Survival strategy of photosynthetic organisms. 1. Variability of the extent of light-harvesting pigment aggregation as a structural factor optimizing the function of oligomeric photosynthetic antenna. Model calculations

In accordance with our concept of rigorous optimization of photosynthetic machinery by a functional criterion, this series of papers continues purposeful search in natural photosynthetic units (PSU) for the basic principles of their organization that we predicted theoretically for optimal model light-harvesting systems. This approach allowed us to determine the basic principles for the organization of a PSU of any fixed size. This series of papers deals with the problem of structural optimization of light-harvesting antenna of variable size controlled in vivo by the light intensity during the growth of organisms, which accentuates the problem of antenna structure optimization because optimization requirements become more stringent as the PSU increases in size. In this work, using mathematical modeling for the functioning of natural PSUs, we have shown that the aggregation of pigments of model light-harvesting antenna, being one of universal optimizing factors, furthermore allows controlling the antenna efficiency if the extent of pigment aggregation is a variable parameter. In this case, the efficiency of antenna increases with the size of the elementary antenna aggregate, thus ensuring the high efficiency of the PSU irrespective of its size; i.e., variation in the extent of pigment aggregation controlled by the size of light-harvesting antenna is biologically expedient.     

Size variability of the unit building block of peripheral light-harvesting antennas as a strategy for effective functioning of antennas of variable size that is controlled in vivo by light intensity

This work continuous a series of studies devoted to discovering principles of organization of natural antennas in photosynthetic microorganisms that generate in vivo large and highly effective light-harvesting structures. The largest antenna is observed in green photosynthesizing bacteria, which are able to grow over a wide range of light intensities and adapt to low intensities by increasing of size of peripheral BChl c/d/e antenna. However, increasing antenna size must inevitably cause structural changes needed to maintain high efficiency of its functioning. Our model calculations have demonstrated that aggregation of the light-harvesting antenna pigments represents one of the universal structural factors that optimize functioning of any antenna and manage antenna efficiency. If the degree of aggregation of antenna pigments is a variable parameter, then efficiency of the antenna increases with increasing size of a single aggregate of the antenna. This means that change in degree of pigment aggregation controlled by light-harvesting antenna size is biologically expedient. We showed in our previous work on the oligomeric chlorosomal BChl c superantenna of green bacteria of the Chloroflexaceae family that this principle of optimization of variable antenna structure, whose size is controlled by light intensity during growth of bacteria, is actually realized in vivo. Studies of this phenomenon are continued in the present work, expanding the number of studied biological materials and investigating optical linear and nonlinear spectra of chlorosomes having different structures. We show for oligomeric chlorosomal superantennas of green bacteria (from two different families, Chloroflexaceae and Oscillochloridaceae) that a single BChl c aggregate is of small size, and the degree of BChl c aggregation is a variable parameter, which is controlled by the size of the entire BChl c superantenna, and the latter, in turn, is controlled by light intensity in the course of cell culture growth.     

Pathways of energy flow through the light-harvesting antenna of the photosynthetic purple bacterium rhodobacter sphaeroides

Using low intensity picosecond absorption spectroscopy with independently tunable excitation and probing infrared pulses, we have studied the pathways of energy transport through the light-harvesting antenna pigments of the photosynthetic purple bacterium Rhodobacter sphaeroides. From the observed excited-state rise time of the red-most pigment B896 as a function of excitation wavelength it is concluded that the B850 pigment of LH2 is spectrally heterogeneous. For excitations originating in the B850 pigment this results in a fast channel (9 ps) that is mainly excited in the peak of the B850 absorption band, and a slow channel (35 ps) that is predominantly excited at ~840 nm. Upon excitation of B800, more than 90% of the excitations follow the fast path. From the observed kinetics it is concluded that the majority of the LH2 → LH1 energy transfer takes place within at most a few picoseconds. The rate-limiting step in the whole energy transfer sequence appears to be the B896 → reaction center transfer. The origin of the B850 heterogeneity and the slow 35-ps component is at the moment unclear. Possibly it represents a highly extended form of LH2 in which transfer to LH1 takes a relatively long time, due to a large number of transfer steps.     

Self-aggregates of natural and modified chlorophylls as photosynthetic light-harvesting antenna systems: substituent effect on the B-ring.

Extramembranous light-harvesting antennae called 'chlorosomes' are the main sunlight-absorbing and energy-migrating systems in photosynthetic green bacteria. In a chlorosome, specific chlorophyllous pigments self-aggregate in hydrophobic environments surrounded by a lipid monolayer to form large oligomers. The self-aggregates of chlorosomal chlorophylls possessing a chlorin pi-system absorb sunlight and can emit near-infrared light, which is transferred to a bacteriochlorin pigment situated in the chlorosomal surface membrane. In vivo and in vitro self-aggregates of natural chlorosomal chlorophylls and their models have been investigated by electronic absorption analysis. Here their self-aggregation is reviewed from the viewpoint of substituent effect on the pyrrolic B-ring. Substituents at the 7- and 8-positions did not disturb the formation of their self-aggregates but affected their absorption bands.     

The size of the photosynthetic unit in purple bacteria

Pigment analysis was performed by means of normal phase HPLC on a number of bacteriochlorophyll a and b containing species of purple bacteria that contain a core antenna only. At least 99% of the bacteriochlorophyll in Rhodobacter sphaeroides R26, Rhodopseudomonas viridis and Thiocapsa pfennigii was esterified with phytol (BChl a _p and BChl b _p, respectively). Rhodospirillum rubrum contained only BChl a esterified with geranyl-geraniol (BChl a _GG). Rhodospirillum sodomense and Rhodopseudomonas marina contained, in addition to BChl a _p, small amounts of BChl a _GG, and presumably also of BChl a esterified with dihydro and tetrahydro geranyl-geraniol (2,10,14-phytatrienol and probably 2,14-phytadienol). In all species bacteriopheophytin (BPhe) esterified with phytol was present. The BChl/BPhe ratio indicated that in these species a constant number of 25 ± 3 antenna BChls is present per reaction centre. This number supports a model in which the core antenna consists of 12 - heterodimers surrounding the reaction centre. Determination of the in vivo extinction coefficient of BChl in the core-reaction centre complex yielded a value of ca. 140 mM^–1 cm^–1 for BChl a containing species and of 130 mM^–1 cm^–1 for Rhodopseudomonas viridis.Abbreviations BChl bacteriochlorophyll - BPhe bacteriopheophytin - GG geranyl-geraniol - LHI and LHII core and peripheral antenna complexes - P phytol - RC reaction centre Dedicated to the memory of Professor D.I. Arnon.     

Energy migration in the light-harvesting antenna of the photosynthetic bacterium Rhodospirillum rubrum studied by time-resolved excitation annihilation at 77 K.

The intensity dependence of picosecond kinetics in the light-harvesting antenna of the photosynthetic bacterium Rhodospirillum rubrum is studied at 77 K. By changing either the average excitation intensity or the pulse intensity we have been able to discriminate singlet-singlet and singlet-triplet annihilation. It is shown that the kinetics of both annihilation types are well characterized by the concept of percolative excitation dynamics leading to the time-dependent annihilation rates. The time dependence of these two types of annihilation rates is qualitatively different, whereas the dependencies can be related through the same adjustable parameter-a spectral dimension of fractal-like structures. The theoretical dependencies give a good fit to the experimental kinetics if the spectral dimension is equal to 1.5 and the overall singlet-singlet annihilation rate is close to the value obtained at room temperature. The percolative transfer is a consequence of spectral inhomogeneous broadening. The effect is more pronounced at lower temperatures because of the narrowing of homogeneous spectra.     

Disordered exciton model for the core light-harvesting antenna of Rhodopseudomonas viridis.

In this work we explain the spectral heterogeneity of the absorption band (. Biochim. Biophys. Acta. 1229:373-380), as well as the spectral evolution of pump-probe spectra for membranes of Rhodopseudomonas (Rps.) viridis. We propose an exciton model for the LH1 antenna of Rps. viridis and assume that LH1 consists of 24-32 strongly coupled BChl b molecules that form a ring-like structure with a 12- or 16-fold symmetry. The orientations and pigment-pigment distances of the BChls were taken to be the same as for the LH2 complexes of BChl a-containing bacteria. The model gave an excellent fit to the experimental results. The amount of energetic disorder necessary to explain the results could be precisely estimated and gave a value of 440-545 cm(-1) (full width at half-maximum) at low temperature and 550-620 cm(-1) at room temperature. Within the context of the model we calculated the coherence length of the steady-state exciton wavepacket to correspond to a delocalization over 5-10 BChl molecules at low temperature and over 4-6 molecules at room temperature. Possible origins of the fast electronic dephasing and the observed long-lived vibrational coherence are discussed.     

Photoinhibitory damage is modulated by the rate of photosynthesis and by the photosystem II light-harvesting chlorophyll antenna size

We investigated the effect of photosynthetic electron transport and of the photosystem II (PSII) chlorophyll (Chl) antenna size on the rate of PSII photoinhibitory damage. To modulate the rate of photosynthesis and the light-harvesting capacity in the unicellular chlorophyte Dunaliella salina Teod., we varied the amount of inorganic carbon in the culture medium. Cells were grown under high irradiance either with a limiting supply of inorganic carbon, provided by an initial concentration of 25 mM NaHCO₃, or with supplemental CO₂ bubbled in the form of 3% CO₂ in air. The NaHCO₃-grown cells displayed slow rates of photosynthesis and had a small PSII light-harvesting Chl antenna size (60 Chl molecules). The half-time of PSII photodamage was 40 min. When switched to supplemental CO₂ conditions, the rate of photodamage was retarded to a t_1/2 = 70 min. Conversely, CO₂-supplemented cells displayed faster rates of photosynthesis and a larger PSII light-harvesting Chl antenna size (500 Chl molecules). They also showed a rate of photodamage with t_1/2 = 40 min. When depleted of CO₂, the rate of photodamage was accelerated (t_1/2  = 20 min). These results indicate that the in-vivo susceptibility to photodamage is modulated by the rate of forward electron transport through PSII. Moreover, a large Chl antenna size enhances the rate of light absorption and photodamage and, therefore, counters the mitigating effect of forward electron transport. We propose that under steady-state photosynthesis, the rate of light absorption (determined by incident light intensity and PS Chl antenna size) and the rate of forward electron transport (determined by CO₂ availability) modulate the oxidation/reduction state of the primary PSII acceptor Q_A, which in turn defines the low/high probability for photodamage in the PSII reaction center. Received: 14 August 1997 / Accepted: 26 September 1997     

Elucidation of structure-function relationships in photosynthetic light-harvesting antenna complexes by non-linear polarization spectroscopy in the frequency domain (NLPF)

Photosynthetically active pigments are usually organized into pigment-protein complexes. These include light-harvesting antenna complexes (LHCs) and reaction centers. Site energies of the bound pigments are determined by interactions with their environment, i.e., by pigment-protein as well as pigment-pigment interactions. Thus, resolution of spectral substructures of the pigment-protein complexes may provide valuable insight into structure-function relationships. By means of conventional (linear) and time-resolved spectroscopic techniques, however, it is often difficult to resolve the spectral substructures of complex pigment-protein assemblies. Nonlinear polarization spectroscopy in the frequency domain (NLPF) is shown to be a valuable technique in this regard. Based on initial experimental work with purple bacterial antenna complexes as well as model systems NLPF has been extended to analyse the substructure(s) of very complex spectra, including analyses of interactions between chlorophylls and "optically dark" states of carotenoids in LHCs. The paper reviews previous work and outlines perspectives regarding the application of NLPF spectroscopy to disentangle structure-function relationships in pigment-protein complexes.     

Dynamics of excitation energy transfer in the LH1 and LH2 light-harvesting complexes of photosynthetic bacteria.

Photosynthetic light harvesting is a unique life process that occurs with amazing efficiency. Since the discovery of the structure of the bacterial peripheral light-harvesting complex (LH2), this process has been studied using a variety of advanced laser spectroscopic methods. We are now in a position to discuss the physical origins of excitation energy transfer and trapping in the LH2 and LH1 antennae of photosynthetic purple bacteria. We demonstrate that the time evolution of the state created by the light is determined by the combined action of excitonic pigment-pitment interactions, energetic disorder, and coupling to nuclear motion in a pigment-protein complex. A quantitative fit of experimental data using Redfield theory allowed us to determine the pathways and time scales of exciton and vibrational relaxation and analyze separately different contributions to the measured transient absorption dynamics. Furthermore, these dynamics were observed to be strongly dependent on the excitation wavelength. A numerical fit of this dependence turns out to be extremely critical to a variation of the structure and disorder parameters and, therefore, can be used as a test for different antenna models (disordered ring, elliptical deformations, correlated disorder, etc.). The calculated equilibration dynamics in the exciton basis allow a visualization of the exciton motion using a density matrix picture in real space.     

Modulation of the light-harvesting chlorophyll antenna size in Chlamydomonas reinhardtii by TLA1 gene over-expression and RNA interference

Truncated light-harvesting antenna 1 (TLA1) is a nuclear gene proposed to regulate the chlorophyll (Chl) antenna size in Chlamydomonas reinhardtii. The Chl antenna size of the photosystems and the chloroplast ultrastructure were manipulated upon TLA1 gene over-expression and RNAi downregulation. The TLA1 over-expressing lines possessed a larger chlorophyll antenna size for both photosystems and contained greater levels of Chl b per cell relative to the wild type. Conversely, TLA1 RNAi transformants had a smaller Chl antenna size for both photosystems and lower levels of Chl b per cell. Western blot analyses of the TLA1 over-expressing and RNAi transformants showed that modulation of TLA1 gene expression was paralleled by modulation in the expression of light-harvesting protein, reaction centre D1 and D2, and VIPP1 genes. Transmission electron microscopy showed that modulation of TLA1 gene expression impacts the organization of thylakoid membranes in the chloroplast. Over-expressing lines showed well-defined grana, whereas RNAi transformants possessed loosely held together and more stroma-exposed thylakoids. Cell fractionation suggested localization of the TLA1 protein in the inner chloroplast envelope and potentially in association with nascent thylakoid membranes, indicating a role in Chl antenna assembly and thylakoid membrane biogenesis. The results provide a mechanistic understanding of the Chl antenna size regulation by the TLA1 gene.     

Strong orientational ordering of the near-infrared transition moment vectors of light-harvesting antenna bacterioviridin in chromatophores of the green photosynthetic bacterium Chlorobium limicola

The direction of the transition moments of chlorosome pigments in chromatophores of the green photosynthetic bacterium Chlorobium limicola was studied by linear dichroism. Orientation of chromatophores was achieved by stretching a polyacrylamide gel in which they were packed. It was shown that in each individual chromatophore the Q_y transition moment vectors of the whole chlorosome bacterioviridin are parallel to each other and are practically ideally oriented along the chlorosome long axis. The exact value of the angle α between the bacterioviridin transition moments and the long axis of the chlorosome is calculated to be α = 0°, the mean square deviation being 7°.     

Regulation of building unit size in the house building of the caddis larva Lepidostoma hirtum

When the normally staggered relationship between the four sides at the anterior house rim of Lepidostoma hirtum was removed by cutting all the sides level, larvae were found to replace panels so as to regain the stagger. This was achieved by cutting panel lengths over a greater size range than normal but keeping mean panel length the same. The length of an individual panel was shown to be influenced by the site it was to occupy. Panel width was found to be positively correlated with panel length. The only rule for positioning a panel was found to be that of fitting to the most posterior gap on the anterior house rim. The rate of panel replacement was found to be increased not only by increase in amount of house removed but also by cutting all sides level.     

Dynamics of energy transfer and trapping in the light-harvesting antenna of Rhodopseudomonas viridis.

By low intensity picosecond absorption spectroscopy it is shown that the exciton lifetime in the light-harvesting antenna of Rhodopseudomonas (Rps.) viridis membranes with photochemically active reaction centers at room temperature is 60 +/- 10 ps. This lifetime reflects the overall trapping rate of the excitation energy by the reaction center. With photochemically inactive reaction centers, in the presence of P+, the exciton lifetime increases to 150 +/- 15 ps. Prereducing the secondary electron acceptor QA does not prevent primary charge separation, but slows it down from 60 to 90 +/- 10 ps. Picosecond kinetics measured at 77 K with inactive reaction centers indicates that the light-harvesting antenna is spectrally homogeneous. Picosecond absorption anisotropy measurements show that energy transfer between identical Bchlb molecules occurs on the subpicosecond time scale. Using these experimental results as input to a random-walk model, results in strict requirements for the antenna-RC coupling. The model analysis prescribes fast trapping (approximately 1 ps) and an approximately 0.5 escape probability from the reaction center, which requires a more tightly coupled RC and antenna, as compared with the Bchla-containing bacteria Rhodospirillum (R.) rubrum and Rhodobacter (Rb.) sphaeroides.     

The light-harvesting chlorpohyll-protein complex of photosystem II. Its location in the photosynthetic membrane

We have investigated the structure of the photosynthetic membrane in a mutant of barley known to lack a chlorophyll-binding protein. This protein is thought to channel excitation energy to photosystem II, and is known as the "light-harvesting chlorophyll-protein complex." Extensive stacking of thylakoids into grana occurs in both mutant and wild-type chloroplasts. Examination of membrane internal structure by freeze-fracturing indicates that only slight differences exist between the fracture faces of mutant and wild-type membranes. These differences are slight reductions in the size of particles visible on the EFs fracture face, and in the number of particles seen on the PFs fracture face. No differences can be detected between mutant and wild-type on the etched out surface of the membrane. In contrast, tetrameric particles visible on the etched inner surface of wild-type thylakoids are extremely difficult to recognize on similar surfaces of the mutant. These particles can be recognized on inner surfaces of the mutant membranes when they are organized into regular lattices, but these lattices show a much closer particle-to-particle spacing than similar lattices in wild-type membranes. Although several interpretations of these data are possible, these observations are consistent with the proposal that the light-harvesting chlorophyll-protein complex of photosystem II is bound to the tetramer (which is visible on the EFs face as a single particle) near the inner surface of the membrane. The large tetramer, which other studies have shown to span the thylakoid membrane, may represent an assembly of protein, lipid, and pigment comprising all the elements of the photosystem II reaction. A scheme is presented which illustrates one possibility for the light reaction across the photosynthetic membrane.