Primary Charge Separation In Photosystem Research Articles

Current state of the primary charge separation mechanism in photosystem I of cyanobacteria.

This review analyzes new data on the mechanism of ultrafast reactions of primary charge separation in photosystem I (PS I) of cyanobacteria obtained in the last decade by methods of femtosecond absorption spectroscopy. Cyanobacterial PS I from many species harbours 96 chlorophyll a (Chl a) molecules, including six specialized Chls denoted Chl1A/Chl1B (dimer P700, or PAPB), Chl2A/Chl2B, and Chl3A/Chl3B arranged in two branches, which participate in electron transfer reactions. The current data indicate that the primary charge separation occurs in a symmetric exciplex, where the special pair P700 is electronically coupled to the symmetrically located monomers Chl2A and Chl2B, which can be considered together as a symmetric exciplex Chl2APAPBChl2B with the mixed excited (Chl2APAPBChl2B)* and two charge-transfer states P700 +Chl2A - and P700 +Chl2B -. The redistribution of electrons between the branches in favor of the A-branch occurs after reduction of the Chl2A and Chl2B monomers. The formation of charge-transfer states and the symmetry breaking mechanisms were clarified by measuring the electrochromic Stark shift of β-carotene and the absorption dynamics of PS I complexes with the genetically altered Chl 2B or Chl 2A monomers. The review gives a brief description of the main methods for analyzing data obtained using femtosecond absorption spectroscopy. The energy levels of excited and charge-transfer intermediates arising in the cyanobacterial PS I are critically analyzed.

Mechanism of adiabatic primary electron transfer in photosystem I: Femtosecond spectroscopy upon excitation of reaction center in the far-red edge of the QY band

The ultrafast primary charge separation in Photosystem I (PS I) excited by femtosecond pulses centered at 720 and 760nm was studied by pump-to-probe laser spectroscopy. The absorbance in the red edge of PS I absorption spectrum has an unusual exponential dependence on wavelength. The cutoff of short wavelength components of 760nm pulse allows direct excitation of reaction center chlorophyll molecules without involvement of light-harvesting antenna. The transient spectrum manifests the features of the primary ion-radical pair P700+A0− at time delay <180fs, followed by formation of the secondary pair P700+A1− with a characteristic time of 26ps. The obtained data are rationalized in the framework of adiabatic three-state model that includes the chlorophyll dimer P700 and two symmetrically arranged nearest chlorophyll molecules of A0. The arrangement of chlorophylls results in strong electronic coupling between P700 and A0. Excitation in the maximum of P700 absorption generates electronic states with the highest contribution from P700*, whereas excitation in the far-red edge predominantly generates charge transfer state P700+A0− in both branches of redox-cofactors. The three-level model accounts for a flat-bottomed potential surface of the excited state and adiabatic character of electron transfer between P700 and A0, providing a microscopic explanation of the ultrafast formation of P700+A0− and exponential decline of PS I absorption.

Effect of the P700 pre-oxidation and point mutations near A 0 on the reversibility of the primary charge separation in Photosystem I from Chlamydomonas reinhardtii

Time-resolved fluorescence studies with a 3-ps temporal resolution were performed in order to: (1) test the recent model of the reversible primary charge separation in Photosystem I (Müller et al., 2003; Holwzwarth et al., 2005, 2006), and (2) to reconcile this model with a mechanism of excitation energy quenching by closed Photosystem I (with P700 pre-oxidized to P700 +). For these purposes, we performed experiments using Photosystem I core samples isolated from Chlamydomonas reinhardtii wild type, and two mutants in which the methionine axial ligand to primary electron acceptor, A 0, has been change to either histidine or serine. The temporal evolution of fluorescence spectra was recorded for each preparation under conditions where the “primary electron donor,” P700, was either neutral or chemically pre-oxidized to P700 +. For all the preparations under study, and under neutral and oxidizing conditions, we observed multiexponential fluorescence decay with the major phases of ∼ 7 ps and ∼ 25 ps. The relative amplitudes and, to a minor extent the lifetimes, of these two phases were modulated by the redox state of P700 and by the mutations near A 0: both pre-oxidation of P700 and mutations caused slight deceleration of the excited state decay. These results are consistent with a model in which P700 is not the primary electron donor, but rather a secondary electron donor, with the primary charge separation event occurring between the accessory chlorophyll, A, and A 0. We assign the faster phase to the equilibration process between the excited state of the antenna/reaction center ensemble and the primary radical pair, and the slower phase to the secondary electron transfer reaction. The pre-oxidation of P700 shifts the equilibrium between the excited state and the primary radical pair towards the excited state. This shift is proposed to be induced by the presence of the positive charge on P700 +. The same charge is proposed to be responsible for the fast A +A 0 − → AA 0 charge recombination to the ground state and, in consequence, excitation quenching in closed reaction centers. Mutations of the A 0 axial ligand shift the equilibrium in the same direction as pre-oxidation of P700 due to the up-shift of the free energy level of the state A +A 0 −.

Temperature-Dependent Energy Gap of the Primary Charge Separation in Photosystem I: Study of Delayed Fluorescence at 77−268 K

The dynamics of fluorescence decay and charge recombination were studied in the ether-extracted photosystem I reaction center isolated from spinach with picosecond resolution over a wide time range up to 100 ns. At all temperatures from 268 to 77 K, a slow fluorescence decay component with a 30-40 ns lifetime was detected. This component was interpreted as a delayed fluorescence emitted from the singlet excited state of the primary donor P700*, which is repopulated through charge recombination that was increased by the lack of secondary acceptor phylloquinone in the sample. Analysis of the fluorescence kinetics allowed estimation of the standard free-energy difference -DeltaG between P700* and the primary radical pair (P700(+)A0(-)) state over a wide temperature range. The values of -DeltaG were estimated to be 160/36 meV at 268/77 K, indicating its high sensitivity to temperature. A temperature-dependent -DeltaG value was also estimated in the delayed fluorescence of the isolated photosystem I in which the secondary acceptor quinone was partially prereduced by preillumination in the presence of dithionite. The results revealed that the temperature-dependent -DeltaG is a universal phenomenon common with the purple bacterial reaction centers, photosystem II and photosystem I reaction centers.

Sensitization of the Primary Charge Separation in Photosystem I to Green Light by an Amphiphilic Polymer Bearing Rhodamine 6G

Abstract An amphiphilic polymer carrying an artificial fluorescent dye, Rhodamine 6G (Rh6G), acts as a good solubilizing agent for a hydrophobic membrane protein, the photosystem (PS) I complex from Spirulina platensis, and at the same time as an efficient sensitizing agent in the green wavelength region, resulting in an enhancement of the primary charge separation within the complex by ca. 1.5-fold.

The analysis of site-directed mutants in the glutamate 189 and histidine 190 in the D1 protein of photosystem II

The primary charge separation in photosystem (PS) II takes place within 3 ps, generating highly oxidative P680+. Site-directed mutagenesis has been proven to be a valuable tool for elucidating the function of each amino acid residue in PSII. Several mutants at glutamate-189 and histidine-190 in the putative cd-helix of the D1 protein were constructed in Chlamydomonas reinhardtii, and the effects of mutation on PS II functions were studied. In E189Q and E189L mutants, they were able to grow photoautotrophycally, but the oxygen evolution activity decreased to a half of the wild type. On the other hand, E189D and H190R mutants lost the oxygen evolution activity, which led to the non-photoautotrophical phenotype. In order to obtain further insights on the effects of the mutations, two-dimensional fluorescence photon counting experiments were carried out. The results showed that the fluorescence lifetime at 77K was markedly prolonged in all mutants, suggesting that the yield of the charge separation becomes to be lower due to the decrease in redox potential of P680 and/or Yz. In PS II reaction center, isolated from His-tagged versions of these mutants, the activity of pheophytin photoacummulation was reduced to 60-70 % of the control. Therefore, it is indicated that these two amino acids exist in the close vicinity of P680 and play important role(s) in the charge separation, in addition to their involvement to the donor side function as revealed E189D and H190R mutants.

Transient Absorption Studies of the Primary Charge Separation in Photosystem II

Femtosecond transient absorption studies of the primary charge separation in photosystem II (PSII) are presented. A careful study of the dependence of the observed signal on laser intensity demonstrates that the multiple excitation of reaction centers produces additional fast components not observed at low excitation energy. In the regime where the observed signals are linear with excitation energy, a 20 ± 2 ps rise of the pheophytin anion absorption, bleach of the pheophytin Qx absorption, and appearance of the chlorophyll cation absorption are observed. Three different protocols, involving varying exposure of the PSII complex to the detergent Triton X−100, are used to prepare D1-D2-Cyt b559 complexes from spinach. The kinetic signals are independent of the method of sample preparation.

Primary Photochemical Processes in P700-Enriched Photosystem I Particles: Trap-Limited Excitation Decay and Primary Charge Separation

The energy transfer and primary charge separation in photosystem I (PS I) reaction center (RC) particles with an antenna size of 12 chlorophyll/P700 were studied by subpicosecond transient absorption spectroscopy. Upon excitation of the particles at 638 nm under the donor chlorophyll (P700)-neutral conditions, the transition from the excited state of chlorophylls to the charge-separated state of P700[sup +]A[sub 0][sup [minus]] (A[sub 0]; acceptor chlorophyll [alpha]) proceeded with a time constant of 6.5 ps at [approximately]278 K. This time constant was obtained from the absorption increase of P700[sup +]A[sub 0][sup [minus]] at 740 ([+-]10) nm, where excited chlorophylls gave only a negligible absorbance change. The distribution of electronic excitation energy among different chlorophyll forms, which was monitored in the wavelength region 650-720 nm, did not show and significant change from 500 fs to 12 ps under both the P700-preoxidized and -neutral conditions. Comparison of the experimental distribution with simulated ones suggests that the excitation energy is distributed within 500 fs highly on P700 and also among other higher-energy chlorophyll forms. 55 refs., 9 figs.

Primary charge separation in photosystem I: a two-step electrogenic charge separation connected with P700+A0- and P700+A1- formation.

A PS I membrane preparation from a PS II deficient mutant of Synechocystis sp. PCC 6803 (psb DI/DII/C) was investigated by picosecond photovoltage and fluorescence measurements. The photovoltage kinetics show two distinct phases. At low excitation energies the fast phase correlates with the fluorescence decay time constant of 22 +/- 4 ps. This phase is ascribed to the trapping of excitons as described by the reaction (AntiP700)* A0-->AntiP700+A0-. In addition to this phase, the photovoltage displays a second rising phase of smaller amplitude with a time constant of 50 +/- 15 ps. We assign the latter phase to further electron transfer from A0 to the secondary acceptor, A1. Assuming the protein as a homogeneous dielectric, our results suggest that the transmembrane distance A0-A1 spans only a small part (20 +/- 8%) of the distance P700-A1.

INVARIABLE TRAPPING TIMES IN PHOTOSYSTEM I UPON EXCITATION OF MINOR LONG‐WAVELENGTH‐ABSORBING PIGMENTS

AbstractThe primary charge separation in photosystem (PS) I was measured on stacked pea thylakoids using the light‐gradient photovoltage technique. Upon 532 nm excitation with picosecond flashes, a trapping time of 80 ± 10 ps for PS I was found, which is in close agreement with literature data. In the wavelength range between 700 nm and 717 nm the trapping time was essentially the same although there was an indication for a slight decrease. To further analyze the data we performed a spectral decomposition of PS I with Chi a and b solvent spectra. This procedure yielded bands at around 682 nm, 690 nm, 705 nm and 715 nm. According to this decomposition, a selective excitation of long‐wavelength antenna pigments at wavelengths Λ &gt; 710 nm is possible, because the direct excitation of the main 682 nm band is small compared to the excitation of the two most red‐shifted bands. The invariability of the trapping time of the excitation wavelength suggests thermal equilibration of the excitation energy among all antenna pigments according to their excited state energy levels and their abundance. Hence, we conclude that trapping in PS I is essentially rate‐limited by the primary charge separation much as it is the case in PS II. Then, according to our spectral decomposition in a time constant of2–3 ps is predicted for the primary charge separation in PS I.

Photoinhibition Causes Loss of Photochemical Activity Without Degradation of D1 Protein

Illumination of isolated thylakoids or intact leaves with excess light resulted in a decline in photosynthetic activity measured as primary charge separation in photosystem II (ΔA320), photosystem II- dependent electron transport, or leaf oxygen evolution. It is concluded that the primary damage causing photoinhibition involves inactivation of the reaction centre function, and that degradation of Dl may be a consequence of that event.

Primary charge separation in photosystem II involves two electrogenic steps

Similarities in the piginent and subunit composition and sequence homologies suggest a common evolutionary origin for the reaction centers (RCs) of photosynthetic purple bacteria and photosystem II (PS II) of green plants [1]. Besides these similarities, significant functional differences exist that make comparative studies between both types of RCs interesting. We report here (i) on the electric detection of two kinetically distinct phases of the primary charge separation in PS II with 100 and 500 ps reflecting trapping and charge stabilization, and (ii) the determination of the dielectrically weighted transmembrane distances between the primary donor (P), the pheophytin intermediary acceptor (I), and the first quinoic acceptor (QA). These data are compared with those for purple bacteria. In PS II the reduction of QA is 2–3-times slower, although the dielectric distance between I and Q A appears to be significantly shorter than in purple bacteria.

Trapping and annihilation in the antenna system of Photosystem I

The primary charge separation in Photosystem I of pea chloroplasts was measured as a photovoltage in the pico- and nanosecond time range by applying laser flashes at 532 nm of variable energy and different duration (12 ns and 30 ps, respectively). Contributions to the photovoltage from Photosystem II was eliminated by addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination. The dependence of the photovoltage amplitude on the excitation energy could be described by an exponential saturation law when the excitation flash had a duration of 12 ns. Nearly the same dependence was found when the excitation source was the train of a mode-locked laser (approx. ten 30-ps flashes spaced by 7 ns; highest energy of a single flash, 80 μJ / cm −2). Even with single 30-ps flashes the photovoltage was only slightly smaller than the one elicited by 12-ns flashes of the same energy. These findings demonstrate that trapping of excitation energy by the reaction center of Photosystem I is much more effective than losses by annihilation and other loss processes. The photovoltage yield was nearly independent of the fraction of closed traps, thus demonstrating that the absorption cross section of Photosystem I is not altered by the closing of its reaction centers. By recording the rise time of the photovoltage with our highest time resolution we found that the trapping rate of the excitation energy in Photosystem I depended on the energy of the 30-ps flashes: at low excitation energies (less than 10 14 photons / cm 2 per pulse) trapping occurred within 90 ± 15 ps and at high excitation energy (10 15 photons / cm 2 per pulse) trapping and charge stabilization occurred within the time resolution of the apparatus, i.e., up to 50 ps. The trapping rate at low energies is in agreement with the one determined by fluorescence decay kinetics. Up to 50 ns there was no further detectable electrogenic phase (neither forward nor backward reactions). This demonstrates that all the electrogenicity, produced by the charge separation, takes place in less than 50 ps.

Isolation of a photosystem II reaction center consisting of D-1 and D-2 polypeptides and cytochrome b -559

A photosystem II reaction center complex consisting of D-1 and D-2 polypeptides and cytochrome b-559 was isolated from spinach grana thylakoids, treated with 4% (wt/vol) Triton X-100, by ion-exchange chromatography using DEAE-Toyopearl 650S. The isolated complex appears to contain five chlorophyll a, two pheophytin a, one beta-carotene, and one or two cytochrome b-559 heme(s) (molar ratio) and exhibits a reversible absorbance change attributable to the photochemical accumulation of reduced pheophytin typical for the intermediary electron acceptor of photosystem II reaction center. These results strongly suggest that the site of primary charge separation in photosystem II is located on the heterodimer composed of D-1 and D-2 subunits.

Non-electrogenic charge recombination in Photosystem II as a source of sub-millisecond luminescence

The luminescence emitted by Photosystem II at times between 1 μs and 1 ms after flash illumination was studied. Membrane potentials of up to 1 V, generated in osmotically swollen chloroplasts by externally applied electrical field pulses, had no effect on the decay components of this luminescence. Instead the field induced an additional, much slower luminescence due to a stimulated recombination of the primary charge separation in Photosystem II centers. The field-insensitive luminescence, in contrast to the field-induced luminescence, was independent of the redox state of the primary acceptor Q, and appeared not to originate from the same Photosystem II centers. The decay kinetics consisted mainly of two phases with 10 and 60 μs halftimes. The initial amplitude could be restored at all times by a second flash, indicating that both phases are due to a reversal of the field-insensitive charge separation. In the presence of DCMU and hydroxylamine, no luminescence was observed between 10 μs and 1 ms after the flash. Presumably the reoxidation of the electron acceptor was significantly slowed down, but it was still much faster than that of Q −.

Primary reactions of Photosystem II at low pH. I. Prompt and delayed fluorescence

Prompt and delayed chlorophyll fluorescence have been studied in broken spinach chloroplasts at pH values down to 2.6. No direct effect of low pH on the primary charge separation in Photosystem II was observed. The irreversible inactivation of a secondary electron donor in a narrow pH range around pH 4.5 was demonstrated. At lower pH values the photooxidized form of a more primary electron donor, revealed by its efficient fluorescence quenching, was reduced with a half time of about 200 μs, 25% by another electron donor and 75% by back reaction with the reduced acceptor. The electron donation had a half time of 800 μs and was practically irreversible. The back reaction had a pH dependent half time: about 270 μs at pH 4 and increasing towards lower pH. The competition of both reactions resulted in a net efficiency of the charge separation at pH 4 of 25%, increasing towards lower pH.