to the 850-nm absorbing pigment (B850) of the LH2 peripheral antenna complex and from LH2 to the core antenna complex (LH1) in Rhodobacter (Rb.) sphaeroides. The B800 to B850 process was studied in membranes from a LH2-reaction center (no LH1) mutant of Rb. sphaeroides and the LH2 to LH1 transfer was studied in both the wild-type species and in LH2 mutants with blue-shifted B850. The measurements were performed by using approximately 100-fs pulses to probe the formation of acceptor excitations in a two-color pump-probe measurement. Our experiments reveal a B800 to B850 transfer time of approximately 0.7 ps at 296 K and energy transfer from LH2 to LH1 is characterized by a time constant of approximately 3 ps at 296 K and approximately 5 ps at 77 K. In the blue-shifted B850 mutants, the transfer time from B850 to LH1 becomes gradually longer with increasing blue-shift of the B850 band as a result of the decreasing spectral overlap between the antennae. The results have been used to produce a model for the association between the ring-like structures that are characteristic of both the LH2 and LH1 antennae.

Vibrational equilibration in absorption difference spectra of chlorophyll a

We describe Franck-Condon simulations of vibrational cooling effects on absorption difference spectra in chlorophyll a (Chl a). The relative contributions of vibrational equilibration in the electronic ground and excited states depend on the pump and probe wavelengths. For Franck-Condon-active vibrational modes exhibiting small Huang-Rhys factors (S < 0.1, characteristic in Chl a pigments), vibrational thermalization causes essentially no spectral changes when the origin band is excited. Significant spectral evolution does occur for S < 0.1 when the 0-1 and 1.0 (hot) vibronic bands are excited. However, vibrational equilibration in these cases causes no spectral shifting in the empirical photobleaching/stimulated emission band maximum. This result bears on the interpretation of time-resolved absorption difference spectra of Chl a-containing antennae such as the Chl a/b light-harvesting peripheral antenna of photosystem II.

Energy migration in Rhodobacter sphaeroides mutants altered by mutagenesis of the peripheral LH2 complex or by removal of the core LH1 complex

The photosynthetic apparatus of the purple bacterium Rhodobacter sphaeroides is organised so that light energy absorbed by the peripheral antenna (LH2) complexes migrates towards the core (LH1) complex, before being trapped by the reaction centre (RC). This migration and trapping process has been studied in mutants where the energy levels of the LH2 BChls have been raised by mutagenesis of the C-terminal aromatic residues (Fowler, G.J.S., Visschers, R.W., Grief, G.G., Van Grondelle, R. and Hunter, C.N. (1992) Nature 355, 848–850), and in a mutant which lacks the core complex. In the former case, the alterations to the LH2 complexes did not prevent efficient energy transfer to the LHI-RC complex, but fluorescence emission spectra indicated that the equilibrium of energy within the system was affected so that back transfer from the LH1-RC core is minimised. This mimics the situation found in some other bacteria such as Rhodopseudomonas acidophila and Rps. cryptolactis. In the mutant lacking LH1, energy is transferred from LH2 directly to the RC, despite the absence of the core antenna. Energy transfer efficiencies for carotenoids and LH2 to LH1 were measured for the blue-shifted LH2 mutants, and were found to be high (70%) in each case. These data, together with measurements of excitation annihilation as a function of incident excitation energy, were used to estimate the domain sizes for energy transfer in these mutants. In the LH2 mutants, domains of about 50 to 170 core BChls were found, depending on the type of mutation. One effect of the removal of LH1 appears to be the reorganisation of the peripheral LH2 antenna to form domains of at least 250 BChls.

Energy transfer in the photosynthetic antenna system of the purple non-sulfur bacterium Rhodopseudomonas cryptolactis

Energy transfer in the antenna of the purple non-sulfur bacterium Rhodopseudomonas cryptolactis was studied by means of fluorescence and transient absorption measurements. Different growth conditions resulted in three cultures which differed in the composition of the peripheral antenna (B800-820, B800-850 or a mixture of both). Oxidation of the primary electron donor caused an increase of the fluorescence yield of the core complex, B880, and of B800-850, but not of B800-820, indicating energy transfer from B880 to B800-850, but not to B800-820. Singlet-singlet annihilation measurements showed that the number of bacteriochlorophylls of B880 between which energy transfer can occur corresponds to one or two photosynthetic units only (40 to 70 BChls), for all the three cultures studied. These results are explained by a model for Rps. cryptolactis in which each 13880 unit is associated with its own complement of B800-850. The B800-850 complexes do not appear to form an interconnecting network. Time-resolved transient absorption measurements showed trapping rates by open and closed reaction centres of (50 ps) −1 and (200 ps) −1, respectively. Time constants of energy transfer from B800-850 to B880 or from B800-820 to other complexes were 12–15 ps.

Theory of fluorescence induction in photosystem II: derivation of analytical expressions in a model including exciton-radical-pair equilibrium and restricted energy transfer between photosynthetic units

The theoretical relationships between the fluorescence and photochemical yields of PS II and the fraction of open reaction centers are examined in a general model endowed with the following features: i) a homogeneous, infinite PS II domain; ii) exciton-radical-pair equilibrium; and iii) different rates of exciton transfer between core and peripheral antenna beds. Simple analytical relations are derived for the yields and their time courses in induction experiments. The introduction of the exciton-radical-pair equilibrium, for both the open and closed states of the trap, is shown to be equivalent to an irreversible trapping scheme with modified parameters. Variation of the interunit transfer rate allows continuous modulation from the case of separated units to the pure lake model. Broadly used relations for estimating the relative amount of reaction centers from the complementary area of the fluorescence kinetics or the photochemical yield from fluorescence levels are examined in this framework. Their dependence on parameters controlling exciton decay is discussed, allowing assessment of their range of applicability. An experimental induction curve is analyzed, with a discussion of its decomposition into alpha and beta contributions. The sigmoidicity of the induction kinetics is characterized by a single parameter J related to Joliot's p, which is shown to depend on both the connectivity of the photosynthetic units and reaction center parameters. On the other hand, the relation between J and the extreme fluorescence levels (or the deviation from the linear Stern-Volmer dependence of 1/phi f on the fraction of open traps) is controlled only by antenna connectivity. Experimental data are consistent with a model of connected units for PS II alpha, intermediate between the pure lake model of unrestricted exciton transfer and the isolated units model.

Energy transfer between antenna complexes in the purple sulfur bacteria Chromatium tepidum and Chromatium vinosum

Abstract Energy transfer between antenna complexes was studied by means of time resolved absorption spectroscopy in the purple sulfur bacteria Chromatium tepidum and C. vinosum. The first species contains only one peripheral antenna complex, B800-B50, the other one has two, B800-850 and B800-820. Results obtained with chromatophores of C. tepidum indicated two time constants for energy transfer from B800-850 to the core complex, of 10 ps and a smaller one of 30 ps, suggesting non-uniform distances between the peripheral and core complexes. Similar results were obtained with C. vinosum, where time constants of 7 and 30 ps were found. Energy transfer from B800-820 to B800-850 was significantly faster. These results show that the rates of energy transfer from bacteriochlorophyll 850 to the core complex in the purple sulfur bacteria studied are quite similar to those found in purple non-sulfur bacteria. This may seem remarkable in view of the fact that the core antenna in C. tepidum absorbs at the unusually long wavelength of 918 nm, but a calculation indicates that the overlap integral for energy transfer to the core is not dramatically less than in C. vinosum.

Diurnal fluctuation in the composition of chlorophyll a/b light harvesting antenna of photosystem II in young wheat leaves

We investigated the diurnal fluctuation in the composition of the light harvesting chlorophyll a/b antenna of photosystem II in young wheat (Triticum aestivum) leaves grown under periodic day/night irradiation. By means of gel electrophoresis of the polypeptides of thylakoid membranes, we determined the amount of 25 kDa and 27 kDa polypeptides, which are the main components of the peripheral and inner antenna subpopulations, respectively. Our data show a preferential fluctuation in the amount of the 25 kDa protein relative to the 27 kDa polypeptide, in parallel to the fluctuation in the amount of chlorophyll a/b antenna of photosystem II, which suggests that the peripheral antenna plays a role in the diurnal adjustment of the antenna size.

FDMR spectroscopy of peridinin-chlorophyll-a protein from Amphidinium carterae

Fluorescence detected magnetic resonance (FDMR) is used to investigate peridinin-chlorophyll-a protein (PCP), the peripheral antenna of the photosystem of Amphidinium carterae. Peridinin triplet resonances are obtained monitoring chlorophyll-a fluorescence in agreement with efficient energy transfer between the two pigments. Two sites are revealed for peridinin having slightly different triplet energy (about 190 cm −1), and D values averaging 0.045 cm −1. Spin exchange occurs between the two sites at a rate of about 5 × 10 7 exp (−67/ T) s −1.

Chlorophyll in a Synechocystis sp. PCC 6803 mutant without photosystem I and photosystem II core complexes. Evidence for peripheral antenna chlorophylls in cyanobacteria.

The chlorophyll protein organization has been investigated in thylakoid membranes from mutants of the cyanobacterium Synechocystis sp. PCC 6803, in which the photosystem II (PS II) genes psbB and/or psbC (coding for CP47 and CP43, respectively) were inactivated together with the psaAB operon (coding for the photosystem I (PS I) core complex) and the apcE gene (coding for the phycobilisome anchor protein). Lack of the CP43 protein led to a significant decrease of the D1, D2, and CP47 proteins and a decrease in the 77 K fluorescence emission peak at 685 nm. In the absence of the CP47 protein, no PS II reaction center assembly was detected and the 77 K fluorescence emission peak at 695 nm was lost. The psbB-/psbC-/PS I-less/apcE- mutant had no assembly of the D1, D2, CP47, and CP43 proteins, had lost the 77 K fluorescence emission peaks at 685 and 695 nm, but retained about 15% of the chlorophyll present in the PS I-less/apcE- background strain. A broad 77 K fluorescence emission band with a maximum at 678 nm was displayed in the PS II-less, PS I-less mutant upon excitation of the remaining chlorophyll. A 678 nm shoulder was observed in the 77 K fluorescence emission spectrum of thylakoids from the psbB-/PS I-less/apcE- mutant, which still contains CP43 but no PS II reaction center. This shoulder was absent in thylakoids from the psbC-/PS I-less/apcE- mutant, which contain some PS II reaction center complexes. These results are consistent with the chlorophyll associated with the 678 nm emission to serve as peripheral antenna to PS II. The fluorescence emission characteristics of this chlorophyll are different from those of an accessory chlorophyll-binding protein expressed under iron-stress conditions in cyanobacteria. The chlorophyll remaining in the absence of PS II and PS I is indicative of a new chlorophyll-binding protein in cyanobacterial thylakoids.

Low-Energy Exciton Level Structure and Dynamics in LightHarvesting Complex II Trimers from the Chl a/b Antenna Complex of Photosystem II

Nonphotochemical hole-burned spectra obtained as a function of burn wavelength at 4.2 K are reported for the isolated LHC II peripheral antenna complex of photosystem II. The lowest-energy state of the trimer complex is shown to lie at 680 nm, 4 nm below the most intense Chl a band at 676 nm. The linear electron-phonon coupling for the 680-nm state is characterized and used to predict that its fluorescence origin should lie at 681 nm, precisely coincident with the observed origin at 4.2 K. The 680-nm band carries the equivalent absorption strength of about one chlorophyll a molecule per C[sub 3] trimer complex, which contains about 27 chlorophyll a molecules. The 680-nm absorption band possesses an inhomogeneous width of [approximately] 120 cm[sup [minus]1], and its zero-phonon line distribution function is largely uncorrelated with those of the higher-energy states. Zero-phonon hole widths are used to determine that the fluorescent 680-nm state dephases in 10 ps at 4.2 K. An interpretation of this dephasing is given in terms of the trimer of subunits structure. Based on the satellite hole structure observed upon hole burning into the 680-nm state, two new states at 674 and 678 nm are identified. 56 refs., 5 figs.

Purification and characterization of the peripheral antenna of the purple-sulfur bacterium Chromatium purpuratum: Evidence of an unusual pigment-protein composition

The purification and characterization of the peripheral antenna and the preliminary characterization of a carotenoid-protein complex from the purple-sulfur bacterium Chromatium purpuratum are described. The peripheral antenna of C. purpuratum is unusual among purple bacteria in that it can be resolved by SDS-PAGE into six subunits, the largest number observed thus far for a spectrally pure antenna complex. N-terminal sequence analyses of these subunits suggest that they may have an additional bacteriochlorophyll-binding site located outside the transmembrane domain. The results of pigment-protein quantification are also consistent with additional pigment-binding sites in the C. purpuratum LH2. Furthermore, CD measurements and sequence analysis suggest the presence of considerable beta-type in addition to alpha-helical secondary structure. Thus, the secondary and quaternary structures of this complex differ significantly from light-harvesting complexes of other purple photosynthetic bacteria. A carotenoid-protein complex is also described; it is an apparent association of three proteins and carotenoid and is closely associated with the peripheral antenna. The purple-sulfur bacteria are evolutionarily older than the relatively better characterized purple-nonsulfur organisms. The phenotypic features described here of the C. purpuratum photosynthetic apparatus are related to those of other purple bacteria and green-sulfur bacteria and may reflect the evolutionary position of this organism.

Cloning of a new antenna gene cluster and expression analysis of the antenna gene family of Rhodopseudomonas palustris.

The genome of Rhodopseudomonas palustris contains five antenna gene clusters, alpha beta a, alpha beta b, alpha beta c, alpha beta d and alpha beta e, which encode the light-harvesting peripheral antenna complex II polypeptides. The isolation and characterisation of the gene which encodes the alpha e and beta e polypeptides are reported. The primary structure of the beta e polypeptide is identical to that of beta b whilst the structure of alpha e is different from the other alpha subunits so far characterised. All five of the gene clusters were transcribed under high-light conditions while under low-light conditions only three were transcribed (alpha beta b, alpha beta d and alpha beta e). Furthermore, Northern-blot analysis showed that the gene clusters encode RNA transcripts of either 500 or 650 nucleotides. Individual members of the gene family showed a differential response in terms of the regulation of abundance of mRNA upon growth under either high-light or low-light intensities. Possible promoter sequences and operator sites upstream of the alpha beta b, alpha beta d and alpha beta e genes were located. Furthermore using puc-lacZ fusions in trans in R. palustris, we were able to examine the positions of the promoter of the gene clusters. The significance of these observations with respect to the regulation, organization and role of the peripheral antenna is discussed.

CHANGES IN SPECTRAL PROPERTIES OF CHLOROPHYLL DURING GREENING OF BARLEY LEAVES

Abstract— In order to study the formation of photochemical systems in the early phase of greening of barley leaves, we examined the changes in chlorophyll (Chl) spectral components of the absorption spectrum of greening tissues using a curvc analysis method. Peak positions of the fourth‐derivative spectrum were changing during the first 30 min of illumination hut became stable thereafter. Results of curve analysis showed that about half of the spectral components of mature leaves were already found at 30 min after onset of illumination, and the rest appeared by 4 h. After 30 min the half widths of some spectral componcnts changed slightly. The 683 nm form of Chl a of the photosystem 11 (PSII) core was present after 30 min of illumination and increased up to 2 h, followed by a decrease. The 641 nm form of Chl u of photosystem I (PSI) appeared at 30 min and increased until 2.5 h. The 699 nm form of Chl a also appeared at 1.5 h. The 640 nm form of Chl b appeared at 2 h, while the 649 nm Chl b form was found earlier (1.5 h). Both components were increasing with prolonged time of illumination. Finally, the 704 nm form of Chl a characteristic of the PSI peripheral antenna appeared at 4 h of greening. Ruorescence of PSI and PSII began to appear at 1 and 2 h, respectively. These spectral changes were discussed in relation to the formation of chlorophyll‐protein complexes and the development of photochemical activities.

Picosecond dynamics of excitations in light-harvesting complex B800-850 from Chromatium minutissimum studied using fluorescence spectrochronography

The picosecond dynamics of excitations in the isolated B800-850 light-harvesting complex of the purple sulfur bacterium Chromatium minutissimum has been studied using picosecond fluorescence spectrochronography. A short-lived component of about 20 ps lifetime has been found at 77K at the short wavelength part of the B850 fluorescence spectrum similar to that previously described for the core antenna bacteriochlorophyll band B880 of Rhodospirillum rubrum. Evidence has been presented indicating that this component is likely to reflect excitation energy relaxation step(s) involving both photoexcited bacteriochlorophyll and the protein environment. A new kinetic scheme of excitation transfer from the peripheral antenna to the photoreceptor units in purple bacteria is suggested which takes into account these findings.

Protein engineering of bacterial light-harvesting complexes

The photosynthetic apparatus of the bacterium Rhodobacter sphueroides contains three types of pigment protein: the photochemical reaction centre, and the LH1 and LH2 light-harvesting (LH) complexes. LH1 surrounds and interconnects the reaction centres, forming an LHl-reaction centre (RC) ‘core’ which receives excitation energy from the peripheral LH2 antenna [l]. This arrangement of complexes presents an experimentally attractive model system for the study of the transfer and trapping of light energy in photosynthesis. There are several reasons for this. First, there is a crystallographically determined structure for the RC [2-41. Second, a wealth of spectroscopic data are available on the RC and I,H complexes. Finally, there is a full range of genetic tools that can be used to produce site-directed mutations in the genes encoding these complexes [5, 61. Light energy is harvested by IJH1 and LH2 complexes, which are both composed of highly aggregated states of a and p polypeptides in close association with bacteriochlorophyll and carotenoid pigments. Secondary-structure predictions indicate that these polypeptides span the membrane once, and form an interconnecting network of several hundred pigments which increase the surface area and range of wavelengths over which light energy can be absorbed. Although this network contains only one type of tetrapyrrole pigment, bacteriochlorophyll a, the antenna system is populated with a series of these molecules which absorb at progressively red-shifted wavelengths so that an excited state at the periphery of a photosynthetic ‘unit’ migrates in an energetically ‘downhill’ fashion towards the RC where the energy is trapped and photochemistry takes place. Spectroscopic studies on the antenna of Rb. sphaeroides have been extremely useful in defining the rates of energy transfer between these pigments, and the orientations of these pigments with respect to the plane of the membrane. The availability of mutant strains has also simplified the interpretations of complicated and overlapping spectroscopic signals by contributing a series of strains in which LH2, LH1 or indeed the RC, can be examined in

The effect of changes in light intensity and temperature on the peripheral antenna of Rhodopseudomonas acidophila.

The effect of changes in light intensity and temperature on the peripheral antenna of Rhodopseudomonas acidophila.

Changes in the Carotenoid Pattern During the Synchronous Life Cycle of Scenedesmus

AbstractThe wild type (WT) of Scenedesmus obliquus and a mutant lacking chlorophyll b and the light‐harvesting complexes (WT‐LHC1) were synchronized by a light‐dark regime. Both cultures contained the same type of carotenoids. However, concentrations and patterns of carotenoids were different during their synchronous life cycles. The concentration of total carotenoids followed more or less that of chlorophyll. The WT contained more carotenoids per cell mass, but slightly less per chlorophyll. It is discussed that part of the carotenoids of the mutant, lacking the peripheral antenna of PSII, might be located in the chlorophyll b‐less apoprotein or in an enlarged core antenna of PSII.During the life cycle of Scenedesmus the carotenes are initially synthesized and most of the α‐carotene is immediately oxidized to lutein which is inserted in the antennae systems of PSII and PSI. The further oxidation of lutein to loroxanthin seems to depend on both the change from dark to light, and on stages of the life cycle itself. Although the major part of β‐carotene appears to be inserted in the reaction centers, a fraction of the total pool is rapidly converted to violaxanthin, following the onset of illumination. The conversion may serve to protect against photooxidation. Further conversion of violaxanthin to neoxanthin occurs to a greater extent in the mutant, WT‐LHC1. The results demonstrate (1) the close connection between the carotenoid pattern and the modulation of the photosynthetic apparatus during the life cycle of Scenedesmus and (2) the flexibility of the organism in compensating for the absence of the light‐harvesting complexes of photosystems II by adjusting the carotenoid distribution.

Structure, function and organization of antenna polypeptides and antenna complexes from the three families of Rhodospirillaneae

Comparative primary structural analysis of polypeptides from antenna complexes from species of the three families of Rhodospirillaneae indicates the structural principles responsible for the formation of spectrally distinct light-harvesting complexes. In many of the characterized antenna systems the basic structural minimal unit is an α/β polypeptide pair. Specific clusters of amino acid residues, in particular aromatic residues in the C-terminal domain, identify the antenna polypeptides to specific types of antenna systems, such as B880 (strong circular dichroism (CD)), B870 (weak CD), B800–850 (high), B800–850 (low) or B800–820. The core complex B880 (B1020) of species from Ectothiorhodospiraceae and Chromatiaceae apparently consists of four (α 1α 2β 1β 2) or three (2αβ 1β 2) chemically dissimilar antenna polypeptides respectively. There is good evidence that the so-called variable antenna complexes, such as the B800–850 (high), B800–850 (low) or B800–820 of Rp. acidophila, Rp. palustris and Cr. vinosum, are comprised of multiple forms of peripheral light-harvesting polypeptides. Structural similarities between prokaryotic and eukaryotic antenna polypeptides are discussed in terms of similar pigment organization. The structural basis for the strict organization of pigment molecules (bacteriochlorophyll (BChl) cluster) in the antenna system of purple bacteria is the hierarchical organization of the α- and β-antenna polypeptides within and between the antenna complexes. On the basis of the three-domain structure of the antenna polypeptides with the central hydrophobic domain, forming a transmembrane α helix, possible arrangements of the antenna polypeptides in the three-dimensional structure of core and peripheral antenna complexes are discussed. Important structural and functional features of these polypeptides and therefore of the BChl cluster are the α/β heterodimers, the α 2,β 2 basic units and cyclic arrangements of these basic units. Equally important for the formation of the antenna complexes or the entire antenna are polypeptide-polypeptide, pigment-pigment and pigment-polypeptide interactions.

Fluorescence studies of photoregulation in the chrysophyte Ochromonas danica

Three physiological states brought about by different preillumination conditions in Ochromonas danica (state 1, state 2, and dark adapted) have been investigated using steady-state and time-resolved fluorescence at 77 K. The former reveals that the photosystem 1 (PS1) emission amplitude is the same in states 1 and 2. The photosystem 2 (PS2) emission amplitude depends on preillumination, state 1 having a higher peak than state 2 and dark adapted cells. Emission decay curves from PS1 are independent of preillumination, while decay curves from PS2 are identical for state-2 and dark adapted cells (1/e time 310 ps), state 1 showing a slower decay (1/e time 390 ps). No evidence suggests a preillumination-dependent redistribution of excitation energy from PS2 to PS1. Variation in the amplitude of a long-lifetime component appears to be sufficient to explain the state-dependent changes in emission of the PS2 peak. This is consistent with the hypothesis that some peripheral antenna is energetically decoupling without redistributing the excitation energy upon the state 2 to state 1 transition, causing increased emission in the 670–700 nm region.

Antenna organization of Rhodopseudomonas acidophila: a study of the excitation migration

Transfer of excitation energy was studied in three different cultures of the purple bacterium Rhodopseudomonas acidophila , which all contained the core antenna complex B880, but differed with respect to the type of peripheral antenna complex, which was either B800–820, B800–850, or both. Contrary to that of B800–850, the fluorescence emitted by B800–820 did not increase upon light-induced oxidation of P-870. Singlet-singlet annihilation measurements showed that in cells containing only B880 and B800–820 energy transfer can take place within a domain of 50 ± 20 bacteriochlorophyll (BChl) molecules, which number equals that of BChls per reaction center. In cells containing B800–850 energy transfer can take place over at least 200 BChls of B880. Our results are explained by a model where small clusters of B880, the size of one photosynthetic unit, are separated from each other by a more or less continuous array of B800–820 or B800–850 complexes. B800–820, but not B800–850, acts as an energy barrier between different B880 units.