Defining the Far-red Limit of Photosystem I: THE PRIMARY CHARGE SEPARATION IS FUNCTIONAL TO 840 nm*

The far-red limit of photosystem I (PS I) photochemistry was studied by EPR spectroscopy using laser flashes between 730 and 850 nm. In manganese-depleted spinach thylakoid membranes, the primary donor in PS I, P₇₀₀, was oxidized simultaneously with tyrosine Z, the secondary donor in PS II. It was found that at 295 K PS I photochemistry, observed as P₇₀₀⁺ formation, was functional up to 840 nm. This is 30 nm further to the red region than was reported for PS II photochemistry (Thapper, A., Mamedov, F., Mokvist, F., Hammarström, L., and Styring, S. (2009) Plant Cell 21, 2391–2401). The same far-red limit for the P₇₀₀⁺ formation was observed in a PS I reaction center core preparation from Nostoc punctiforme. The reduction of the acceptor side of PS I, observed as reduction of the iron-sulfur centers F_A and F_B by low temperature EPR measurements, was also functional at 15 K with light up to >830 nm. Taken together, these results, obtained from both plants and cyanobacteria, most likely rule out involvement of the red-absorbing antenna chlorophylls in this reaction. Instead we propose the existence of weak charge transfer bands absorbing in the far-red region in the ensemble of excitonically coupled chlorophyll a molecules around P₇₀₀ similar to what has been found in the reaction center of PS II. These charge transfer bands could be responsible for the far-red light absorption leading to PS I photochemistry at wavelengths up to 840 nm.     

Far-red photosynthesis: Two charge separation pathways exist in plant Photosystem II reaction center

An alternative charge separation pathway in Photosystem II under the far-red light was proposed by us on the basis of electron transfer properties at 295 K and 5 K. Here we extend these studies to the temperature range of 77–295 K with help of electron paramagnetic resonance spectroscopy. Induction of the S₂ state multiline signal, oxidation of Cytochrome b₅₅₉ and Chlorophyll_Z was studied in Photosystem II membrane preparations from spinach after application of a laser flashes in visible (532 nm) or far-red (730–750 nm) spectral regions. Temperature dependence of the S₂ state signal induction after single flash at 730–750 nm (T_inhibition ~ 240 K) was found to be different than that at 532 nm (T_inhibition ~ 157 K). No contaminant oxidation of the secondary electron donors cytochrome b₅₅₉ or chlorophyll_Z was observed. Photoaccumulation experiments with extensive flashing at 77 K showed similar results, with no or very little induction of the secondary electron donors. Thus, the partition ratio defined as (yield of Y_Z/CaMn₄O₅-cluster oxidation):(yield of Cytb₅₅₉/Chl_Z/Car_D2 oxidation) was found to be 0.4 at under visible light and 1.7 at under far-red light at 77 K. Our data indicate that different products of charge separation after far-red light exists in the wide temperature range which further support the model of the different primary photochemistry in Photosystem II with localization of hole on the Chl_D1 molecule.     

Flash photolysis-electron spin resonance study of the effect of o-phenanthroline and temperature on the decay time of the ESR signal B1 in reaction-center preparations and chromatophores of mutant and wild strains of Rhodopseudomonas spheroides and Rhodospirillum rubrum

We have studied the effect of o-phenanthroline and temperature on the decay rate of Signal B1 in reaction-center preparations and in chromatophores from Rhodopseudomonas spheroides and Rhodospirillum rubrum. We have shown that o-phenanthroline binds specifically to the reaction center protein (the binding center is probably at the iron) and when so bound inhibits the transfer of electrons from primary to secondary acceptors. We have also shown that the direct return decay time (A^? → P865⁺) increases with increasing temperature above approx. 150 K. This phenomenon has been interpreted within a quantum mechanical tunnelling model in which the distance of closest approach between P865⁺ and A^? increases about 2 Å between approx. 150 and 300 K.     

A possible new mechanism of temperature dependence of electron transfer in photosynthetic systems

A new theory for the electron transfer by the non-adiabatic process is formulated taking into account the origin shift and the frequency change of the vibration. The resultant formulas are quite similar to those of Jortner (Jortner, J. (1976) J. Chem. Phys. 64, 4860–4867) except that the free energy gap ΔG is used instead of the energy gap ΔE. By applying this theory to the photosynthetic electron transfer, the role of the remarkable temperature dependence of the electron transfer from cytochrome to P⁺ in Chromatium vinosum and the experimental data were reproduced very well using a small value of the coupling strength in contrast with the previous theory. This implies that proteins play a role to exclude many of the solvent molecules from the region of the electron transfer reaction between the donor and acceptor molecules. The negative activation process in the back electron transfer from Q^?_A to P⁺, the very slow back electron transfer from I^? to P⁺ and the solvent isotope effect on the cytochrome oxidation are also successfully explained by this new theory. It is shown that even a qualitative conclusion as to the molecular parameters obtained from the temperature dependence of the electron transfer is different between the present theory and that of Jortner.     

Specificity of the transhydrogenase factor for chromatophores of Rhodopseudomonas spheroides and Rhodospirillum rubrum

Extensive washing of chromatophores of Rhodospirillum rubrum and Rhodopseudomonas spheroides with dilute buffer results in a complete loss of the energylinked transhydrogenase activities of Rsp. rubrum but only a partial loss of the light-driven reaction in chromatophores of Rps. spheroides. It was not possible to reactivate the Rps. spheroides transhydrogenation with the Rsp. rubrum transhydrogenase factor nor with a protein fraction of Rps. spheroides isolated by procedures identical to that used for the isolation of the Rsp. rubrum transhydrogenase factor. The Rsp. rubrum factor is highly specific and cannot be replaced by a number of sulfhydryl compounds tested for reconstitution of Rsp. rubrum transhydrogenation. A published procedure for the isolation of a “transhydrogenase factor” from Rps. spheroides chromatophores yields a preparation having energy-dependent transhydrogenation when supplemented with dithiothreitol in the absence of added chromatophores.     

The primary charge separation,cytochrome oxidation and triplet formation in preparations from the green photosynthetic bacterium Prosthecochloris aestuarii

Flash-induced absorbance changes were measured in intact cells and subcellular preparations of the green photosynthetic bacterium Prosthecochloris aestuarii. In Complex I, a membrane vesicle preparation, photooxidation of the primary electron donor, P-840, and of cytochrome c-553 was observed. Flash excitation of the photosystem pigment complex caused in addition the generation of a bacteriochlorophyll a triplet. Triplet formation was the only reaction observed after flash excitation in the reaction center pigment -protein complex. The triplet had a lifetime of 90 μs at 295 K and of 165 μs at 120 K. The amount of triplet formed in a flash increased upon cooling from 295 to 120 K from 0.2 and 0.5 per reaction center to 0.45 and nearly 1 per reaction center in the photosystem pigment and reaction center pigment-protein complex, respectively. Measurements of absorbance changes in the near infrared in the reaction center pigment-protein complex indicate that the triplet is formed in the reaction center and that the reaction center bacteriochlorophyll a triplet is that of P-840. Formation of a carotenoid triplet did not occur in our preparations.Illumination with continuous light at 295 K of the reaction center pigment-protein complex produced a stable charge separation (with oxidation of P-840 and cytochrome c-553) in each reaction center, but with a low efficiency. This low efficiency, and the high yield of triplet formation is probably due to damage of the electron transport chain at the acceptor side of the reaction center of the reaction center pigment-protein complex.The halftime for cytochrome c-553 oxidation in Complex I and the photosystem pigment complex was 90 μs at 295 K; below 220 K no cytochrome oxidation occurred. At 120 K P-840⁺ was rereduced with a halftime of 20 ms, presumably by a back reaction with a reduced acceptor.     

Periplasmic electron carriers and photo-induced electron transfer in the photosynthetic bacterium Ectothiorhodospira sp.

A detailed analysis of the periplasmic electron carriers of the photosynthetic bacterium Ectothiorhodospira sp. has been performed. Two low mid-point redox potential electron carriers, cytochrome c′ and cytochrome c, are detected. A high potential iron–sulfur protein is the only high mid-point redox potential electron transfer component present in the periplasm. Analysis of light-induced absorption changes shows that this high potential iron–sulfur protein acts in vivo as efficient electron donor to the photo-oxidized high potential heme of the Ectothiorhodospira sp. reaction center. This revised version was published online in June 2006 with corrections to the Cover Date.     

Delayed fluorescence observed in the nanosecond time region at 77 K originates directly from the photosystem II reaction center

The excited-state dynamics of delayed fluorescence in photosystem (PS) II at 77 K were studied by time-resolved fluorescence spectroscopy and decay analysis on three samples with different antenna sizes: PS II particles and the PS II reaction center from spinach, and the PS II core complexes from Synechocystis sp. PCC 6803. Delayed fluorescence in the nanosecond time region originated from the 683-nm component in all three samples, even though a slight variation in lifetimes was detected from 15 to 25 ns. The relative amplitude of the delayed fluorescence was higher when the antenna size was smaller. Energy transfer from the 683-nm pigment responsible for delayed fluorescence to antenna pigment(s) at a lower energy level was not observed in any of the samples examined. This indicated that the excited state generated by charge recombination was not shared with antenna pigments under the low-temperature condition, and that delayed fluorescence originates directly from the PS II reaction center, either from chlorophyll a_D1 or P680. Supplemental data on delayed fluorescence from spinach PS I complexes are included.     

Multiple pathways of charge recombination revealed by the temperature dependence of electron transfer kinetics in cyanobacterial photosystem I

The kinetics of charge recombination in Photosystem I P₇₀₀-F_A/F_B complexes and P₇₀₀-F_X cores lacking the terminal iron?sulfur clusters were studied over a temperatures range of 310 K to 4.2 K. Analysis of the charge recombination kinetics in this temperature range allowed the assignment of backward electron transfer from the different electron acceptors to P₇₀₀⁺. The kinetic and thermodynamic parameters of these recombination reactions were determined. The kinetics of all electron transfer reactions were activation-less below 170 K, the glass transition temperature of the water-glycerol solution. Above this temperature, recombination from [F_A/F_B]^? in P₇₀₀-F_A/F_B complexes was found to proceed along two pathways with different activation energies (E_a). The charge recombination via A_1A has an E_a of ~290 meV and is dominant at temperatures above ~280 K, whereas the direct recombination from F_X^? has an E_a of 22 meV and is prevalent in the 200 K to 270 K temperature range. Charge recombination from the F_X cluster becomes highly heterogeneous at temperatures below 200 K. The conformational mobility of Photosystem I was studied by molecular dynamics simulations. The F_X cluster was found to ‘swing’ by ~30° along the axis between the two sulfur atoms proximal to F_A/F_B. The partial rotation of F_X is accompanied by significant changes of electric potential within the iron?sulfur cluster, which may induce preferential electron localization at different atoms of the F_X cluster. These effects may account for the partial arrest of forward electron transfer and for the heterogeneity of charge recombination observed at the glass transition temperature.     

Kinetic model of primary energy transfer and trapping in photosynthetic membranes

The picosecond time-domain incoherent singlet excitation transfer and trapping kinetics in core antenna of photosynthetic bacteria are studied in case of low excitation intensities by numerical integration of the appropriate master equation in a wide temperature range of 4-300 K. The essential features of our two-dimensional-lattice model are as follows: Förster excitation transfer theory, spectral heterogeneity of both the light-harvesting antenna and the reaction center, treatment of temperature effects through temperature dependence of spectral bands, inclusion of inner structure of the trap, and transition dipole moment orientation. The fluorescence kinetics is analyzed in terms of distributions of various kinetic components, and the influence of different inhomogeneities (orientational, spectral) is studied.

A reasonably good agreement between theoretical and experimental fluorescence decay kinetics for purple photosynthetic bacterium Rhodospirillum rubrum is achieved at high temperatures by assuming relatively large antenna spectral inhomogeneity: 20 nm at the whole bandwidth of 40 nm. The mean residence time in the antenna lattice site (it is assumed to be the aggregate of four bacteriochlorophyll a molecules bound to proteins) is estimated to be ~12 ps. At 4 K only qualitative agreement between model and experiment is gained. The failure of quantitative fitting is perhaps due to the lack of knowledge about the real structure of antenna or local heating and cooling effects not taken into account.

     

A new methodical approach to determine the quantum yield of the conversion of electronic excitation in the reaction centers of purple bacteria

A new methodical approach is proposed for precise determination (better than ± 2%) of the quantum yield of charge separation in the reaction centers of purple bacteria; for Rhodospirillum rubrum this value proves to be 93.5%.     

Coupling of collective motions of the protein matrix to vibrations of the non-heme iron in bacterial photosynthetic reaction centers

Non-heme iron is a conservative component of type II photosynthetic reaction centers of unknown function. We found that in the reaction center from Rba. sphaeroides it exists in two forms, high and low spin ferrous states, whereas in Rsp. rubrum mostly in a low spin state, in line with our earlier finding of its low spin state in the algal photosystem II reaction center (Burda et al., 2003). The temperature dependence of the non-heme iron displacement studied by Mössbauer spectroscopy shows that the surrounding of the high spin iron is more flexible (Debye temperature ~ 165 K) than that of the low spin atom (~ 207 K). Nuclear inelastic scattering measurements of the collective motions in the Rba. sphaeroides reaction center show that the density of vibrational states, originating from non-heme iron, has well-separated modes between lower (4-17 meV) and higher (17-25 meV) energies while in the one from Rsp. rubrum its distribution is more uniform with only little contribution of low energy (~ 6 meV) vibrations. It is the first experimental evidence that the fluctuations of the protein matrix in type II reaction center are correlated to the spin state of non-heme iron. We propose a simple mechanism in which the spin state of non-heme iron directly determines the strength of coupling between the two quinone acceptors (Q_A and Q_B) and fast collective motions of protein matrix that play a crucial role in activation and regulation of the electron and proton transfer between these two quinones. We suggest that hydrogen bond network on the acceptor side of reaction center is responsible for stabilization of non-heme iron in different spin states.     

On the state of carotenoids bound to reaction centers of photosynthetic bacteria: A resonance raman study

The carotenoids bound to reaction centers of wild, Ga and GlC strains of Rhodopseudomonas spheroides, of Rhodospirillum rubrum, strain S1 and of Rhodopseudomonas viridis, yield very similar, but unusual resonance Raman spectra. Through a comparison with resonance Raman spectra of 15,15′-cis-β-carotene, these carotenoids are shown to assume cis conformations, while the corresponding chromatophores contain all-trans forms only. These cis conformations likely are identical for all the carotenoids studied. They remain unaffected by variations of temperature from 20 to 300 K as well as by the redox state of $\text{P-870}$. They are unstable, being rapidly isomerised towards the all-trans forms when extracted from the reaction centers. The possible nature of these conformers is discussed on the basis of their electronic and vibrational spectra.     

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     

psbA based molecular analysis of cross-feeding experiments suggests that Dinophysis acuta does not harbour permanent plastids

Toxic marine dinoflagellate species of the genus Dinophysis Ehrenberg are obligate mixotrophs that require feeding on the ciliate Mesodinium rubrum and light to achieve growth. It is now well known that they harbour plastids of cryptophyte origin, particularly of the genus Teleaulax, Plagioselmis or Geminigera group (TPG clade). Nevertheless, whether these plastids are permanent, or periodically acquired from M. rubrum prey, need additional studies in different phototrophic Dinophysis species. The origin of plastids from Dinophysis acuta Ehrenberg, one of the main agents of diarrhetic shellfish poisoning (DSP) outbreaks in Western Europe, was investigated here. Cross feeding-starvation experiments were carried out with cultures of D. acuta using M. rubrum as prey, the latter fed with two cryptophyte species, Teleaulax amphioxeia Hill and Teleaulax gracilis, belonging to the TPG clade in addition to Falcomonas sp. and Hemiselmis sp. The fate of cryptophyte plastids transferred to D. acuta through its ciliate prey was investigated using the plastid psbA gene as a tracer.     

Kinetic and Energetic Model for the Primary Processes in Photosystem II

A detailed model for the kinetics and energetics of the exciton trapping, charge separation, charge recombination, and charge stabilization processes in photosystem (PS) II is presented. The rate constants describing these processes in open and closed reaction centers (RC) are calculated on the basis of picosecond data (Schatz, G. H., H. Brock, and A. R. Holzwarth. 1987. Proc. Natl. Acad. Sci. USA. 84:8414-8418) obtained for oxygen-evolving PS II particles from Synechococcus sp. with ~80 chlorophylls/P₆₈₀. The analysis gives the following results. (a) The PS II reaction center donor chlorophyll P₆₈₀ constitutes a shallow trap, and charge separation is overall trap limited. (b) The rate constant of charge separation drops by a factor of ~6 when going from open (Q-oxidized) to closed (Q-reduced) reaction centers. Thus the redox state of Q controls the yield of radical pair formation and the exciton lifetime in the Chl antenna. (c) The intrinsic rate constant of charge separation in open PS II reaction centers is calculated to be ~2.7 ps^-1. (d) In particles with open RC the charge separation step is exergonic with a decrease in standard free energy of ~38 meV. (e) In particles with closed RC the radical pair formation is endergonic by ~12 meV. We conclude on the basis of these results that the long-lived (nanoseconds) fluorescence generally observed with closed PS II reaction centers is prompt fluorescence and that the amount of primary radical pair formation is decreased significantly upon closing of the RC.     

Comparative study of reaction centers from purple photosynthetic bacteria: Isolation and optical spectroscopy

Reaction centers from two species of purple bacteria, Rhodospirillum rubrum and Rhodospirillum centenum, have been characterized and compared to reaction centers from Rhodobacter sphaeroides and Rhodobacter capsulatus. The reaction centers purified from these four species can be divided into two classes according to the spectral characteristics of the primary donor. Reaction centers from one class have a donor optical band at a longer wavelength, 865 nm compared to 850 nm, and an optical absorption band associated with the oxidized donor at 1250 nm that has a larger oscillator strength than reaction centers from the second class. Under normal buffering conditions, reaction centers isolated from Rb. sphaeroides and Rs. rubrum exhibit characteristics of the first class while those from Rb. capsulatus and Rs. centenum exhibit characteristics of the second class. However, the reaction centers can be converted between the two groups by the addition of charged detergents. Thus, the observed spectral differences are not due to intrinsic differences between reaction centers but represent changes in the electronic structure of the donor due to interactions with the detergents as has been confirmed by recent ENDOR measurements (Rautter J, Lendzian F, Lubitz W, Wang S and Allen JP (1994) Biochemistry 33: 12077–12084). The oxidation midpoint potential for the donor has values of 445 mV, 475 mV, 480 mV and 495 mV for Rs. rubrum, Rs. centenum, Rb. capsulatus, and Rb. sphaeroides, respectively. Despite this range of values for the midpoint potential, the decay rates of the stimulated emission are all fast with values of 4.1 ps, 4.5 ps. 5.5 ps and 6.1 ps for quinone-reduced RCs from Rs. rubrum, Rb. capsulatus, Rs. centenum, and Rb. sphaeroides, respectively. The general spectral features of the initial charge separated state are essentially the same for the four species, except for differences in the wavelengths of the absorption changes due to the different donor band positions. The pH dependence of the charge recombination rates from the primary and secondary quinones differ for reaction centers from the four species indicating different interactions between the quinones and ionizable residues. A different mechanism for charge recombination from the secondary quinone, that probably is direct recombination, is proposed for RCs from Rs. centenum.Abbreviations RC reaction center - P bacteriochlorophyll dimer - H bacteriopheophytin - Q quinone - Rb Rhodobacter - Rs Rhodospirillum - Rps Rhodopseudomonas - EDTA (ethylenediamine)tetraaceticacid - LDAO N,N-dimethyl-dodecylamine-N-oxide - CTAB cetyltrimethylammonium bromide - DOC deoxycholate - Tris Tris-(hydroxymethyl)aminomethane - ns nanosecond - ps picosecond - fs femtosecond     

Defining the Far-Red Limit of Photosystem II in Spinach

The far-red limit of photosystem II (PSII) photochemistry was studied in PSII-enriched membranes and PSII core preparations from spinach (Spinacia oleracea) after application of laser flashes between 730 and 820 nm. Light up to 800 nm was found to drive PSII activity in both acceptor side reduction and oxidation of the water-oxidizing CaMn₄ cluster. Far-red illumination induced enhancement of, and slowed down decay kinetics of, variable fluorescence. Both effects reflect reduction of the acceptor side of PSII. The effects on the donor side of PSII were monitored using electron paramagnetic resonance spectroscopy. Signals from the S₂-, S₃-, and S₀-states could be detected after one, two, and three far-red flashes, respectively, indicating that PSII underwent conventional S-state transitions. Full PSII turnover was demonstrated by far-red flash-induced oxygen release, with oxygen appearing on the third flash. In addition, both the pheophytin anion and the Tyr Z radical were formed by far-red flashes. The efficiency of this far-red photochemistry in PSII decreases with increasing wavelength. The upper limit for detectable photochemistry in PSII on a single flash was determined to be 780 nm. In photoaccumulation experiments, photochemistry was detectable up to 800 nm. Implications for the energetics and energy levels of the charge separated states in PSII are discussed in light of the presented results.