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.     

Electrochromic detection of a coherent component in the formation of the charge pair P(+)H(L)(-) in bacterial reaction centers

We demonstrate coupling of an intraprotein electron transfer reaction to coherent vibrational motions. The kinetics of charge separation toward the radical pair state P(+)H(L)(-) were studied in reaction centers of Rhodobacter sphaeroides at 15 K. The electrochromic shift of the bacteriochlorophyll monomers is the most prominent spectral feature associated with this charge displacement. The newly reported absolute absorption spectrum of the P(+)H(L)(-) state is discussed in terms of this shift. In wild-type reaction centers, the rise kinetics of the electrochromic shift display a small but significant 30 cm(-)(1) periodic modulation (period of approximately 1 ps). This modulation is also present in FL181Y mutant reaction centers, where overall charge separation is somewhat more rapid than in the wild-type reaction center. In contrast, in YM210L mutant reaction centers, where the charge separation is much slower, the modulation is absent. The conclusion that the motion along the reaction coordinate has a 30 cm(-)(1) coherent component is discussed in light of possible mechanisms of electron transfer.     

The nature and magnitude of the charge-separation reactions of ubiquinol cytochrome c2 oxidoreductase

The transdielectric charge separation reaction catalyzed by the ubiquinol-cytochrome c2 oxidoreductase is achieved in two fractional steps. We present a detailed analysis which addresses the nature of the charge transferred, the redox groups directly involved in charge separation and the contributions of each to the full charge separation catalyzed by the enzyme. Accounting for light saturation effects, reaction centers unconnected to cytochrome c2 and the fraction of total cytochrome bc1 turning over per flash permits detailed quantitation of: (1) the red carotenoid bandshift associated with electron transfer between ubiquinol at site Qz and the high- (2Fe2S center, cytochrome c1) and low-potential (cytochrome bL, cytochrome bH) components of cytochrome bc1; (2) the blue bandshift accompanying reduction of cytochrome bH by ubiquinol via site Qc (the reverse of the physiological reaction); and (3) the effect of delta psi on the Qc-cytochrome bH redox equilibrium. Studies were performed at pH values above and below the redox-linked pK values of the redox centers known to be involved in each reaction at equilibrium. The conclusions of this study may be summarized as follows: (1) there is no transdielectric charge separation apparent in the redox reactions between Qz and cytochrome bL, 2Fe2S and cytochrome c1 (in agreement with Glaser, E. and Crofts, A.R. (1984) Biochim. Biophys. Acta 766, 223-235), i.e., charge separation accompanies electron transfer between cytochrome bL and cytochrome bH; (2) the redox reactions between cytochrome bL and cytochrome bH and between cytochrome bH and Qc constitute the full electrogenic span; (3) electron transfer between cytochrome bL and cytochrome bH contributes approx. 60% of this span; (4) electron transfer between cytochrome bH and Qc contributes 45-55% as calculated from the blue bandshift or the delta psi-dependent equilibrium shift; (5) there is no discernable pH dependence of the Qz-cytochrome bH or Qc-cytochrome bH charge-separation reactions; (6) cytochrome bL, Qz, 2Fe2S, and cytochrome c1 are on the periplasmic side out of the low dielectric part of the membrane while cytochrome bH is buried in the low dielectric medium; (7) electron transfer is the predominant if not the sole contributor to charge separation; (8) Qz and Qc are on opposite sides of the membrane dielectric profile.     

High throughput engineering to revitalize a vestigial electron transfer pathway in bacterial photosynthetic reaction centers

Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B branch cofactors, but the best overall electron transfer yields are still low. In an attempt to better realize the architectural and energetic factors that underlie the directionality and yields of electron transfer, sites within the protein-cofactor complex were targeted in a directed molecular evolution strategy that implements streamlined mutagenesis and high throughput spectroscopic screening. The polycistronic approach enables efficient construction and expression of a large number of variants of a heteroligomeric complex that has two intimately regulated subunits with high sequence similarity, common features of many prokaryotic and eukaryotic transmembrane protein assemblies. The strategy has succeeded in the discovery of several mutant reaction centers with increased efficiency of the B pathway; they carry multiple substitutions that have not been explored or linked using traditional approaches. This work expands our understanding of the structure-function relationships that dictate the efficiency of biological energy-conversion reactions, concepts that will aid the design of bio-inspired assemblies capable of both efficient charge separation and charge stabilization.     

Evolution of uphill electron transfer

The evolution of photosynthetic energy storage is considered. The primary event in primordial inorganic or organic photoreceptors was charge separation at the expense of light quantum energy. The subsequent improvement of energy storage was attained by separately channeling electrons and “holes” to prevent back reactions. The anisotropic arrangement of photoreceptors in the primary membrane caused a coupling of photochemical charge separation to subsequent ion dislocation and was a prerequisite of primary photophosphorylation. The gradual improvement of the molecular organization of photoreceptor units resulted in antenna and reaction center development. The “hole” was primary located on a peculiar photoreceptor form and the electron passed by tunneling through the chain of intermediate carriers (chlorophylls and pheophytins); thus long-lived charge separation was achieved. The use of the electrons and the “holes” stored in reaction centers for the functioning of the photosynthetic electron transfer chain was realized by cyclic and non-cyclic pathways when the coupling of two photochemical events became the more perfect mechanism to use water molecule as an ultimate electron donor. The appearance of primitive cells inevitably required the coupling of the solar energy conversion mechanism to the reproduction mechanism which used stored solar energy.     

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 mus, 25% by another electron donor and 75% by back reaction with the reduced acceptor. The electron donation had a half time of 800 mus and was practically irreversible. The back reaction had a pH dependent half time: about 270 mus 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.     

Electron transfer in photosystem II

The picture presently emerging from studies on the mechanism of photosystem II electron transport is discussed. The reactions involved in excitation trapping, charge separation and stabilization of the charge pair in the reaction center, followed by the reactions with the substrates, plastoquinone reduction and water oxidation, are described successively. Finally, a brief discussion on photosystem II heterogeneity is presented.     

Coupled electron transfers in artificial photosynthesis

Light-induced charge separation in molecular assemblies has been widely investigated in the context of artificial photosynthesis. Important progress has been made in the fundamental understanding of electron and energy transfer and in stabilizing charge separation by multi-step electron transfer. In the Swedish Consortium for Artificial Photosynthesis, we build on principles from the natural enzyme photosystem II and Fe-hydrogenases. An important theme in this biomimetic effort is that of coupled electron-transfer reactions, which have so far received only little attention. (i) Each absorbed photon leads to charge separation on a single-electron level only, while catalytic water splitting and hydrogen production are multi-electron processes; thus there is the need for controlling accumulative electron transfer on molecular components. (ii) Water splitting and proton reduction at the potential catalysts necessarily require the management of proton release and/or uptake. Far from being just a stoichiometric requirement, this controls the electron transfer processes by proton-coupled electron transfer (PCET). (iii) Redox-active links between the photosensitizers and the catalysts are required to rectify the accumulative electron-transfer reactions, and will often be the starting points of PCET.     

Rate constants of primary reversible reactions of electron transfer do not depend on temperature. Numerical experiment

The process of irreversible photochemical charge separation in photosynthetic bacterial reaction centers is proposed to be characterized by the effective rate constant. A formula to compute this effective rate constant is derived. Similar rate constant was previously considered by R.A. Marcus (Marcus R.A. 1956. J. Chem. Phys. 24, 966–978) in order to describe nonphotochemical intermolecular electron transfer. The effective rate constant of the irreversible charge separation in photosynthetic bacterial reaction centers is shown to depend on the temperature. In contrast, rate constants of the forward electron transfer from the excited singlet primary donor to the bacteriochlorophyll and from its ion-radical to the bacteriopheophytin acceptor do not depend on temperature.     

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.     

Primary photochemistry of reaction centers from the photosynthetic purple bacteria

Photosynthetic organisms transform the energy of sunlight into chemical potential in a specialized membrane-bound pigment-protein complex called the reaction center. Following light activation, the reaction center produces a charge-separated state consisting of an oxidized electron donor molecule and a reduced electron acceptor molecule. This primary photochemical process, which occurs via a series of rapid electron transfer steps, is complete within a nanosecond of photon absorption. Recent structural data on reaction centers of photosynthetic bacteria, combined with results from a large variety of photochemical measurements have expanded our understanding of how efficient charge separation occurs in the reaction center, and have changed many of the outstanding questions.Abbreviations BChl bacteriochlorophyll - P a dimer of BChl molecules - BPh bacteriopheophytin - Q_A and Q_B quinone molecules - L, M and H light, medium and heavy polypeptides of the reaction center     

Theoretical study on primary reaction of photosynthetic bacteria

Theoretical calculation was carried out on the primary electron donor P_(870) of photosynthetic bacteria. The results show that: (ⅰ) the bimolecular structure of the primary electron donor is more advantageous in energy than monomolecular structure; (ⅱ) the initial configuration of primary electron donor is no longer stable and changes to the configuration with lower energy and chemical reactivity after the charge separation. In the P_(870), such structural change is completed through the rotation of C_3 acetyl, so the oxygen atom of acetyl interacts with the magnesium atom of another bacterio-chlorophyll molecule, and the total energy and chemical reactivity are reduced evidently. It is suggested that the structural change of the primary electron donor is important in preventing the occurrence of charge recombination during the primary reaction and maintaining the high efficiency of the conversion of sun-light to chemical energy. A new mechanism of primary reaction has been proposed, which can give r     

A comparative look at the first few milliseconds of the light reactions of photosynthesis

This mini-review describes the current state of our understanding of the structure and function of the photosynthetic light-harvesting and reaction centres. A comparative approach is used in order to highlight the underlying principles that must be satisfied for efficient energy-transfer (light-harvesting) and electron transfer (charge separation in the reaction centres).     

Femtosecond charge separation in organized assemblies: free-radical reactions with pyridine nucleotides in micelles

Femtosecond laser UV pulse-induced charge separation and electron transfer across a polar interface have been investigated in anionic aqueous micells (sodium lauryl sulfate) containing an aromatic hydrocarbon (phenothiazine). The early events of the photoejection of the electron from the micellized chromophore and subsequent reaction of electron with the aqueous perimicellar phase have been studied by ultrafast infrared and visible absorption spectroscopy. The charge separation (chromophore +...e-) inside the micelle occurs in less than 10(-13) s (100 fs). The subsequent thermalization and localization of the photoelectron in the aqueous phase are reached in 250 fs. This results in the appearance of an infrared band assigned to a nonrelaxed solvated electron (presolvated state). This transient species relaxes toward the fully solvated state of the electron in 270 fs. In anionic aqueous micelles containing pyridine dinucleotides at high concentration (0.025-0.103 M), a single electron transfer can be initiated by femtosecond photoionization of phenothiazine. The one-electron reduction of the oxidized pyridine dinucleotide leads to the formation of a free pyridinyl radical. The bimolecular rate constant of this electron transfer depends on both the pH of the micellar system and the concentration of oxidized acceptor. The free-radical reaction is analyzed in terms of the time dependence of a diffusion-controlled process. In the first 2 ps following the femtosecond photoionization of PTH inside the micelle, an early formation of a free pyridinyl radical is observed. This suggests that an ultrafast free-radical reaction with an oxidized form of pyridine nucleotide can be triggered by a single electron transfer in less than 5 X 10(11) s-1.     

Asymmetric electron transfer in cyanobacterial Photosystem I: charge separation and secondary electron transfer dynamics of mutations near the primary electron acceptor A0

Point mutations were introduced near the primary electron acceptor sites assigned to A0 in both the PsaA and PsaB branches of Photosystem I in the cyanobacterium Synechocystis sp. PCC 6803. The residues Met688PsaA and Met668PsaB, which provide the axial ligands to the Mg2+ of the eC-A3 and eC-B3 chlorophylls, were changed to leucine and asparagine (chlorophyll notation follows Jordan et al., 2001). The removal of the ligand is expected to alter the midpoint potential of the A0/A0- redox pair and result in a change in the intrinsic charge separation rate and secondary electron transfer kinetics from A0- to A1. The dynamics of primary charge separation and secondary electron transfer were studied at 690 nm and 390 nm in these mutants by ultrafast optical pump-probe spectroscopy. The data reveal that mutations in the PsaB branch do not alter electron transfer dynamics, whereas mutations in the PsaA branch have a distinct effect on electron transfer, slowing down both the primary charge separation and the secondary electron transfer step (the latter by a factor of 3-10). These results suggest that electron transfer in cyanobacterial Photosystem I is asymmetric and occurs primarily along the PsaA branch of cofactors.     

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     

Light-induced charge separation in Photosystem I at low temperature is not influenced by vitamin K-1

The photoreduction of iron-sulfur centers was studied at low temperature in Photosystem I particles from spinach and the cyanobacterium Synechocystis 6803, which contain various amounts of vitamin K-1 (recently tentatively identified as the acceptor A1). The irreversible charge separation that was progressively induced at low temperature between P-700 and FA (or FB) by successive laser flashes was studied at 15 K. Its maximum amount after a large number of flashes was shown to be fairly independent of the number (0, 1 or 2) of vitamins K-1 per reaction center. Moreover, the first flash yield of this charge separation was diminished by only about 50% when vitamin K-1 was completely absent from the particles by comparison with particles containing one or two vitamin K-1 per reaction center. When FA and FB were prereduced, the iron-sulfur center FX was also reversibly photoreduced at 9 K in the absence of vitamin K-1. The implications of these results for the electron pathways of Photosystem I are discussed and it is proposed that a direct electron transfer from A0- to the iron-sulfur centers is highly efficient at low temperature.     

Excitation energy transfer and charge separation in the isolated Photosystem II reaction center

The nature of excitation energy transfer and charge separation in isolated Photosystem II reaction centers is an area of considerable interest and controversy. Excitation energy transfer from accessory chlorophyll a to the primary electron donor P680 takes place in tens of picoseconds, although there is some evidence that thermal equilibration of the excitation between P680 and a subset of the accessory chlorophyll a occurs on a 100-fs timescale. The intrinsic rate for charge separation at low temperature is accepted to be ca. (2 ps)^–1, and is based on several measurements using different experimental techniques. This rate is in good agreement with estimates based on larger sized particles, and is similar to the rate observed with bacterial reaction centers. However, near room temperature there is considerable disagreement as to the observed rate for charge separation, with several experiments pointing to a ca. (3 ps)^–1 rate, and others to a ca. (20 ps)^-1 rate. These processes and the experiments used to measure them will be reviewed.Abbreviations Chl chlorophyll - FWHM full-width at half-maximum - Pheo pheophytin - PS II Photosystem II - P680 primary electron donor of the Photosystem II reaction center - RC reaction center The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Characterization of a semi-stable,charge-separated state in reaction centers from Rhodobacter sphaeroides

In reaction centers from Rhodobacter sphaeroides, subjected to continuous illumination in the presence of an inhibitor of the Q_A to Q_B electron transfer, the oxidation of P870 consisted of several kinetic phases with a fast initial reaction followed by very slow accumulation of P870⁺ with a halftime of several minutes. When the light was turned off, a phase of fast charge recombination was followed by an equally slow reduction of P870⁺. In reaction centers depleted of Q_B, where forward electron transfer from Q_A is also prevented, the slow reactions were also observed but with different kinetic properties. The kinetic traces of accumulation and decay of P870⁺ could be fitted to a simple three-state model where the initial, fast charge separation is followed by a slow reversible conversion to a long-lived, charge-stabilized state. Spectroscopic examination of the charge-separated, semi-stable state, using optical absorbance and EPR spectroscopy, suggests that the unpaired electron on the acceptor side is located in an environment significantly different from normal. The activation parameters and enthalpy and entropy changes, determined from the temperature dependence of the slow conversion reaction, suggest that this might be coupled to changes in the protein structure of the reaction centers, supporting the spectroscopic results. One model that is consistent with the present observations is that reaction centers, after the primary charge separation, undergo a slow, light-induced change in conformation affecting the acceptor side. This revised version was published online in June 2006 with corrections to the Cover Date.