Time-resolved absorption and emission show that the CP43' antenna ring of iron-stressed synechocystis sp. PCC6803 is efficiently coupled to the photosystem I reaction center core

Excitation energy transfer and trapping processes in an iron stress-induced supercomplex of photosystem I from the cyanobacterium Synechocystis sp. PCC6803 were studied by time-resolved absorption and fluorescence spectroscopy on femtosecond and picosecond time scales. The data provide evidence that the energy transfer dynamics of the CP43'-PSI supercomplex are consistent with energy transfer processes that occur in the Chl a network of the PSI trimer antenna. The most significant absorbance changes in the CP43'-PSI supercomplex are observed within the first several picoseconds after the excitation into the spectral region of CP43' absorption (665 nm). The difference time-resolved spectra (DeltaDeltaA) resulting from subtraction of the PSI trimer kinetic data from the CP43'-PSI supercomplex data indicate three energy transfer processes with time constants of 0.2, 1.7, and 10 ps. The 0.2 ps kinetic phase is tentatively interpreted as arising from energy transfer processes originating within or between the CP43' complexes. The 1.7 ps phase is interpreted as possibly arising from energy transfer from the CP43' ring to the PSI trimer via closely located clusters of Chl a in CP43' and the PSI core, while the slower 10 ps process might reflect the overall excitation transfer from the CP43' ring to the PSI trimer. These three fast kinetic phases are followed by a 40 ps overall excitation decay in the supercomplex, in contrast to a 25 ps overall decay observed in the trimer complex without CP43'. Excitation of Chl a in both the CP43'-PSI antenna supercomplex and the PSI trimer completely decays within 100 ps, resulting in the formation of P700(+). The data indicate that there is a rapid and efficient energy transfer between the outer antenna ring and the PSI reaction center complex.     

Pigment organization and energy transfer dynamics in isolated photosystem I (PSI) complexes from Arabidopsis thaliana depleted of the PSI-G, PSI-K, PSI-L, or PSI-N subunit

Green plant photosystem I (PSI) consists of at least 18 different protein subunits. The roles of some of these protein subunits are not well known, in particular those that do not occur in the well characterized PSI complexes from cyanobacteria. We investigated the spectroscopic properties and excited-state dynamics of isolated PSI-200 particles from wild-type and mutant Arabidopsis thaliana plants devoid of the PSI-G, PSI-K, PSI-L, or PSI-N subunit. Pigment analysis and a comparison of the 5 K absorption spectra of the various particles suggests that the PSI-L and PSI-H subunits together bind approximately five chlorophyll a molecules with absorption maxima near 688 and 667 nm, that the PSI-G subunit binds approximately two red-shifted beta-carotene molecules, that PSI-200 particles without PSI-K lack a part of the peripheral antenna, and that the PSI-N subunit does not bind pigments. Measurements of fluorescence decay kinetics at room temperature with picosecond time resolution revealed lifetimes of ~0.6, 5, 15, 50, 120, and 5000 ps in all particles. The 5- and 15-ps phases could, at least in part, be attributed to the excitation equilibration between bulk and red chlorophyll forms, though the 15-ps phase also contains a contribution from trapping by charge separation. The 50- and 120-ps phases predominantly reflect trapping by charge separation. We suggest that contributions from the core antenna dominate the 15-ps trapping phase, that those from the peripheral antenna proteins Lhca2 and Lhca3 dominate the 50-ps phase, and that those from Lhca1 and Lhca4 dominate the 120-ps phase. In the PSI-200 particles without PSI-K or PSI-G protein, more excitations are trapped in the 15-ps phase and less in 50- and 120-ps phases, which is in agreement with the notion that these subunits are involved in the interaction between the core and peripheral antenna proteins.     

The High Efficiency of Photosystem I in the Green Alga Chlamydomonas reinhardtii Is Maintained after the Antenna Size Is Substantially Increased by the Association of Light-harvesting Complexes II

Photosystems (PS) I and II activities depend on their light-harvesting capacity and trapping efficiency, which vary in different environmental conditions. For optimal functioning, these activities need to be balanced. This is achieved by redistribution of excitation energy between the two photosystems via the association and disassociation of light-harvesting complexes (LHC) II, in a process known as state transitions. Here we study the effect of LHCII binding to PSI on its absorption properties and trapping efficiency by comparing time-resolved fluorescence kinetics of PSI-LHCI and PSI-LHCI-LHCII complexes of Chlamydomonas reinhardtii. PSI-LHCI-LHCII of C. reinhardtii is the largest PSI supercomplex isolated so far and contains seven Lhcbs, in addition to the PSI core and the nine Lhcas that compose PSI-LHCI, together binding ∼320 chlorophylls. The average decay time for PSI-LHCI-LHCII is ∼65 ps upon 400 nm excitation (15 ps slower than PSI-LHCI) and ∼78 ps upon 475 nm excitation (27 ps slower). The transfer of excitation energy from LHCII to PSI-LHCI occurs in ∼60 ps. This relatively slow transfer, as compared with that from LHCI to the PSI core, suggests loose connectivity between LHCII and PSI-LHCI. Despite the relatively slow transfer, the overall decay time of PSI-LHCI-LHCII remains fast enough to assure a 96% trapping efficiency, which is only 1.4% lower than that of PSI-LHCI, concomitant with an increase of the absorption cross section of 47%. This indicates that, at variance with PSII, the design of PSI allows for a large increase of its light-harvesting capacities.     

Excitation dynamics in Photosystem I from Chlamydomonas reinhardtii. Comparative studies of isolated complexes and whole cells

Identical time-resolved fluorescence measurements with ~ 3.5-ps resolution were performed for three types of PSI preparations from the green alga, Chlamydomonas reinhardtii: isolated PSI cores, isolated PSI–LHCI complexes and PSI–LHCI complexes in whole living cells. Fluorescence decay in these types of PSI preparations has been previously investigated but never under the same experimental conditions. As a result we present consistent picture of excitation dynamics in algal PSI. Temporal evolution of fluorescence spectra can be generally described by three decay components with similar lifetimes in all samples (6–8 ps, 25–30 ps, 166–314 ps). In the PSI cores, the fluorescence decay is dominated by the two fastest components (~ 90%), which can be assigned to excitation energy trapping in the reaction center by reversible primary charge separation. Excitation dynamics in the PSI–LHCI preparations is more complex because of the energy transfer between the LHCI antenna system and the core. The average trapping time of excitations created in the well coupled LHCI antenna system is about 12–15 ps longer than excitations formed in the PSI core antenna. Excitation dynamics in PSI–LHCI complexes in whole living cells is very similar to that observed in isolated complexes. Our data support the view that chlorophylls responsible for the long-wavelength emission are located mostly in LHCI. We also compared in detail our results with the literature data obtained for plant PSI.     

Decay kinetics and quantum yields of fluorescence in photosystem I from Synechococcus elongatus with P700 in the reduced and oxidized state: are the kinetics of excited state decay trap-limited or transfer-limited?

Transfer and trapping of excitation energy in photosystem I (PS I) trimers isolated from Synechococcus elongatus have been studied by an approach combining fluorescence induction experiments with picosecond time-resolved fluorescence measurements, both at room temperature (RT) and at low temperature (5 K). Special attention was paid to the influence of the oxidation state of the primary electron donor P700. A fluorescence induction effect has been observed, showing a approximately 12% increase in fluorescence quantum yield upon P700 oxidation at RT, whereas at temperatures below 160 K oxidation of P700 leads to a decrease in fluorescence quantum yield ( approximately 50% at 5 K). The fluorescence quantum yield for open PS I (with P700 reduced) at 5 K is increased by approximately 20-fold and that for closed PS I (with P700 oxidized) is increased by approximately 10-fold, as compared to RT. Picosecond fluorescence decay kinetics at RT reveal a difference in lifetime of the main decay component: 34 +/- 1 ps for open PS I and 37 +/- 1 ps for closed PS I. At 5 K the fluorescence yield is mainly associated with long-lived components (lifetimes of 401 ps and 1.5 ns in closed PS I and of 377 ps, 1.3 ns, and 4.1 ns in samples containing approximately 50% open and 50% closed PS I). The spectra associated with energy transfer and the steady-state emission spectra suggest that the excitation energy is not completely thermally equilibrated over the core-antenna-RC complex before being trapped. Structure-based modeling indicates that the so-called red antenna pigments (A708 and A720, i.e., those with absorption maxima at 708 nm and 720 nm, respectively) play a decisive role in the observed fluorescence kinetics. The A720 are preferentially located at the periphery of the PS I core-antenna-RC complex; the A708 must essentially connect the A720 to the reaction center. The excited-state decay kinetics turn out to be neither purely trap limited nor purely transfer (to the trap) limited, but seem to be rather balanced.     

Ultrafast transient absorption studies on Photosystem I reaction centers from Chlamydomonas reinhardtii. 1. A new interpretation of the energy trapping and early electron transfer steps in Photosystem I

The energy transfer and charge separation kinetics in core Photosystem I (PSI) particles of Chlamydomonas reinhardtii has been studied using ultrafast transient absorption in the femtosecond-to-nanosecond time range. Although the energy transfer processes in the antenna are found to be generally in good agreement with previous interpretations, we present evidence that the interpretation of the energy trapping and electron transfer processes in terms of both kinetics and mechanisms has to be revised substantially as compared to current interpretations in the literature. We resolved for the first time i), the transient difference spectrum for the excited reaction center state, and ii), the formation and decay of the primary radical pair and its intermediate spectrum directly from measurements on open PSI reaction centers. It is shown that the dominant energy trapping lifetime due to charge separation is only 6-9 ps, i.e., by a factor of 3 shorter than assumed so far. The spectrum of the first radical pair shows the expected strong bleaching band at 680 nm which decays again in the next electron transfer step. We show furthermore that the early electron transfer processes up to approximately 100 ps are more complex than assumed so far. Several possibilities are discussed for the intermediate redox states and their sequence which involve oxidation of P700 in the first electron transfer step, as assumed so far, or only in the second electron transfer step, which would represent a fundamental change from the presently assumed mechanism. To explain the data we favor the inclusion of an additional redox state in the electron transfer scheme. Thus we distinguish three different redox intermediates on the timescale up to 100 ps. At this level no final conclusion as to the exact mechanism and the nature of the intermediates can be drawn, however. From comparison of our data with fluorescence kinetics in the literature we also propose a reversible first charge separation step which has been excluded so far for open PSI reaction centers. For the first time an ultrafast 150-fs equilibration process, occurring among exciton states in the reaction center proper, upon direct excitation of the reaction center at 700 nm, has been resolved. Taken together the data call for a fundamental revision of the present understanding of the energy trapping and early electron transfer kinetics in the PSI reaction center. Due to the fact that it shows the fastest trapping time observed so far of any intact PSI particle, the PSI core of C. reinhardtii seems to be best suited to further characterize the electron transfer steps and mechanisms in the reaction center of PSI.     

Ultrafast primary processes in PS I from Synechocystis sp. PCC 6803: roles of P700 and A(0)

The excitation transport and trapping kinetics of core antenna-reaction center complexes from photosystem I of wild-type Synechocystis sp. PCC 6803 were investigated under annihilation-free conditions in complexes with open and closed reaction centers. For closed reaction centers, the long-component decay-associated spectrum (DAS) from global analysis of absorption difference spectra excited at 660 nm is essentially flat (maximum amplitude <10(-5) absorbance units). For open reaction centers, the long-time spectrum (which exhibits photobleaching maxima at approximately 680 and 700 nm, and an absorbance feature near 690 nm) resembles one previously attributed to (P700(+) - P700). For photosystem I complexes excited at 660 nm with open reaction centers, the equilibration between the bulk antenna and far-red chlorophylls absorbing at wavelengths >700 nm is well described by a single DAS component with lifetime 2.3 ps. For closed reaction centers, two DAS components (2.0 and 6.5 ps) are required to fit the kinetics. The overall trapping time at P700 ( approximately 24 ps) is very nearly the same in either case. Our results support a scenario in which the time constant for the P700 --> A(0) electron transfer is 9-10 ps, whereas the kinetics of the subsequent A(0) --> A(1) electron transfer are still unknown.     

Chlorophyll-protein complexes of barley photosystem I

Photosystem I (PSI) preparations with a chlorophyll a/b ratio of 6.0 were isolated from barley thylakoids using two different methods. The high-molecular-mass complex (CP1a) which is resolved by non-denaturing gel electrophoresis had the same properties as a PSI preparation (PSI-200) isolated by Triton X-100 solubilisation of thylakoids followed by sucrose gradient ultracentrifugation. This material had a chlorophyll:P700 ratio of 208:1 and was composed of three different chlorophyll-protein complexes which could be separated from each other by solubilising the PSI preparation in dodecyl maltoside followed by sucrose gradient ultracentrifugation. Approximately half of the chlorophyll, including all the chlorophyll b, was located in two antenna complexes designated LHCI-680 and LHCI-730, which were identified by their characteristic low-temperature fluorescence emission spectra. The rest of the chlorophyll a was associated with the PSI reaction centre, P700 Chla-P1, which fluoresced at 720 nm. Each chlorophyll-protein complex had a unique polypeptide composition and characteristic circular dichroic and absorption spectra. The use of dodecyl maltoside instead of dodecyl sulphate resulted in a less denatured form of LHCI-680, which fluoresced at 690 nm at 77 K. One of the sucrose gradient fractions contained a complex consisting of only LHCI-730 and P700 Chla-P1 which fluoresced at 731 nm, indicating that LHCI-730 is structurally associated with P700 Chla-P1 and quenches its fluorescence. Approximately three-quarters of the light-harvesting antenna chlorophyll was in LHCI-730, but only about one-quarter of the normal complement of LHCI-730 was required to quench the reaction centre. By reducing the amount of Triton relative to the chlorophyll concentration, a PSI preparation (chlorophyll a/b ratio of 3.5) with a chlorophyll:P700 ratio of 300:1 was isolated. It contained no photosystem II, but a significant amount of LHCII which was functionally connected to the PSI reaction centre. Reconstitution studies demonstrated that excitation energy transfer from LHCII to PSI requires the presence of LHCI-680, and we propose that, in PSI, the following linear excitation energy transfer sequence occurs: LHCII----LHCI-680----LHCI-730----P700 Chla-P1.     

Fluorescence decay and spectral evolution in intact photosystem I of higher plants

A photosystem I preparation from maize, containing its full antenna complement (PSI-200) and in which detergent effects on chlorophyll coupling are almost completely absent, has been studied by time-resolved fluorescence techniques with approximately 5 ps resolution at 280 and 170 K in the wavelength interval of 690-780 nm. The data have been analyzed in terms of both the decay-associated spectra (DAS) and the time-resolved emission spectra (TRES). As in a previous room temperature study [Turconi, S., Weber, N., Schweitzer, D., Strotmann, H., and Holzwarth, A. R. (1994) Biochim. Biophys. Acta 1187, 324-334], the 280 K decay is well described by three DAS components in the 11-130 ps time range, the fastest of which displays both positive and negative amplitudes characteristic of excitation transfer from the bulk to the red antenna forms. Both the 57 and 130 ps components have all positive amplitudes and describe complex decay and equilibration processes involving the red forms. At 170 K, four major components in the 10-715 ps time range are required to describe the decay. The fastest represents bulk to red form transfer processes, while the 55, 216, and 715 ps decays, with all positive amplitudes, have maxima near 720, 730, and 740 nm, respectively, in accord with previous steady-state fluorescence measurements. The width and asymmetry of these DAS indicate that they are spectrally complex and represent decay and equilibration processes involving the red forms. Spectral evolution during the fluorescence decay process was analyzed in terms of the TRES. The red shifting of the TRES was analyzed in terms of the first central spectral moment (mean spectral energy) which is biexponential at both temperatures. The slower component, which describes equilibration between the red forms, leads to spectral red shifting during the entire fluorescence decay process, and the mean lifetimes of the spectral moments at 280 and 170 K (86 and 291 ps, respectively) are similar to the mean lifetimes of the fluorescence decays (119 and 384 ps, respectively). Thus, both spectral evolution and the trapping-associated fluorescence decay occur on a similar time scale, and both processes display a very similar temperature sensitivity. On the basis of these data, it is concluded that trapping in PSI-200 is to a large extent rate-limited by excitation diffusion in the antenna and in particular by the slow "uphill" transfer from the low-energy forms to the bulk and/or inner core chlorophyll molecules.     

On the spectral properties and excitation dynamics of long-wavelength chlorophylls in higher-plant photosystem I

In higher-plant Photosystem I (PSI), the majority of “red” chlorophylls (absorbing at longer wavelengths than the reaction centre P₇₀₀) are located in the peripheral antenna, but contradicting reports are given about red forms in the core complex. Here we attempt to clarify the spectroscopic characteristics and quantify the red forms in the PSI core complex, which have profound implication on understanding the energy transfer and charge separation dynamics. To this end we compare the steady-state absorption and fluorescence spectra and picosecond time-resolved fluorescence kinetics of isolated PSI core complex and PSI–LHCI supercomplex from Pisum sativum recorded at 77 K. Gaussian decomposition of the absorption spectra revealed a broad band at 705 nm in the core complex with an oscillator strength of three chlorophylls. Additional absorption at 703 nm and 711 nm in PSI–LHCI indicated up to five red chlorophylls in the peripheral antenna. Analysis of fluorescence emission spectra resolved states emitting at 705, 715 and 722 nm in the core and additional states around 705–710 nm and 733 nm in PSI–LHCI. The red states compete with P₇₀₀ in trapping excitations in the bulk antenna, which occurs on a timescale of ~20 ps. The three red forms in the core have distinct decay kinetics, probably in part determined by the rate of quenching by the oxidized P₇₀₀. These results affirm that the red chlorophylls in the core complex must not be neglected when interpreting kinetic experimental results of PSI.     

PMS: Photosystem I electron donor or fluorescence quencher

Light energy harvested by the pigments in Photosystem I (PSI) is used for charge separation in the reaction center (RC), after which the positive charge resides on a special chlorophyll dimer called P700. In studies on the PSI trapping kinetics, P700(+) is usually chemically reduced to re-open the RCs. So far, the information available about the reduction rate and possible chlorophyll fluorescence quenching effects of these reducing agents is limited. This information is indispensible to estimate the fraction of open RCs under known experimental conditions. Moreover, it would be important to understand if these reagents have a chlorophyll fluorescence quenching effects to avoid the introduction of exogenous singlet excitation quenching in the measurements. In this study, we investigated the effect of the commonly used reducing agent phenazine methosulfate (PMS) on the RC and fluorescence emission of higher plant PSI-LHCI. We measured the P700(+) reduction rate for different PMS concentrations, and show that we can give a reliable estimation on the fraction of closed RCs based on these rates. The data show that PMS is quenching chlorophyll fluorescence emission. Finally, we determined that the fluorescence quantum yield of PSI with closed RCs is 4% higher than if the RCs are open.     

Excitation energy transfer in native and unstacked thylakoid membranes studied by low temperature and ultrafast fluorescence spectroscopy

In this work, the transfer of excitation energy was studied in native and cation-depletion induced, unstacked thylakoid membranes of spinach by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission spectra at 5 K show an increase in photosystem I (PSI) emission upon unstacking, which suggests an increase of its antenna size. Fluorescence excitation measurements at 77 K indicate that the increase of PSI emission upon unstacking is caused both by a direct spillover from the photosystem II (PSII) core antenna and by a functional association of light-harvesting complex II (LHCII) to PSI, which is most likely caused by the formation of LHCII-LHCI-PSI supercomplexes. Time-resolved fluorescence measurements, both at room temperature and at 77 K, reveal differences in the fluorescence decay kinetics of stacked and unstacked membranes. Energy transfer between LHCII and PSI is observed to take place within 25 ps at room temperature and within 38 ps at 77 K, consistent with the formation of LHCII-LHCI-PSI supercomplexes. At the 150–160 ps timescale, both energy transfer from LHCII to PSI as well as spillover from the core antenna of PSII to PSI is shown to occur at 77 K. At room temperature the spillover and energy transfer to PSI is less clear at the 150 ps timescale, because these processes compete with charge separation in the PSII reaction center, which also takes place at a timescale of about 150 ps.     

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.     

Combined fluorescence and photovoltage studies on chlorosome containing bacteria

Energy transfer kinetics, primary charge separation, antenna size and excitonic connectivity of photosynthetic units (PSU) in whole cells of Chloroflexus aurantiacus were studied at room temperature by ps-fluorescence and ps-photovoltage as well as by stationary fluorescence-spectroscopy and fluorescence induction measurements. The fluorescence decay kinetics measured at different wavelengths are in accordance with the currently accepted sequential energy transfer from the chlorosomes via the baseplates to the B808–866 complexes and the final trapping in the RC with time constants of 19 ± 2 ps, 40 ± 4 ps and 90 ± 9 ps, respectively. However, the quantitative analysis of fluorescence spectra and the occurrence of slow phases in the fluorescence decays reveal that in whole cells a significant fraction of BChl c in the chlorosome and of BChl a in the baseplate is unconnected. The photovoltage kinetics consisted of two electrogenic phases with time constants of 118 ± 5 ps and 326 ± 35 ps and comparable electrogenicities. The first phase is ascribed to trapping from the B808-866 complexes by P+H_A- formation and the second one to charge stabilization on a quinone acceptor. Fluorescence induction curves displayed a pronounced sigmoidicity, indicating efficient lateral energy transfer between neighbored PSUs and a dense packing of 19 reaction centers (RC) beneath one chlorosome. A quantitative analysis of the fluorescence-induction curves at different excitation wavelengths allows the estimation of pigment stoichiometries (i.e. antenna sizes): BChl c/RC 794 and B808/RC 15.     

The State of Photosystem I Complexes in the Central Regions of Pea Granal Thylakoids

Grana-core and grana-margin fragments were obtained from pea (Pisum sativum L.) thylakoids, and both fractions contained photosystem I (PSI) complexes. The yield of these fractions exhibited variations for the plants grown during various periods of the summer season. Low-temperature fluorescence spectra, excitation spectra of long-wave fluorescence, and P700 kinetic characteristics were recorded for these fractions. PSI complexes in central granal regions were associated with PSII and the light-harvesting complexes of PSII, which followed from the excitation spectra of long-wave fluorescence and the kinetic characteristics of P700 light oxidation and dark reduction. The characteristics of the margin regions were changed depending on the fraction yield. If the yield was low, marginal fragments contained mainly PSI complexes. When the yield increased, PSI associates with PSII appeared. A spatial distribution and state of PSI complexes in granal thylakoids are discussed as related to the size and composition of the light-harvesting antenna.     

Excitation energy transfer in thylakoid membranes from Chlamydomonas reinhardtii lacking chlorophyll b and with mutant Photosystem I

Energy trapping in Photosystem I (PS I) was studied by time-resolved fluorescence spectroscopy of PS II-deleted Chl b-minus thylakoid membranes isolated from site-directed mutants of Chlamydomonas reinhardtii with specific amino acid substitutions of a histidine ligand to P₇₀₀. In vivo the fluorescence of the PS I core antenna in mutant thylakoids with His-656 of PsaB replaced by asparagine, serine or phenylalanine is characterized by an increase in the lifetime of the fast decay component ascribed to the energy trapping in PS I (25 ps in wild type PS I with intact histidine-656, 50 ps in the mutant PS I with asparagine-656 and 70 ps in the mutant PS I with phenylalanine-656). Assuming that the excitation dynamics in the PS I antenna are trap-limited, the increase in the trapping time suggests a decrease in the primary charge separation rate. Western blot analysis showed that the mutants accumulate significantly less PS I than wild type. Spectroscopically, the mutations lead to a decrease in relative quantum yield of the trapping in the PS I core and increase in relative quantum yield of the fluorescence decay phase ascribed to uncoupled chlorophyll–protein complexes which suggests that improper assembly of PS I and LHC in the mutant thylakoids may result in energy uncoupling in PS I.     

PSI-SMALP,a Detergent-free Cyanobacterial Photosystem I,Reveals Faster Femtosecond Photochemistry

Cyanobacterial photosystem I (PSI) functions as a light-driven cyt c₆-ferredoxin/oxidoreductase located in the thylakoid membrane. In this work, the energy and charge transfer processes in PSI complexes isolated from Thermosynechococcus elongatus via conventional n-dodecyl-β-D-maltoside solubilization (DM-PSI) and a, to our knowledge, new detergent-free method using styrene-maleic acid copolymers (SMA-PSI) have been investigated by pump-to-probe femtosecond laser spectroscopy. In DM-PSI preparations excited at 740 nm, the excitation remained localized on the long-wavelength chlorophyll forms within 0.1–20 ps and revealed little or no charge separation and oxidation of the special pair, P700. The formation of ion-radical pair P700⁺A1⁻ occurred with a characteristic time of 36 ps, being kinetically controlled by energy transfer from the long-wavelength chlorophyll to P₇₀₀. Quite surprisingly, the detergent-free SMA-PSI complexes upon excitation by these long-wave pulses undergo an ultrafast (<100 fs) charge separation in ∼45% of particles. In the remaining complexes (∼55%), the energy transfer to P₇₀₀ occurred at ∼36 ps, similar to the DM-PSI. Both isolation methods result in a trimeric form of PSI, yet the SMA-PSI complexes display a heterogenous kinetic behavior. The much faster rate of charge separation suggests the existence of an ultrafast pathway for charge separation in the SMA-PSI that may be disrupted during detergent isolation.     

Determination of the excitation migration time in Photosystem II consequences for the membrane organization and charge separation parameters

The fluorescence decay kinetics of Photosystem II (PSII) membranes from spinach with open reaction centers (RCs), were compared after exciting at 420 and 484 nm. These wavelengths lead to preferential excitation of chlorophyll (Chl) a and Chl b, respectively, which causes different initial excited-state populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 4.3+/-1.8 ps slower upon 484 nm excitation for preparations that contain on average 2.45 LHCII (light-harvesting complex II) trimers per reaction center. Using a recently introduced coarse-grained model it can be concluded that the average migration time of an electronic excitation towards the RC contributes approximately 23% to the overall average trapping time. The migration time appears to be approximately two times faster than expected based on previous ultrafast transient absorption and fluorescence measurements. It is concluded that excitation energy transfer in PSII follows specific energy transfer pathways that require an optimized organization of the antenna complexes with respect to each other. Within the context of the coarse-grained model it can be calculated that the rate of primary charge separation of the RC is (5.5+/-0.4 ps)(-1), the rate of secondary charge separation is (137+/-5 ps)(-1) and the drop in free energy upon primary charge separation is 826+/-30 cm(-1). These parameters are in rather good agreement with recently published results on isolated core complexes [Y. Miloslavina, M. Szczepaniak, M.G. Muller, J. Sander, M. Nowaczyk, M. R?gner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].     

Long-wavelength chlorophylls in photosystem I of cyanobacteria: Origin,localization, and functions

The structural organization of photosystem I (PSI) complexes in cyanobacteria and the origin of the PSI antenna long-wavelength chlorophylls and their role in energy migration, charge separation, and dissipation of excess absorbed energy are discussed. The PSI complex in cyanobacterial membranes is organized preferentially as a trimer with the core antenna enriched with long-wavelength chlorophylls. The contents of long-wavelength chlorophylls and their spectral characteristics in PSI trimers and monomers are species-specific. Chlorophyll aggregates in PSI antenna are potential candidates for the role of the long-wavelength chlorophylls. The red-most chlorophylls in PSI trimers of the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus can be formed as a result of interaction of pigments peripherally localized on different monomeric complexes within the PSI trimers. Long-wavelength chlorophylls affect weakly energy equilibration within the heterogeneous PSI antenna, but they significantly delay energy trapping by P700. When the reaction center is open, energy absorbed by long-wavelength chlorophylls migrates to P700 at physiological temperatures, causing its oxidation. When the PSI reaction center is closed, the P700 cation radical or P700 triplet state (depending on the P700 redox state and the PSI acceptor side cofactors) efficiently quench the fluorescence of the long-wavelength chlorophylls of PSI and thus protect the complex against photodestruction.     

Far-red light-regulated efficient energy transfer from phycobilisomes to photosystem I in the red microalga Galdieria sulphuraria and photosystems-related heterogeneity of phycobilisome population

Phycobilisomes (PBS) are the major photosynthetic antenna complexes in cyanobacteria and red algae. In the red microalga Galdieria sulphuraria, action spectra measured separately for photosynthetic activities of photosystem I (PSI) and photosystem II (PSII) demonstrate that PBS fraction attributed to PSI is more sensitive to stress conditions and upon nitrogen starvation disappears from the cell earlier than the fraction of PBS coupled to PSII. Preillumination of the cells by actinic far-red light primarily absorbed by PSI caused an increase in the amplitude of the PBS low-temperature fluorescence emission that was accompanied by the decrease in PBS region of the PSI 77 K fluorescence excitation spectrum. Under the same conditions, fluorescence excitation spectrum of PSII remained unchanged. The amplitude of P700 photooxidation in PBS-absorbed light at physiological temperature was found to match the fluorescence changes observed at 77 K. The far-red light adaptations were reversible within 2-5min. It is suggested that the short-term fluorescence alterations observed in far-red light are triggered by the redox state of P700 and correspond to the temporal detachment of the PBS antenna from the core complexes of PSI. Furthermore, the absence of any change in the 77 K fluorescence excitation cross-section of PSII suggests that light energy transfer from PBS to PSI in G. sulphuraria is direct and does not occur through PSII. Finally, a novel photoprotective role of PBS in red algae is discussed.