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Preliminary X-ray diffraction analysis of the extrinsic PsbP protein of photosystem II from spinach (Spinacia oleracea) was performed using N-terminally His-tagged recombinant PsbP protein overexpressed in Escherichia coli. Recombinant PsbP protein (thrombin-digested recombinant His-tagged PsbP) stored in bis-Tris buffer pH 6.00 was crystallized using the sitting-drop vapour diffusion technique with PEG 550 MME as a precipitant and zinc sulfate as an additive. SDS–PAGE analysis of a dissolved crystal showed that the crystals did not contain the degradation products of recombinant PsbP protein. PsbP crystals diffracted to 2.06 A resolution in space group P2(1)2(1)2(1), with unit-cell parameters a = 38.68, b = 46.73, c = 88.9 A.

Hydrophilic carotenoids, unusual members of an intrinsically hydrophobic family, and their radical ions are important reactants. An all-optical method for generating singly charged radical ions of a hydrophilic carotenoid (Car) is described. It relies on photolyzing an aqueous mixture of Car and a photoionizable auxiliary solute (A), and making conditions conducive to the capture, by Car, of the hydrated electron (e−aq) or the positive hole in A·+ or both. When A is Trolox (TOH), only e−aq can be captured, since TOH·+ deprotonates too rapidly to be a hole donor; when A is Trolox methyl ether (TOMe), both Car·− and Car·+ are formed, since TOMe·+ lives long enough to transfer its positive hole to Car; formation of Car·− is prevented under aerobic conditions.

Filamentous cyanobacteria develop heterocysts in response to deprivation for combined nitrogen under aerobic conditions. The most prominent structural change in heterocysts is the biosynthesis of an envelope that restricts gas permeability, providing an appropriate micro-oxic environment for N2 fixation inside. The additional thickness of the differentiated cells, when compared to vegetative cells, makes filamentous cyanobacteria an attractive biological system to investigate cellular response against femtosecond laser processing. By irradiating the cyanobacterial filaments with 120 fs, 795 nm, 1 kHz pulses focused through a 100× microscope objective with a numerical aperture of 0.85, we have determined that the pulse energy threshold for an apparent disruption of the cell wall of vegetative cells is 13 ± 4 nJ per pulse. A further increase in the pulse energy to 43 ± 13 nJ causes the complete removal of vegetative cells. In contrast, the pulse energy threshold has to be augmented about three-fold for heterocyst envelope disruption or two-fold for complete removal of heterocysts. We propose that the singular cross-linked structure of the glycolipid multilayer of the envelope, required to restrict gas permeability, accounts for the remarked difference in the ablation energy threshold between vegetative cells and heterocysts.

Chlorosomes, the main light-harvesting complexes of green photosynthetic bacteria, contain bacteriochlorophyll (BChl) molecules in the form of self-assembling aggregates. To study the role of esterifying alcohols in BChl aggregation we have prepared a series of bacteriochlorophyllide c (BChlide c) derivatives differing in the length of the esterifying alcohol (C1, C4, C8 and C12). Their aggregation behavior was studied both in polar (aqueous buffer) and nonpolar (hexane) environments and the esterifying alcohols were found to play an essential role. In aqueous buffer, hydrophobic interactions among esterifying alcohols drive BChlide c derivatives with longer chains into the formation of dimers, while this interaction is weak for BChlides with shorter esterifying alcohols and they remain mainly as monomers. All studied BChlide c derivatives form aggregates in hexane, but the process slows down with longer esterifying alcohols due to competing hydrophobic interactions with hexane molecules. In addition, the effect of the length of the solvent molecules (n-alkanes) was explored for BChl c aggregation. With an increasing length of n-alkane molecules, the hydrophobic interaction with the farnesyl chain becomes stronger, leading to a slower aggregation rate. The results show that the hydrophobic interaction is the driving force for the aggregation in an aqueous environment, while in nonpolar solvents it is the hydrophilic interaction.

Chlorosomes are light-harvesting complexes of green photosynthetic bacteria. Chlorosomes contain bacteriochlorophyll (BChl) c, d, or e aggregates that exhibit strong excitonic coupling. The short-range order, which is responsible for the coupling, has been proposed to be augmented by pigment arrangement into undulated lamellar structures with spacing between 2 and 3 nm. Treatment of chlorosomes with hexanol reversibly converts the aggregated chlorosome chlorophylls into a form with spectral properties very similar to that of the monomer. Although this transition has been extensively studied, the structural basis remains unclear due to variability in the obtained morphologies. Here we investigated hexanol-induced structural changes in the lamellar organization of BChl c in chlorosomes from Chlorobium tepidum by a combination of X-ray scattering, electron cryomicroscopy, and optical spectroscopy. At a low hexanol/pigment ratio, the lamellae persisted in the presence of hexanol while the short-range order and exciton interactions between chlorin rings were effectively eliminated, producing a monomer-like absorption. The result suggested that hexanol hydroxyls solvated the chlorin rings while the aliphatic tail partitioned into the hydrophobic part of the lamellar structure. This partitioning extended the chlorosome along its long axis. Further increase of the hexanol/pigment ratio produced round pigment-hexanol droplets, which lost all lamellar order. After hexanol removal the spectral properties were restored. In the samples treated under the high hexanol/pigment ratio, lamellae reassembled in small domains after hexanol removal while the shape and long-range order were irreversibly lost. Thus, all the interactions required for establishing the short-range order by self-assembly are provided by BChl c molecules alone. However, the long-range order and overall shape are imposed by an external structure, e.g., the proteinaceous chlorosome baseplate.

Aggregation of bacteriochlorophyll (BChl) c from chlorosomes, the main light-harvesting complex of green bacteria, has been studied in aqueous buffer. Unlike other chlorophyll-like molecules, BChl c is rather soluble in aqueous buffer, forming dimers. When BChl c is mixed with carotenoids (Car), the BChl c Qy transition is further redshifted, in respect to that of monomers and dimers. The results suggest that Car are incorporated in the aggregates and induce further aggregation of BChl c. The redshift of the BChl c Qy band is proportional to the Car concentration. In contrast, the mixture of bacteriochlorophyllide (BChlide) c, which lacks the nonpolar esterifying alcohol, does not form aggregates with Car in aqueous buffer or nonpolar solvents. Instead, the position of the BChlide c Qy transition remains unshifted in respect to that of the monomeric molecule, and Car precipitates with the course of time in aqueous buffer. Similar effects on both BChl c and BChlide c are also observed when monogalactosyl diglyceride (MGDG), which forms the monolayer envelope of chlorosomes, is used instead of (or together with) Car. The results show that the hydrophobic interactions of the BChl c esterifying alcohols with themselves and the nonpolar carbon skeleton of Car, or the fatty acid tails of MGDG, are essential driving forces for BChl aggregation in chlorosomes.

Multichannel flash spectroscopy (with microsecond time resolution) has been applied to carotenoid (Car)-containing and Car-less reaction centers (RC) of Rhodobacter sphaeroides with a view to investigate the interaction between the Car and its neighboring pigments at room temperature. Under neutral redox potential conditions, where the primary quinone acceptor (QA) is oxidized, the light-induced spectral changes in the 350-1000 nm region are attributed to the photochemical oxidation of the special pair (denoted here as P870), the generation of P870(+)QA(-), and the attendant electrochromism of adjacent chromophores. A bathochromic shift of <1 nm in the visible absorption region of Car reveals the sensitivity of Car to the P870 photooxidation. Under low redox potential conditions, where QA is reduced, P870 triplets (P870(+)) are formed. The time-resolved triplet-minus-singlet (TmS) spectrum of Car-less RC shows a deep bleaching at 870 nm, which belongs to P870(+), and additional (but smaller) bleaching at 800 nm; the entire spectrum decays at the same rate (with a lifetime of about 50 micros). The bleaching at 800 nm arises from the pigment interaction between P870(+) and the accessory bacteriochlorophylls on A and B branches (BA,B). In Car-containing RC, the TmS spectra of Car are accompanied by two smaller, negative signals--a sharp peak at 809 +/- 2 nm and a broad band at 870 nm--which decay at the same rate as the TmS spectrum of Car (ca 10 micros). The former is ascribed to the perturbation, by Car(+), of the absorption spectrum of BB; the latter, to the TmS spectrum of P870(+), a species that appears to be in approximate thermal equilibrium with Car(+). These assignments are consistent with the absorption-detected magnetic resonance spectra obtained by other workers at low temperatures.

The excited-state relaxation within bacteriochlorophyll (BChl) e and a in chlorosomes of Chlorobium phaeobacteroides has been studied by femtosecond transient absorption spectroscopy at room temperature. Singlet-singlet annihilation was observed to strongly influence both the isotropic and anisotropic decays. Pump intensities in the order of 1011 photons x pulse-1 x cm-2 were required to obtain annihilation-free conditions. The most important consequence of applied very low excitation doses is an observation of a subpicosecond process within the BChl e manifold (200–500 fs), manifesting itself as a rise in the red part of the Qy absorption band of the BChl e aggregates. The subsequent decay of the kinetics measured in the BChl e region and the corresponding rise in the baseplate BChl a is not single-exponential, and at least two components are necessary to fit the data, corresponding to several BChl eBChl a transfer steps. Under annihilation-free conditions, the anisotropic kinetics show a generally slow decay within the BChl e band (10–20 ps) whereas it decays more rapidly in the BChl a region (1 ps). Analysis of the experimental data gives a detailed picture of the overall time evolution of the energy relaxation and energy transfer processes within the chlorosome. The results are interpreted within an exciton model based on the proposed structure.

Previously, the spatial arrangement of the carotenoid and bacteriochlorophyll molecules in the peripheral light-harvesting (LH2) complex from Rhodopseudomonas acidophila strain 10050 has been determined at high resolution. Here, we have time resolved the energy transfer steps that occur between the carotenoid's initial excited state and the lowest energy group of bacteriochlorophyll molecules in LH2. These kinetic data, together with the existing structural information, lay the foundation for understanding the detailed mechanisms of energy transfer involved in this fundamental, early reaction in photosynthesis. Remarkably, energy transfer from the rhodopin glucoside S2 state, which has an intrinsic lifetime of ~120 fs, is by far the dominant pathway, with only a minor contribution from the longer-lived S1 state.

A laser in situ scattering and transmissometry (Lisst-100) probe has been used for estimating the particle-size distribution of phytopankton, purple photosynthetic sulphur bacteria (Chromatiaceae), and suspended inorganic sediments in different lakes. Results from Lisst-100 have been compared to laboratory measurements, such as those obtained by using a Galai laser size analyzer (GL), an optical microscope (OM), and a flow cytometer (FC). Although all of these instruments were shown to provide reliable values of the particle number concentration for the given populations, the Lisst-100 was the fastest and most reliable instrument because it did not require manipulation of the samples—which is not the case of GL, OM and FC instruments—and avoided the tedious procedure of microscopic counts. The total particle volume concentration results obtained with Lisst-100 differed from those obtained with GL for populations with large and porous aggregates, such as phytoplankton cells. The difference was attributed to the breakage of fragile algal aggregates resulting from the measuring procedure used by GL. Although for suspended sediment particles both instruments gave the same results for the total particle volume concentration, the particle-size distribution obtained with GL was found always shifted to smaller diameters than with Lisst-100, probably because inorganic sediment particles present compact aggregates. When these aggregates break, they split into a high number of small particles that contribute the same to the total volume concentration as the previous aggregates. Finally, results of the total particle volume concentration with Lisst-100 were in accordance with those obtained with GL for the Chromatiaceae population, because cells remained in a dispersed phase. A good correlation was found between the total particle volume concentration of Chromatiaceae measured with Lisst-100 and the concentration of bacteriochlorophyll a (BChl a), which is the parameter habitua

Previously, the spatial arrangement of the carotenoid and bacteriochlorophyll molecules in the peripheral light-harvesting (LH2) complex from Rhodopseudomonas acidophila strain 10050 has been determined at high resolution. Here, we have time resolved the energy transfer steps that occur between the carotenoid's initial excited state and the lowest energy group of bacteriochlorophyll molecules in LH2. These kinetic data, together with the existing structural information, lay the foundation for understanding the detailed mechanisms of energy transfer involved in this fundamental, early reaction in photosynthesis. Remarkably, energy transfer from the rhodopin glucoside S2 state, which has an intrinsic lifetime of ~120 fs, is by far the dominant pathway, with only a minor contribution from the longer-lived S1 state.

We have studied the effect of the absence of carotenoids on the organization of bacteriochlorophylls (BChls) in chlorosomes of Chlorobium (Chl.) phaeobacteroides strain CL1401. Carotenoid-depleted chlorosomes were obtained by means of 2-hydroxybiphenyl–supplemented cultures. In the presence of the inhibitor, isorenieratene (Isr) and β-Isr biosynthesis were inhibited to more than 95%, leading to an accumulation of the colorless precursor phytoene inside the chlorosomes. In addition, there was a 30–40% decrease in the baseplate BChl a content. The absorption spectrum of the carotenoid-depleted chlorosomes showed a 10 nm blue shift in the BChl e Qy absorption peak. Under reducing conditions, a decrease in the BChl a/BChl e fluorescence emission ratio was observed in carotenoid-depleted chlorosomes relative to that in control chlorosomes, caused mainly by the decrease in the BChl a content. The steady-state fluorescence emission anisotropy in the BChl e region dropped from 0.24 for native chlorosomes to 0.14 for carotenoid-depleted ones, indicating reorganization of BChl e. The circular dichroism (CD) signal of the carotenoid-depleted chlorosomes was increased two times in the BChl e Qy region. A simple model based on the structure proposed was used to explain the observed effects. Carotenoids might affect the angle between the direction of the BChl e Qy transition and the axis of the rod. The orientation of BChl a in the baseplate remains unchanged in carotenoid-depleted chlorosomes, although there is a partial loss of BChl a as a consequence of a decrease in the baseplate size. The carotenoids are most likely rather close to the BChls and appear to be important for the aggregate structure in Chl. phaeobacteroides.

The major light-absorbing pigments in purple photosynthetic bacteria are the bacteriochlorophylls (a and b) (BChl) and the carotenoids. These pigments are noncovalently attached to two types of integral membrane protein forming the reaction centers and the light-harvesting or antenna complexes (Hawthornthwaite and Cogdell, 1991; Hunter, 1995; Zuber and Cogdell, 1995). Photosynthesis in purple bacteria usually begins with the absorption of a photon in the light-harvesting system. The absorbed energy is then rapidly (in less than ~100 ps) and efficiently transferred to the reaction center (~95% quantum efficiency). In the reaction center this energy is used to drive the initial charge separation reaction and the energy is then "trapped" (Feher and Okamura, 1978; Feher et al., 1989; Deisenhofer et al., 1995). The combination of antenna complexes with a reaction center constitutes the photosynthetic unit (PSU). For most commonly studied purple bacteria the number of PSUs per cell and their size are variable. Depending on such factors as the light-intensity at which cells are grown, the size of the PSU can vary from about 30 BChls per reaction center up to 200-300 BChls per reaction center (Aagaard and Sistrom, 1972; Drews, 1985). This arrangement of reaction centers surrounded by an antenna system ensures that each reaction center is kept well supplied with incoming solar energy and effectively acts to increase their cross-sectional area for photon capture. It is interesting to note that in most species the same pigments are found in both reaction centers and antenna complexes, and it is the protein that determines which function a given pigment is destined to fulfil., When BChl a is dissolved in an organic solvent such as 7:2 v/v acetone:methanol its NIR absorption band is located at 772 nm. This is the typical Qy absorption band of monomeric BChl a. However, when the BChl a is non-covalently bound into an antenna complex, this NIR absorption band is red shifted between 800-940 nm, depending on the species (Fig. 1) (Thornber et al., 1978; Hawthornthwaite and Cogdell, 1991). In most species this red shift is associated with an increase in spectral complexity, with several peaks/shoulders clearly visible in the in vivo absorption spectrum. This red shift arises from pigment-pigment and pigment-protein interactions within the antenna complexes and is regularly used to both identify them and judge their integrity., Since they are integral membrane proteins, the isolation of a purple bacterial antenna complex begins with the solubilization of the photosynthetic membrane with a suitable detergent (Cogdell and Thorber, 1979; Hawthornthwaite and Cogdell, 1991). Very often the solubilized complexes are then initially fractionated by sucrose gradient centrifugation (Fig. 2). In most species this fractionation reveals two types of antenna complex, called LH1 and LH2. LH1 forms the so called "core" complex. It is closely associated with the reaction center and forms a stoichiometric complex with it (usually ~32 BChls per reaction center (Gall, 1995; Karrasch et al., 1995; Zuber and Cogdell, 1995). LH2, also sometimes called the "variable" or "peripheral" antenna complex, is the topic for the remainder of this review. Readers who want to obtain more information on the overall subject of the structure and function of the bacterial PSU should consult the following general reviews (Somsen et al., 1993; Blankenship et al., 1995; Loach and Parkes-Loach, 1995; Cogdell et al., 1996; Papiz et al., 1996).

Copper is an essential micronutrient for higher plants and algae and has a direct impact on photosynthesis. It is a constituent of the primary electron donor in photosystem I, the Cu-protein plastocyanin. Many authors have also described Cu as a constituent of photosystem II (PSII). However, high Cu concentrations inhibit the photosynthetic electron transport, especially in PSII. In addition, both Cu deficiency and Cu toxicity interfere with pigment and lipid biosynthesis and, consequently, with chloroplast ultrastructure thus negatively influencing the photosynthetic efficiency.

El Cu es un micronutriente esencial para el crecimiento y desarrollo de las plantas superiores, observándose que pequeñas variaciones en su concentración óptima en el medio producen graves alteraciones en el proceso fotosintético. Esta Tesis Doctoral se ha centrado principalmente en el complejo del fotosistema II, integrado en la membrana tilacoidal del cloroplasto, y que se caracteriza por ser el complejo mas sensible del transporte electrónico a situaciones adversas, a la vez que puede generar mayores mecanismos de defensa. En nuestro estudio hemos puesto de manifiesto que el Cu no es un elemento estructural del fotosistema II, habiendo conseguido previamente para ello una nueva preparación de dicho fotosistema con un mayor grado de pureza. Para determinar su efecto tóxico ha sido necesario el uso de técnicas biofísicas que han permitido determinar que el Cu bloquea el transporte electrónico entre la Tirosina Z y el centro de reacción P680 y que, como consecuencia, un nuevo donador reduce al P680.

In conventional photosystem II preparation high amounts of Cu are found. After fractionation by centrifugation, Cu can be completely removed from photosystem II without affecting either its photosynthetic activity or the composition of its specific proteins. We could demonstrate that the Cu was associated with nuclear contaminants in the starch fraction. Among the contaminants, several histones were identified by specific antisera and by N-terminal sequencing. In order to obtain homogeneous BBY preparations of PSII a procedure is employed that involves a 10000 g centrifugation step and which eliminates non-specifically bound metals, nucleic acids and histones with the starch pellet. The resulting starch-free BBY (BBYs-), which is free of these nuclear contaminants, is an appropriate preparation for biophysical studies or for those of metal interactions with PSII.

Measurements of flash-induced absorption changes at 325, 436, and 830 nm and of oxygen evolution were performed in order to analyze in detail the inhibition of photosystem II (PS II) by Cu(II) in PS II membrane fragments from spinach. (a) The kinetics of P680+ reduction become markedly slower in the presence of 100 microM CuSO4. (b) The CuSO4- induced kinetics of P680+ reduction are dominated by a 140-160- microsecond decay. (c) The extent of these 140-160-microsecond kinetics, normalized to the overall decay, remains virtually unaffected by addition of the exogenous PS II donor, NH2OH. (d) In thoroughly dark- adapted samples the CuSO4-induced 140-160-microsecond kinetics are already observed after the first flash and remain unchanged by a train of excitation flashes. (e) The extent of P680+ and QA- formation under repetitive flash excitation is not diminished by addition of 100 microM CuSO4. (f) The induction of microsecond kinetics of P680+ reduction at the expense of ns kinetics and the inhibition of the saturation rate of oxygen evolution exhibit the same dependence on CuSO4 concentration. (g) CuSO4 also transforms the 10-20-microsecond reduction of P680+ by TyrZ in Tris-washed PS II membrane fragments into 140-160-microsecond kinetics without any effect on the extent of flash-induced P680+ formation. These results unambiguously show that Cu(II) does not affect the charge separation (P680+QA-), but instead specifically modifies TyrZ and/or its micro environment so that the electron transfer to P680+ becomes blocked.

The determination of total Cu is not often correlated with states of deficiency in plant material. This fact makes it necessary to look for biologically active Cu. Suspensions of thylakoid membranes and photosystem II particles, properly diluted with 13 mM nitric acid, were used for this purpose. The presence of a minute quantity of an antifoaming agent, such as 1-octanol, is essential when an aliquot of the slurry is injected into the graphite furnace of the atomic absorption spectrophotometer. Good agreement was obtained between our results and those obtained by a classical dry combustion method. Reproducibility was better than 5% when expressed as relative standard deviation.