Comparison of bacterial reaction centers and photosystem II

In photosynthetic organisms, the utilization of solar energy to drive electron and proton transfer reactions across membranes is performed by pigment-protein complexes including bacterial reaction centers (BRCs) and photosystem II. The well-characterized BRC has served as a structural and functional model for the evolutionarily-related photosystem II for many years. Even though these complexes transfer electrons and protons across cell membranes in analogous manners, they utilize different secondary electron donors. Photosystem II has the unique ability to abstract electrons from water, while BRCs use molecules with much lower potentials as electron donors. This article compares the two complexes and reviews the factors that give rise to the functional differences. Also discussed are the modifications that have been performed on BRCs so that they perform reactions, such as amino acid and metal oxidation, which occur in photosystem II.     

A general model for regulation of photosynthetic unit function by protein phosphorylation

We propose that regulatory effects of membrane protein phosphorylation in photosynthetic systems result in all cases from simultaneous phosphorylation by a single kinase of the polypeptides of two intrinsic pigment-protein complexes, with phosphorylation leading to their mutual electrostatic repulsion in a direction parallel to the membrane plane and therefore to decreased excitation energy transfer between them. One complex is a peripheral light-harvesting complex and the other is bound to the reaction centre and functions as a link in excitation energy transfer. Immediate effects of phosphorylation are therefore decreased absorption cross-section together with decreased cooperativity of photosynthetic units. This general model applies equally to photosystem II of green plants, algae and cyanobacteria, as well as to the single photosystem of purple bacteria. Special cases of this general model permit increased excitation energy transfer to one type of reaction centre at the expense of another, and this may occur even in laterally homogeneous membranes that are uniformly unappressed.     

Comparative study of B890 pigment-lipoprotein complexes from sulfur (Chromatium minutissimum) and non-sulfur (Rhodopseudomonas palustris) purple photosynthesizing bacteria]

Pigment-lipoprotein B890 complexes containing reaction center and "light-focusing" bacteriochlorophyll a were isolated from photosynthetic membranes of sulfur (Chromatium minutissimum) and non-sulfur (Rhodopseudomonas palustris) purple bacteria after the treatment with Triton X-100. The molecular weights of complexes were evaluated using several methods (200 000-300 000). By means of electron microscopy the sizes of complexes were found to be about 80 A. On the air-water interface hexagonal packing of complexes was observed. The chemical compositions of complexes are very similar except bacteriochlorophyll a whose specific content is somewhat higher in Chromatium minutissimum. The protein composition of complexes was studied and the molecular weights of proteins were estimated by SDS-gel electrophoresis. The results obtained show significant similarities in molecular organization of B890 complexes isolated from sulfur (Chromatium minutissimum) and non-sulfur (Rhodopseudomonas palustris) purple bacteria.     

Effect of modification of histidine residues on organization of the pigment--protein complexes of chromatium minutissimum

The effect of diethyl pyrocarbonate on chromatophores and isolated pigment--protein complexes of Chromatium minutissimum was studied. It is shown that modification of histidine residues results in the destruction of the core antenna LHI (B880) and in a spectral shift from 850 to 830 nm in the peripheral antenna LHII (B800-850). In the purple sulfur bacterium Chromatium minutissimum the pigment--protein complexes B800-B850 (peripheral antenna, LHII) and B880 (core antenna, LHI) collect and transmit the absorbed light energy to the reaction centers. The composition of pigments and proteins as well as primary structure of the majority of polypeptides in both types of complexes from various photosynthetic bacteria have been determined.     

Excitation energy transfer kinetics and efficiency in phototrophic green sulfur bacteria

A series of spectroscopic measurements were performed on membrane fractions and detergent-solubilized complexes from the green sulfur bacterium (GSB) Chlorobaculum (Cba.) tepidum. The excitation migration through the entire GSB photosynthetic apparatus cannot be observed upon excitation of membranes in the chlorosome region at 77?K. In order to observe energy transfer from the Fenna-Matthews-Olson (FMO) protein to the reaction center (RC), FMO was directly excited at ~800?nm in transient absorption experiments. However, interpretation of the results is complicated by the spectral overlap between FMO and the RC. The availability of the Y16F FMO mutant, whose absorption spectrum is drastically different from that of the WT, has enabled the selection of spectral regions where either only FMO or the RC contributes. The application of a directed kinetic modeling approach, or target analysis, revealed the various decay and energy transfer pathways within the pigment-protein complexes. The calculated FMO-to-RC excitation energy transfer efficiencies are approximately 25% and 48% for the Y16F and WT samples, respectively.     

Energy trapping and detrapping by wild type and mutant reaction centers of purple non-sulfur bacteria

Time-correlated single photon counting was used to study energy trapping and detrapping kinetics at 295 K in Rhodobacter sphaeroides chromatophore membranes containing mutant reaction centers. The mutant reaction centers were expressed in a background strain of Rb. sphaeroides which contained only B880 antenna complexes and no B800-850 antenna complexes. The excited state decay times in the isolated reaction centers from these strains were previously shown to vary by roughly 15-fold, from 3.4 to 52 ps, due to differences in the charge separation rates in the different mutants (Allen and Williams (1995) J Bioenerg Biomembr 27: 275–283). In this study, measurements were also performed on wild type Rhodospirillum rubrum and Rb. sphaeroides B880 antenna-only mutant chromatophores for comparison. The emission kinetics in membranes containing mutant reaction centers was complex. The experimental data were analyzed in terms of a kinetic model that involved fast excitation migration between antenna complexes followed by reversible energy transfer to the reaction center and charge separation. Three emission time constants were identified by fitting the data to a sum of exponential decay components. They were assigned to trapping/quenching of antenna excitations by the reaction center, recombination of the P⁺H^– charge-separated state of the reaction center reforming an emitting state, and emission from uncoupled antenna pigment-protein complexes. The first varied from 60 to 160 ps, depending on the reaction center mutation; the second was 200–300 ps, and the third was about 700 ps. The observed weak linear dependence of the trapping time on the primary charge separation time, together with the known sub-picosecond exciton migration time within the antenna, supports the concept that it is energy transfer from the antenna to the reaction center, rather than charge separation, that limits the overall energy trapping time in wild type chromatophores. The component due to charge recombination reforming the excited state is minor in wild type membranes, but increases substantially in mutants due to the decreasing free energy gap between the states P^* and P⁺H^–.Abbreviations PSU photosynthetic unit - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - P reaction center primary electron donor - RC reaction center - Rb. Rhodobacter - Rs. Rhodospirillum - EDTA (ethylenediamine)tetraacetic acid - Tris tris(hydroxymethyl)aminomethane Author for correspondence     

Kinetics and spectra of photo-induced changes in the absorption of pigment-protein complexes of photosystem 1 in a picosecond range

The energy transfer from the light-harvesting antenna chlorophylls to the reaction center molecules and subsequent charge separation were investigated using a difference picosecond spectrophotometer with selective excitation. The objects were the pigment-protein complexes of photosystem 1 (Chl/P700 = 60) isolated from bean leaves. The difference absorption spectra of the excited states of light-harvesting antenna chlorophylls and the P700 photooxidation were measured. It was shown that the excited states of antenna chlorophylls were generated within 10 ps and deactivated with three-component kinetics: tau 1 = 20--45 ps, tau 2 = 100--300 ps, tau 3 greater than 500 ps. The process of the P700 photooxidation induced by the 650 nm exciting pulse was approximately monoexponential with tau equal to 15--30 ps. It is established that the P700 photooxidation is due to the efficient transfer of excitation energy from antenna chlorophylls to reaction centers.     

Excitation and Emission Spectroscopy of Membranes and Pigment-protein Complexes of an Aerobic Photosynthetic Bacterium, Erythrobacter sp. OCh 114

Emission and excitation spectra of steady-state fluorescencefrom membranes and isolated pigment-protein complexes of anaerobic photosynthetic bacterium, Erythrobacter sp. strain OCh114 indicated high efficiency of energy transfer from Bchl 806to Bchl 870 and from carotenoids to bacteriochlorophyll. Thus,this bacterium has a highly efficient light-harvesting systemtypical of photosynthetic bacteria. (Received August 3, 1989; Accepted January 27, 1990)     

Difference picosecond spectroscopy of pigment-protein complexes of photosystem I from higher plants

Using a difference picosecond spectrophotometer with a time resolution of 10 ps, we investigated excitation energy transfer and charge separation in pigment-protein complexes of Photosystem I from bean leaves (chlorophyll/P-700 = 60). Under 20 ps excitation at 650 or 667 nm, the difference absorption spectra in the spectral region 600–720 nm were measured. They are associated with transition of antenna chlorophylls into singlet excited states and P-700 photooxidation. It was shown that the excited states in the whole inhomogeneous antenna were generated within 10 ps and deactivated with three-component kinetics, the t_1/e values being 20–45, 100–300 and over 500 ps. Formation of P-700⁺ has a rise time of 15–30 ps. The fast component of the depletion of the antenna excited states is suggested to be due to transfer of excitation energy from antenna pigments to reaction centers and its trapping. The kinetics of the fast component is independent of excitation energy and a redox state of P-700.     

Magnetic field effects on the fluorescence of mutant strains of Rhodopseudomonas capsulata

The magnetic field effects on bacteriochlorophyll fluorescence in six strains of Rhodopseudomonas capsulata were investigated. All strains exhibit an increase in fluorescence upon application of a magnetic field. Large magnetic field effects are shown to arise in mutants which contain the B800–850 complex as the only bacteriochlorophyll-containing protein. These fluorescence increases are observed only with carotenoid excitation and are best described by a carotenoid singlet heterofission mechanism. Variations in the magnitudes of the magnetic field effects for the Rps. capsulata strain arise from energy differences in the excited states of the molecules involved in the process. In order to determine the contribution from reaction centers to the magnetic field effects observed in the mutants which contain all three pigment-protein complexes, reaction centers were isolated from these strains. The reaction center contribution to the magnetic field effect on fluorescence in whole cells was determined to be smaller than the antenna contribution when carotenoid excitation was employed.     

Energy transfer and fluorescence mechanisms in photosynthetic membranes

Energy transfer in photosynthetic membranes involves the migration of excitons from light‐harvesting antenna chlorophyll‐protein complexes to the reaction center complexes. Recent efforts have focused on determining the time of arrival of excitons (trapping times) at the reaction centers following excitation with a single picosecond laser pulse. Three different approaches have been utilized: (1) determination of appearance of separated charges within the reaction centers by differential absorbtion spectroscopy, (2) determination of appearance of separated charges by fast photoemf measurements, and (3) kinetics of decay of fluorescence. The first two methods provide more direct information on exciton trapping by reaction centers than fluorescence methods, but are experimentally difficult to realize. Therefore, much activity has centered around the accurate measurement and analysis of fluorescence‐decay profiles by single‐photon counting methods. In green plants, about three different components with lifetimes of about 100 psec, 200 to 500 psec, and >1 nsec, have been reported. The first two components are believed to be related to trapping rates by reaction centers, while the third component is attributed to a charge recombination (Klimov) mechanism. Results from photoemf and exciton‐exciton annihilation experiments are consistent with the interpretation that the first decay component reflects exciton‐trapping rates. A critical analysis and discussion of these fast energy‐transfer phenomena in photosynthetic membranes of green plants are offered in this review.     

In vivo states and functions of carotenoids in an aerobic photosynthetic bacterium,Erythrobacter longus

In vivo states and functions of carotenoids in the membranes and the isolated RC-B865 pigment-protein complexes from an aerobic photosynthetic bacterium, Erythrobacter longus, are investigated by means of fluorescence excitation and resonance Raman (RR) spectra. Erythroxanthin sulfate, a dominant carotenoid species in the membranes (>70%), is found not to transfer the absorbed light energy to bacteriochlorophyll (Bchl), and its RR spectra are similar between the in vivo and in vitro states. These observations indicate that erythroxanthin sulfate does not interact with either Bchl or proteins in the membranes, and suggest that its function may be limited to photoprotection by quenching the harmful singlet oxygen. On the other hand, two other carotenoid species contained in the isolated RC-B865 complexes, zeaxanthin and bacteriorubixanthinal, have a high efficiency of energy transfer to Bchl (88±5%). The RR spectra of these two carotenoids, each of which can be selectively obtained by choosing the excitation wavelength, show some characteristics of interactions with proteins or Bchl.Abbreviations Bchl bacteriochlorophyll a - FWHM full width at half maximum - PAGE polyacrylamide gel electrophoresis - RC reaction center - RR resonance Raman - SDS sodium dodecyl sulfate     

Changes in the composition of the photosynthetic apparatus in the galactolipid-deficient dgd1 mutant of Arabidopsis thaliana.

The glycerolipid digalactosyl diacylglycerol (DGDG) is exclusively associated with photosynthetic membranes and thus may play a role in the proper assembly and maintenance of the photosynthetic apparatus. Here we employ a genetic approach based on the dgd1 mutant of Arabidopsis thaliana to investigate the function of DGDG in thylakoid membranes. The primary defect in the genetically well-characterized dgd1 mutant resulted in a 90% reduction of the DGDG content. The mutant showed a decreased photosystem II (PSII) to photosystem I ratio. In vivo room- and low-temperature (77 K) chlorophyll fluorescence measurements with thylakoid preparations are in agreement with a drastically altered excitation energy allocation to the reaction centers. Quantification of pigment-binding apoproteins and pigments supports an altered stoichiometry of individual pigment-protein complexes in the mutant. Most strikingly, an increase in the amount of peripheral light-harvesting complexes of PSII relative to the inner antenna complexes and the PSII reaction center/core complexes was observed. Regardless of the severe alterations in thylakoid organization, photosynthetic oxygen evolution was virtually not compromised in dgd1 mutant leaves.     

Structural and functional alterations of cyanobacterial phycobilisomes induced by high-light stress

Exposure of cyanobacterial or red algal cells to high light has been proposed to lead to excitonic decoupling of the phycobilisome antennae (PBSs) from the reaction centers. Here we show that excitonic decoupling of PBSs of Synechocystis sp. PCC 6803 is induced by strong light at wavelengths that excite either phycobilin or chlorophyll pigments. We further show that decoupling is generally followed by disassembly of the antenna complexes and/or their detachment from the thylakoid membrane. Based on a previously proposed mechanism, we suggest that local heat transients generated in the PBSs by non-radiative energy dissipation lead to alterations in thermo-labile elements, likely in certain rod and core linker polypeptides. These alterations disrupt the transfer of excitation energy within and from the PBSs and destabilize the antenna complexes and/or promote their dissociation from the reaction centers and from the thylakoid membranes. Possible implications of the aforementioned alterations to adaptation of cyanobacteria to light and other environmental stresses are discussed.     

Pathways for energy transfer in the core light-harvesting complexes CP43 and CP47 of photosystem II

The pigment-protein complexes CP43 and CP47 transfer excitation energy from the peripheral antenna of photosystem II toward the photochemical reaction center. We measured the excitation dynamics of the chlorophylls in isolated CP43 and CP47 complexes at 77 K by time-resolved absorbance-difference and fluorescence spectroscopy. The spectral relaxation appeared to occur with rates of 0.2-0.4 ps and 2-3 ps in both complexes, whereas an additional relaxation of 17 ps was observed only in CP47. Using the 3.8-A crystal structure of the photosystem II core complex from Synechococcus elongatus (A. Zouni, H.-T. Witt, J. Kern, P. Fromme, N. Krauss, W. Saenger, and P. Orth, 2001, Nature, 409:739-743), excitation energy transfer kinetics were calculated and a Monte Carlo simulation of the absorption spectra was performed. In both complexes, the rate of 0.2-0.4 ps can be ascribed to excitation energy transfer within a layer of chlorophylls near the stromal side of the membrane, and the slower 2-3-ps process to excitation energy transfer to the calculated lowest excitonic state. We conclude that excitation energy transfer within CP43 and CP47 is fast and does not contribute significantly to the well-known slow trapping of excitation energy in photosystem II.     

Energy transfer in purple bacterial photosynthetic units from cells grown in various light intensities

Three photosynthetic membranes, called intra-cytoplasmic membranes (ICMs), from wild-type and the ?pucBA_abce mutant of the purple phototrophic bacterium Rps. palustris were investigated using optical spectroscopy. The ICMs contain identical light-harvesting complex 1–reaction centers (LH1–RC) but have various spectral forms of light-harvesting complex 2 (LH2). Spectroscopic studies involving steady-state absorption, fluorescence, and femtosecond time-resolved absorption at room temperature and at 77 K focused on inter-protein excitation energy transfer. The studies investigated how energy transfer is affected by altered spectral features of the LH2 complexes as those develop under growth at different light conditions. The study shows that LH1 → LH2 excitation energy transfer is strongly affected if the LH2 complex alters its spectroscopic signature. The LH1 → LH2 excitation energy transfer rate modeled with the Förster mechanism and kinetic simulations of transient absorption of the ICMs demonstrated that the transfer rate will be 2–3 times larger for ICMs accumulating LH2 complexes with the classical B800–850 spectral signature (grown in high light) compared to the ICMs from the same strain grown in low light. For the ICMs from the ?pucBA_abce mutant, in which the B850 band of the LH2 complex is blue-shifted and almost degenerate with the B800 band, the LH1 → LH2 excitation energy transfer was not observed nor predicted by calculations.     

Energy transfer and bacteriochlorophyll fluorescence in purple bacteria at low temperature

Emission spectra of bacteriochlorophyll a fluorescence and absorption spectra of various purple bacteria were measured at temperatures between 295 and 4 K. For Rhodospirillum rubrum the relative yield of photochemistry was measured in the same temperature region. In agreement with earlier results, sharpening and shifts of absorption bands were observed upon cooling to 77 K. Below 77 K further sharpening occurred. In all species an absorption band was observed at 751-757 nm. The position of this band and its amplitude relative to the concentration of reaction centers indicate that this band is due to reaction center bacteriopheophytin. The main infrared absorption band of Rhodopseudomonas sphaeroides strain R26 is resolved in two bands at low temperature, which may suggest that there are two pigment-protein complexes in this species. Emission bands, like the absorption bands, shifted and sharpened upon cooling. The fluorescence yield remained constant or even decreased in some species between room temperature and 120 K, but showed an increased below 120 K. This increase was most pronounced in species, such as R. rubrum, which showed single banded emission spectra. In Chromatium vinosum three (835, 893 and 934 nm) and in Rps. sphaeroides two (888 and 909 nm) emission bands were observed at low temperature. The temperature dependence of the amplitudes of the short wavelength bands indicated the absence of a thermal equilibrium for the excitation energy distribution in C. vinosum and Rps. sphaeroides. In all species the increased in the yield was larger when all reaction centers were photochemically active than when the reaction centers were closed. In R. rubrum the increase in the fluorescence yield was accompanied by a decrease of the quantum yield of charge separation upon excitation of the antenna but not of the reaction center chlorophyll. Calculation of the F?rster resonance integral at various temperatures indicated that the increase in fluorescence yield and the decrease in the yield of photochemistry may be due to a decrease in the rate of energy transfer between antenna bacteriochlorophyll molecules. The energy transfer from carotenoids to bacteriochlorophyll was independent of the temperature in all species examined. The results are discussed in terms of existing models for energy transfer in the antenna pigment system.     

Effect of the in situ electrochemical oxidation on the pigment-protein arrangement and energy transfer in light-harvesting complex from Rhodobacter sphaeroides 601

The oxidation of bacteriochlorophylls (BChls) in peripheral light-harvesting complexes (LH2) from Rhodobacter sphaeroides was investigated by spectroelectrochemistry of absorption, fluorescence emission, and femtosecond (fs) pump-probe, with the aim obtaining information about the effect of in situ electrochemical oxidation on the pigment-protein arrangement and energy transfer within LH2. The experimental results revealed that: (a) the generation of the BChl radical cation in both B800 and B850 rings dramatically induced bleaching of the characteristic absorption in the NIR region and quenching of the fluorescence emission from the B850 ring for the electrochemical oxidized LH2; (b) the BChl-B850 radical cation might act as an additional channel to compete with the unoxidized BChl-B850 molecules for rapidly releasing the excitation energy, however the B800-B850 energy transfer rate remained almost unchanged during the oxidation process.     

Quantum yield and rate of formation of the carotenoid triplet state in photosynthetic structures

The formation of the triplet state of carotenoids (detected by an absorption peak at 515 nm) and the photo-oxidation of the primary donor of Photosystem II, P-680 (detected by an absorption increase at 820 nm) have been measured by flash absorption spectroscopy in chloroplasts in which the oxygen evolution was inhibited by treatment with Tris. The amount of each transient form has been followed versus excitation flash intensity (at 590 or 694 nm). At low excitation energy the quantum yield of triplet formation (with the Photosystem II reaction center in the state Q⁻) is about 30% that of P-680 photo-oxidation. The yield of carotenoid triplet formation is higher in the state Q⁻ than in the state Q, in nearly the same proportion as chlorophyll a fluorescence. It is concluded that, for excited chlorophyll a, the relative rates of intersystem crossing to the triplet state and of fluorescence emission are the same in vivo as in organic solvent. At high flash intensity the signal of P-680⁺ completely saturates, whereas that of carotenoid triplet continues to increase.

The rate of triplet-triplet energy transfer from chlorophyll a to carotenoids has been derived from the rise time of the absorption change at 515 nm, in chloroplasts and in several light-harvesting pigment-protein complexes. In all cases the rate is very high, around 8 · 10⁷ s⁻¹ at 294 K. It is about 2–3 times slower at 5 K. The transitory formation of chlorophyll triplet has been verified in two pigment-protein complexes, at 5 K.