Excited state dynamics in photosystem I: effects of detergent and excitation wavelength.

Femtosecond transient absorption spectroscopy has been used to investigate the energy transfer and trapping processes in both intact membranes and purified detergent-isolated particles from a photosystem II deletion mutant of the cyanobacterium Synechocystis sp. PCC 6803, which contains only the photosystem I reaction center. Processes with similar lifetimes and spectra are observed in both the membrane fragments and the detergent-isolated particles, suggesting little disruption of the core antenna resulting from the detergent treatment. For the detergent-isolated particles, three different excitation wavelengths were used to excite different distributions of pigments in the spectrally heterogeneous core antenna. Only two lifetimes of 2.7-4.3 ps and 24-28 ps, and a nondecaying component are required to describe all the data. The 24-28 ps component is associated with trapping. The trapping process gives rise to a nondecaying spectrum that is due to oxidation of the primary electron donor. The lifetimes and spectra associated with trapping and radical pair formation are independent of excitation wavelength, suggesting that trapping proceeds from an equilibrated excited state. The 2.7-4.3 ps component characterizes the evolution from the initially excited distribution of pigments to the equilibrated excited state distribution. The spectrum associated with the 2.7-4.3 ps component is therefore strongly excitation wavelength dependent. Comparison of the difference spectra associated with the spectrally equilibrated state and the radical pair state suggests that the pigments in the photosystem I core antenna display some degree of excitonic coupling.     

Regulation of excitation energy distribution in subchloroplast particles: photosystem I

Mono- and divalent cations were found to increase the transfer of excitation energy within Photosystem I from the light-harvesting chlorophyll a molecules to P700. The P700-chlorophyll a protein of Shiozawa et al. (J. A. Shiozawa, R. S. Alberte, and J. P. Thornber, 1974, Arch. Biochem. Biophys.165, 388–397) was used for these studies. Cations stimulated the quantum yields for electron transport when the light-harvesting chlorophyll a molecules were irradiated. They also decreased chlorophyll a fluorescence. Half-maximal effects were observed at 0.5–0.6 mm for divalent cations and at 5–6 mm for monovalent cations. Triton X-100, 0.02%, also increased energy transfer. The increases in energy transfer are due to an intramolecular conformational change in the protein. A structural change is involved, since there is a correlation between the cation-induced changes in energy transfer and increases in 90 ° light scattering. However, there was no change in the molecular weight upon the addition of MgCl₂. The molecular weight, as determined by gel filtration, was 105,000 in the presence of 0.05% Triton X-100. On the other hand, circular dichroism measurements showed an increase in the α-helical content from 51 to 63% when 5 mm MgCl₂ was added. Changes in the absorption spectra were also observed. We believe that the cation regulation of Photosystem I activity provides a fine-tuning mechanism for the regulation of energy transfer.     

Comparison of excitation energy transfer in cyanobacterial photosystem I in solution and immobilized on conducting glass

Excitation energy transfer in monomeric and trimeric forms of photosystem I (PSI) from the cyanobacterium Synechocystis sp. PCC 6803 in solution or immobilized on FTO conducting glass was compared using time-resolved fluorescence. Deposition of PSI on glass preserves bi-exponential excitation decay of ~4–7 and ~21–25 ps lifetimes characteristic of PSI in solution. The faster phase was assigned in part to photochemical quenching (charge separation) of excited bulk chlorophylls and in part to energy transfer from bulk to low-energy (red) chlorophylls. The slower phase was assigned to photochemical quenching of the excitation equilibrated over bulk and red chlorophylls. The main differences between dissolved and immobilized PSI (iPSI) are: (1) the average excitation decay in iPSI is about 11 ps, which is faster by a few ps than for PSI in solution due to significantly faster excitation quenching of bulk chlorophylls by charge separation (~10 ps instead of ~15 ps) accompanied by slightly weaker coupling of bulk and red chlorophylls; (2) the number of red chlorophylls in monomeric PSI increases twice—from 3 in solution to 6 after immobilization—as a result of interaction with neighboring monomers and conducting glass; despite the increased number of red chlorophylls, the excitation decay accelerates in iPSI; (3) the number of red chlorophylls in trimeric PSI is 4 (per monomer) and remains unchanged after immobilization; (4) in all the samples under study, the free energy gap between mean red (emission at ~710 nm) and mean bulk (emission at ~686 nm) emitting states of chlorophylls was estimated at a similar level of 17–27 meV. All these observations indicate that despite slight modifications, dried PSI complexes adsorbed on the FTO surface remain fully functional in terms of excitation energy transfer and primary charge separation that is particularly important in the view of photovoltaic applications of this photosystem.     

Quantum mechanical analysis of excitation energy transfer couplings in photosystem II

We evaluated excitation energy transfer (EET) coupling (J) between all pairs of chlorophylls (Chls) and pheophytins (Pheos) in the protein environment of photosystem II based on the time-dependent density functional theory with a quantum mechanical/molecular mechanics approach. In the reaction center, the EET coupling between Chls P_D1 and P_D2 is weaker (|J(P_D1/P_D2)| = 79 cm^?1), irrespective of a short edge-to-edge distance of 3.6 Å (Mg-to-Mg distance of 8.1 Å), than the couplings between P_D1 and the accessory Chl_D1 (|J(P_D1/Chl_D2)| = 104 cm^?1) and between P_D2 and Chl_D2 (|J(P_D2/Chl_D1)| = 101 cm^?1), suggesting that P_D1 and P_D2 are two monomeric Chls rather than a “special pair”. There exist strongly coupled Chl pairs (|J| > ～100 cm^?1) in the CP47 and CP43 core antennas, which may be candidates for the red-shifted Chls observed in spectroscopic studies. In CP47 and CP43, Chls ligated to CP47-His26 and CP43-His56, which are located in the middle layer of the thylakoid membrane, play a role in the “hub” that mediates the EET from the lumenal to stromal layers. In the stromal layer, Chls ligated to CP47-His466, CP43-His441, and CP43-His444 mediate the EET from CP47 to Chl_D2/Pheo_D2 and from CP43 to Chl_D1/Pheo_D1 in the reaction center. Thus, the excitation energy from both CP47 and CP43 can always be utilized for the charge-separation reaction in the reaction center.     

Reversible inhibition and reactivation of electron transfer in photosystem I

Photosynthesis Research - In photosystem I (PSI) complexes at room temperature electron transfer from A1– to FX is an order of magnitude faster on the B-branch compared to the A-branch. One...     

Phosphorylation of the light-harvesting chlorophyll-protein regulates excitation energy distribution between photosystem II and photosystem I

The kinetics of thylakoid membrane protein phosphorylation in the presence of light and adenosine triphosphate is correlated to an incease in the 77 °K fluorescence emission at 735 nm (F735) relative to that at 685 nm (F685). Analysis of detergent-derived submembrane fractions indicate phosphorylation only of the polypeptides of Photosystem II, and the light-harvesting chlorophyll-protein complex serving Photosystem II (LHC-II). Although several polypeptides are phosphorylated, only the dephosphorylation kinetics of LHC-II follow the kinetics of the decrease of the $\text{F735}\text{F685}$ fluorescence emission ratios. The relative quantum yield of Photosystem II was significantly lower in phosphorylated membranes compared to dephosphorylated membranes. Reversible LHC-II phosphorylation thus provides the physiological mechanism for the control of the distribution of absorbed excitation energy between the two photosystems.     

The role of the individual Lhcas in photosystem I excitation energy trapping

In this work, we have investigated the role of the individual antenna complexes and of the low-energy forms in excitation energy transfer and trapping in Photosystem I of higher plants. To this aim, a series of Photosystem I (sub)complexes with different antenna size/composition/absorption have been studied by picosecond fluorescence spectroscopy. The data show that Lhca3 and Lhca4, which harbor the most red forms, have similar emission spectra (λ_max = 715–720 nm) and transfer excitation energy to the core with a relative slow rate of ∼25/ns. Differently, the energy transfer from Lhca1 and Lhca2, the “blue” antenna complexes, occurs about four times faster. In contrast to what is often assumed, it is shown that energy transfer from the Lhca1/4 and the Lhca2/3 dimer to the core occurs on a faster timescale than energy equilibration within these dimers. Furthermore, it is shown that all four monomers contribute almost equally to the transfer to the core and that the red forms slow down the overall trapping rate by about two times. Combining all the data allows the construction of a comprehensive picture of the excitation-energy transfer routes and rates in Photosystem I.     

Relationship between excitation energy transfer, trapping, and antenna size in photosystem II

We present a systematic study of the effect of antenna size on energy transfer and trapping in photosystem II. Time-resolved fluorescence experiments have been used to probe a range of particles isolated from both higher plants and the cyanobacterium Synechocystis 6803. The isolated reaction center dynamics are represented by a quasi-phenomenological model that fits the extensive time-resolved data from photosystem II reaction centers and reaction center mutants. This representation of the photosystem II "trapping engine" is found to correctly predict the extent of, and time scale for, charge separation in a range of photosystem II particles of varying antenna size (8-250 chlorins). This work shows that the presence of the shallow trap and slow charge separation kinetics, observed in isolated D1/D2/cyt b559 reaction centers, are indeed retained in larger particles and that these properties are reflected in the trapping dynamics of all larger photosystem II preparations. A shallow equilibrium between the antennae and reaction center in photosystem II will certainly facilitate regulation via nonphotochemical quenching, and one possible interpretation of these findings is therefore that photosystem II is optimized for regulation rather than for efficiency.     

Distribution of excitation energy among photosystem I and photosystem II in red algae. I. Action spectra of light reactions I and II

Action spectra of light reaction I and light reaction II from red algae (marine members of Florideae and Bangiales) were measured with 550 nm (light 2) or 699 nm (light 1) background light, using a Teflon-covered platinum electrode for O₂ measurement. Care was taken to ensure that maximum enhancement was reached by the background light.The action spectra of light reaction I, we found under these conditions, are very similar to the thallus absorption, whilst the action spectra of light reaction II show, besides strong bands of the phycobilins, only minor bands of chlorophyll a, which account for only 10–20% of the total chlorophyll.The spectra are discussed on the basis of two main types of models of energy distribution over both photosynthetic systems. If this distribution is considered to be invariable (models 1a and b), one has to assume that almost exactly half of the total chlorophyll is not involved in the supply of the non-cyclic electron transport with excitation energy. This part, however, has to be thought of as incorporated in the thylakoid membrane in a similar manner to the chlorophyll in photosystem I. However, if one supposes an almost complete equilibration in the energy distribution over both systems as long as the primary absorption in photosystem II prevails (models 2a and b), there is no need for the assumption of such photosynthetically ‘inactive’ or less active chlorophyll. Some evidence is shown that strongly supports model 2.     

Chloroplast site-directed mutagenesis of photosystem I in Chlamydomonas: electron transfer reactions and light sensitivity

The photosystem I (PSI) complex is a multisubunit protein-pigment complex embedded in the thylakoid membrane which acts as a light-driven plastocyanin/cytochrome c(6)-ferredoxin oxido-reductase. The use of chloroplast transformation and site-directed mutagenesis coupled with the biochemical and biophysical analysis of mutants of the green alga Chlamydomonas reinhardtii with specific amino acid changes in several subunits of PSI has provided new insights into the structure-function relationship of this important photosynthetic complex. In particular, this molecular-genetic analysis has identified key residues of the reaction center polypeptides of PSI which are the ligands of some of the redox cofactors and it has also provided important insights into the orientation of the terminal electron acceptors of this complex. Finally this analysis has also shown that mutations affecting the donor side of PSI are limiting for overall electron transfer under high light and that electron trapping within the terminal electron acceptors of PSI is highly deleterious to the cells.     

Regulation of excitation energy in Nannochloropsis photosystem II

Photosynthesis Research - Recently, we isolated a complex consisting of photosystem II (PSII) and light-harvesting complexes (LHCs) from Nannochloropsis granulata (Umetani et al. Photosynth Res...     

Excitation energy transfer between photosystem II and photosystem I in red algae: larger amounts of phycobilisome enhance spillover

We examined energy transfer dynamics from the photosystem II reaction center (PSII-RC) in intact red algae cells of Porphyridium cruentum, Bangia fuscopurpurea, Porphyra yezoensis, Chondrus giganteus, and Prionitis crispata. Time resolved fluorescence measurements were conducted in the range of 0-80ns at -196°C. The delayed fluorescence spectra were then determined, where the delayed fluorescence was derived from the charge recombination between P680(+) and pheophytin a in PSII-RC. Therefore, the delayed fluorescence spectrum reflected the energy migration processes including PSII-RC. All samples examined showed prominent distribution of delayed fluorescence in PSII and PSI, which suggests that a certain amount of PSII attaches to PSI to share excitation energy in red algae. The energy transfer from PSII to PSI was found to be dominant when the amount of phycoerythrobilin was increased.     

Redox potential of quinones in both electron transfer branches of photosystem I

The redox potentials of the two electron transfer (ET) active quinones in the central part of photosystem I (PSI) were determined by evaluating the electrostatic energies from the solution of the Poisson-Boltzmann equation based on the crystal structure. The calculated redox potentials are -531 mV for A1A and -686 mV for A1B. From these results we conclude the following. (i) Both branches are active with a much faster ET in the B-branch than in the A-branch. (ii) The measured lifetime of 200-290 ns of reduced quinones agrees with the estimate for the A-branch and corroborates with an uphill ET from this quinone to the iron-sulfur cluster as observed in recent kinetic measurements. (iii) The electron paramagnetic resonance spectroscopic data refer to the A-branch quinone where the corresponding ET is uphill in energy. The negative redox potential of A1 in PSI is primarily because of the influence from the negatively charged FX, in contrast to the positive shift on the quinone redox potential in bacterial reaction center and PSII that is attributed to the positively charged non-heme iron atom. The conserved residue Asp-B575 changes its protonation state after quinone reduction. The difference of 155 mV in the quinone redox potentials of the two branches were attributed to the conformation of the backbone with a large contribution from Ser-A692 and Ser-B672 and to the side chain of Asp-B575, whose protonation state couples differently with the formation of the quinone radicals.     

Light-induced alteration of low-temperature interprotein electron transfer between photosystem I and flavodoxin

Electron paramagnetic resonance (EPR) was used to study light-induced electron transfer in Photosystem I-flavodoxin complexes. Deuteration of flavodoxin enables the signals of the reduced flavin acceptor and oxidized primary donor, P(700)(+), to be well-resolved at X- and D-band EPR. In dark-adapted samples, photoinitiated interprotein electron transfer does not occur at 5 K. However, for samples prepared in dim light, significant interprotein electron transfer occurs at 5 K and a concomitant loss of the spin-correlated radical pair P(+)A(1A)(-) signal is observed. These results indicate a light-induced reorientation of flavodoxin in the PSI docking site that allows a high quantum yield efficiency for the interprotein electron transfer reaction.     

Heat stress induces an inhibition of excitation energy transfer from phycobilisomes to photosystem II but not to photosystem I in a cyanobacterium Spirulina platensis.

The effects of high temperature (30-52.5 degrees C) on excitation energy transfer from phycobilisomes (PBS) to photosystem I (PSI) and photosystem II (PSII) in a cyanobacterium Spirulina platensis grown at 30 degrees C were studied by measuring 77 K chlorophyll (Chl) fluorescence emission spectra. Heat stress had a significant effect on 77 K Chl fluorescence emission spectra excited either at 436 or 580 nm. In order to reveal what parts of the photosynthetic apparatus were responsible for the changes in the related Chl fluorescence emission peaks, we fitted the emission spectra by Gaussian components according to the assignments of emission bands to different components of the photosynthetic apparatus. The 643 and 664 nm emissions originate from C-phycocyanin (CPC) and allophycocyanin (APC), respectively. The 685 and 695 nm emissions originate mainly from the core antenna complexes of PSII, CP43 and CP47, respectively. The 725 and 751 nm band is most effectively produced by PSI. There was no significant change in F725 and F751 during heat stress, suggesting that heat stress had no effects on excitation energy transfer from PBS to PSI. On the other hand, heat stress induced an increase in the ratio of Chl fluorescence yield of PBS to PSII, indicating that heat stress inhibits excitation energy transfer from PBS to PSII. However, this inhibition was not associated with an inhibition of excitation energy transfer from CPC to APC since no significant changes in F643 occurred at high temperatures. A dramatic enhancement of F664 occurring at 52.5 degrees C indicates that excitation energy transfer from APC to the PSII core complexes is suppressed at this temperature, possibly due to the structural changes within the PBS core but not to a detachment of PBS from PSII, resulting in an inhibition of excitation energy transfer from APC to PSII core complexes (CP47 + CP43). A decrease in F685 and F695 in heat-stressed cells with excitation at 436 nm seems to suggest that heat stress did not inhibit excitation energy transfer from the Chl a binding proteins CP47 and CP43 to the PSII reaction center and the decreased Chl fluorescence yields from CP43 and CP47 could be explained by the inhibition of the energy transfer from APC to PSII core complexes (CP47 + CP43).     

Disturbed excitation energy transfer in Arabidopsis thaliana mutants lacking minor antenna complexes of photosystem II

Minor light-harvesting complexes (Lhcs) CP24, CP26 and CP29 occupy a position in photosystem II (PSII) of plants between the major light-harvesting complexes LHCII and the PSII core subunits. Lack of minor Lhcs in vivo causes impairment of PSII organization, and negatively affects electron transport rates and photoprotection capacity. Here we used picosecond-fluorescence spectroscopy to study excitation-energy transfer (EET) in thylakoid membranes isolated from Arabidopsis thaliana wild-type plants and knockout lines depleted of either two (koCP26/24 and koCP29/24) or all minor Lhcs (NoM). In the absence of all minor Lhcs, the functional connection of LHCII to the PSII cores appears to be seriously impaired whereas the “disconnected” LHCII is substantially quenched. For both double knock-out mutants, excitation trapping in PSII is faster than in NoM thylakoids but slower than in WT thylakoids. In NoM thylakoids, the loss of all minor Lhcs is accompanied by an over-accumulation of LHCII, suggesting a compensating response to the reduced trapping efficiency in limiting light, which leads to a photosynthetic phenotype resembling that of low-light-acclimated plants. Finally, fluorescence kinetics and biochemical results show that the missing minor complexes are not replaced by other Lhcs, implying that they are unique among the antenna subunits and crucial for the functioning and macro-organization of PSII.     

Structure and energy transfer pathways of the Dunaliella Salina photosystem I supercomplex

Oxygenic photosynthesis evolved more than 3 billion years ago in cyanobacteria. The increased complexity of photosystem I (PSI) became apparent from the high-resolution structures that were obtained for the complexes that were isolated from various organisms, ranging from cyanobacteria to plants. These complexes are all evolutionarily linked. In this paper, the researchers have uncovered the increased complexity of PSI in a single organism demonstrated by the coexistance of two distinct PSI compositions. The Large Dunaliella PSI contains eight additional subunits, six in PSI core and two light harvesting complexes. Two additional chlorophyll a molecules pertinent for efficient excitation energy transfer in state II transition were identified in PsaL and PsaO. Short distances between these newly identified chlorophylls correspond with fast excitation transfer rates previously reported during state II transition. The apparent PSI conformations could be a coping mechanism for the high salinity.     

Dynamics of electron transfer in photosystem II

Photosystem II, being a constituent of light driven photosynthetic apparatus, is a highly organized pigment-protein-lipid complex. The arrangement of PSII active redox cofactors insures efficiency of electron transfer within it. Donation of electrons extracted from water by the oxygen evolving complex to plastoquinones requires an additional activation energy. In this paper we present theoretical discussion of the anharmonic fluctuations of the protein-lipid matrix of PSII and an experimental evidence showing that the fluctuations are responsible for coupling of its donor and acceptor side. We argue that the fast collective motions liberated at temperatures higher that 200 K are crucial for the two final steps of the water splitting cycle and that one can distinguish three different dynamic regimes of PSII action which are controlled by the timescales of forward electron transfer, which vary with temperature. The three regimes of the dynamical behavior are related to different spatial domains of PSII.