Charge Separation Kinetics Research Articles

Energetics and Mechanism of Primary Charge Separation in Bacterial Photosynthesis. A Comparative Study on Reaction Centers of Rhodobacter sphaeroides and Chloroflexus aurantiacus

The high efficiency of charge separation in photosynthetic reaction centers arises from the interplay of energetics, electronic couplings, and reorganization energies relevant for the fast charge separation and slow recombination processes. All these parameters can be determined unambiguously only from magnetic-field-dependent measurements of the recombination dynamics of the intermediate radical pair P+HA- and the lifetime of the recombination product 3P*. Results obtained on QA-depleted reaction centers of Chloroflexus aurantiacus are compared with those for the well-characterized reaction centers of Rhodobacter sphaeroides. In contrast to Rb. sphaeroides, the magnetic field dependence of the triplet yield in Cf. aurantiacus has a pronounced resonance structure, allowing the direct determination of the exchange interaction of P+HA-, J = 21 G. The recombination rate kT is slightly larger for Cf. aurantiacus and shows a different temperature dependence. All these differences can be explained by the free energy of P+HA-, found to be larger by 0.04 eV in Cf. aurantiacus compared to Rb. sphaeroides. We propose that this different energy arises largely from the different amino acid at position L104, which is glutamic acid in the case of Rb. sphaeroides and glutamine in the case of Cf. aurantiacus. The electronic couplings and the reorganization energies, on the other hand, are very similar in both reaction centers. Implications for the mechanism of primary charge separation are discussed. The pronounced nonexponential kinetics of charge separation in Cf. aurantiacus is explained by the energetic inhomogeneity of the primary radical pair P+BA-.

Photochemical Charge Transfer and Hydrogen Evolution Mediated by Oxide Semiconductor Particles in Zeolite-Based Molecular Assemblies

Two integrated systems for light-induced vectorial electron transfer are described. Both utilize photosensitized semiconductor particles grown in linear channel zeolites as components of the electron transfer chain. One system consists of internally platinized zeolites L and mordenite containing TiO2 particles and methylviologen ions, with a size-excluded photosensitizer, tris(2,2‘-bipyridyl-4,4‘-dicarboxylate)ruthenium (RuL32+), adsorbed on the external surface of the zeolite/TiO2 composite. In the other system, Nb2O5 replaces TiO2. The kinetics of photochemical electron transfer reactions and charge separation were studied by diffuse reflectance flash photolysis. Despite very efficient initial charge separation, the TiO2 system does not generate hydrogen photochemically in the presence of an electrochemically reversible, anionic electron donor, methoxyaniline N,N‘-bis(ethyl sulfonate). Only the Nb2O5-containing composites evolved hydrogen photochemically under these conditions. These results are interpreted in terms of semiconductor band energetics and the irreversibility of electron transfer from Nb2O5 to intrazeolitic platinum particles.

Kinetics of charge separation and energy transfer in photosystem II reaction center

In the present study we investigated the kinetics of the initial charge separation and energy transfer in the PS II reaction center using pico-second and femto-second time-resolved techniques. We conclude that there are energy transfer processes between s-Car or Pheo and P680 in the PSII reaction center. The time constant of energy transfer from Pheo to P680 is less than 100 ps, while that from (s-Car to P680 is about 350 ps. For the initial charge separation in the PS II reaction center, the preliminary finding supported the about 3-ps time constant of charge separation. The possible kinetic scheme in PS II reaction center was proposed. Further experiments are in progress.

Primary charge separation processes in reaction centers of an antenna-free mutant of Rhodobacter capsulatus

The primary charge separation processes of membrane-bound reaction centers from an antenna-free mutant of the purple bacterium Rhodobacter capsulatus have been investigated by picosecond time-resolved fluorescence and femtosecond stimulated emission measurements. Target analysis has been applied to decompose the time-resolved fluorescence emission spectrum into its components for open and closed (quinone-reduced) reaction centers. The reaction center environment, i.e. membrane vs. detergent, exerts a remarkable influence on the primary charge separation kinetics. For fully open reaction centers the main component of the primary electron transfer process from the excited special pair to bacteriopheophytin — measured both in fluorescence and in femtosecond stimulated emission — takes 5–6 ps, which is a factor of nearly two longer than in the corresponding detergent-isolated reaction centers (2.7 ps). For quinone-reduced RCs a very long charge separation time of approx. 10 ps is observed, concomitant with a more than five-fold increase in fluorescence yield. All of these parameters differ substantially from those of detergent-isolated RCs. Possible origins for the observed kinetics are discussed.

A Monte-Carlo Method for Modelling the Photosystem II Operation and Intersystem Electron Transport in Higher Plants

Abstract We suggest a method of numerical modelling of the kinetics of the PSII charge separation and concomitant electron transport between the photosystems PSI and PSII. We employ a dynamic Monte-Carlo procedure with adjustable variable time scale, that enables us to work with a model which has characteristic times ranging over 8 – 10 orders of magnitude. We present the test results for our procedure. We design a simple mathematical macroscopic model of the charge separation and the electron transfer, that is based on several characteristic times. We apply the suggested procedure to calculate the time-dependent kinetics of the model under various conditions, relevant to typical situations in the time-resolved fluoresence experiments.

Solvent effects on the electron transfer reactions in supramolecular systems

The kinetics of charge separation due to the outer-sphere electron transfer processes in triad systems D-A-A in a polar environment were investigated using the stochastic Lioville equations. The solvation phenomena of the triad system in the different electronic states were described using Green's functions in two mutually correlated reaction coordinates and their auto- and cross-correlation functions. The coupling between the sequential and superexchange processes in the triad system was investigated.

Transient kinetics of charge separation influenced by the reactant translational diffusion and solvent rotational relaxation

The purpose of this contribution is a formal analysis of the rate function for the electron transfer in polar solvents in the presence of diffusion in translational and rotational degrees of freedom. Electron transfer processes in polar solvents are investigated for a two-surface system using Zusman kinetic equations. The dynamics in the polarization coordinate approximates the dielectric friction effects on the reaction rates. The solutions of the Zusman equations are given in terms of the appropriate Green functions. The integral equations for the rate functions in the Laplace space have been derived. Limiting cases are considered. An approximate decoupling scheme is proposed to simplify the integral equations for the rate function.

Excitation Wavelength Dependence of Bacterial Reaction Center Photochemistry. 2. Low-Temperature Measurements and Spectroscopy of Charge Separation

Excitation with spectrally narrow (5 nm), temporally short duration (200 fs) laser pulses at a variety of wavelengths between 848 and 903 nm results in substantial excitation wavelength dependent differences in the evolution of the bacterial reaction center absorbance spectrum both before and after charge separation occurs. The transient holes in the initial electron donor band showed a more resolved vibrational band structure at 20 K, when compared to those of earlier room temperature transient hole-burning experiments (Peloquin, J. M.; Lin, S.; Taguchi, A. K. W.; Woodbury, N. W. J. Phys. Chem. 1995, 99, 1349). The dominant vibrational band observed is at 120 cm-1, in agreement with dynamic measurements of coherent oscillations on this time scale (Vos, M. H.; Rappaport, F.; Lambry, J.-C.; Breton, J.; Martin, J.-L. Nature 1993, 363, 320). At both room temperature and low temperature, there is a distribution of P to P* transition energies due to a distribution of the protein conformations in the ground state. At 20 K, one can also see a distribution of P* to P stimulated emission energies. As might be expected, the barriers to conformational interconversion are more easily crossed at room temperature, resulting in a smaller difference between the mean transition energies of the photoselectable subpopulations on the picosecond time scale relative to those at low temperature. At 20 K, much of this conformational interconversion is apparently lost when exciting near the 0−0 transition wavelength of P. More conformational interconversion appears to take place at low temperature when higher energy excitation is used, implying that P* is vibrationally hot for at least hundreds of femtoseconds following excitation on the blue side of its QY band. Of particular interest is the insensitivity of the overall charge separation kinetics to the selection of ground state conformational subpopulations by different excitation wavelengths. There appears to be very little coupling between the nuclear motion that defines the ground state transition distribution and the charge separation reaction itself. Given the apparently slow rate of vibrational relaxation of some of the excited state modes most strongly coupled to the P to P* transition, the lack of excitation wavelength dependence of the charge separation rate also suggests that these modes are not strongly coupled to the charge separation reaction. What the ground state (and possibly excited state) conformational distributions and interconversions do affect is the kinetic complexity of the absorbance changes in the 800 nm region. Specifically, the extent of involvement of fast, multiexponential decay components in this region depends strongly on the wavelength of excitation.

Time-resolved and steady-state spectroscopic analysis of membrane-bound reaction centers from Rhodobacter sphaeroides: comparisons with detergent-solubilized complexes.

The spectroscopic analysis of the antenna-deficient Rhodobacter sphaeroides strain RCO1 has been extended to an investigation of the kinetics and spectroscopy of primary charge separation. Global analysis of time-resolved difference spectra demonstrated that the rate of charge separation in membrane-bound reaction centers is slightly slower than in detergent-solubilized reaction centers from the same strain. A kinetic analysis of the decay of the primary donor excited state at single wavelengths was carried out using a high repetition rate laser system, with the reaction centers being maintained in the open state using a combination of phenazine methosulfate and horse heart cytochrome c. The kinetics of primary charge separation in both membrane-bound and solubilized reaction centers were found to be non-monoexponential, with two exponential decay components required for a satisfactory description of the decay of the primary donor excited state. The overall rate of charge separation in membrane-bound reaction centers was slowed if the primary acceptor quinone was reduced using sodium ascorbate. This slowing was caused, in part, by an increase in the relative amplitude of the slower of the two exponential components. The acceleration in the rate of charge separation observed on removal of the reaction center from the membrane did not appear to be caused by a significant change in the electrochemical properties of the primary donor. The influence of the environment of the reaction center on primary charge separation is discussed together with the origins of the non-monoexponential decay of the primary donor excited state.

Pressure dependence of primary charge separation in a photosynthetic reaction center

Abstract Spectral hole burning is used to study the pressure dependence of the Qy absorption spectrum and primary charge separation kinetics of the D1–D2-cyt b559 reaction center complex of photosystem II. The 4.2 K lifetime of P680∗, the primary donor state, lengthens from 2.0 ps at 0.1 MPa to 7.0 ps at 267 MPa. Importantly, this effect is irreversible (plastic), in sharp contrast with the elastic effects of pressure on the low-temperature absorption and non-line-narrowed hole spectrum of P680. These observations and data which show that the electron-phonon coupling is weakly dependent on pressure, suggest a model that has the plastic behavior of charge separation kinetics due mainly to the pressure dependence of the energy of the acceptor state and of the variance of the P680∗-acceptor energy gap stemming from structural heterogeneity. Nonadiabatic rate expressions, which take into account the distribution of energy gap values, are used to estimate the linear pressure shift of the acceptor state energy for both the superexchange and two-step mechanisms for primary charge separation. For both mechanisms shifts in the vicinity of 1 cm−1/MPa are required to explain the data, a value which is not unreasonable based on pressure dependent studies of other systems. The results point to the marriage of hole burning and high pressures as having considerable potential for the study of primary transport dynamics in reaction center and antenna complexes.

Initial charge separation kinetics of bacterial photosynthetic reaction centers in oriented Langmuir-Blodgett films in an applied electric field

Abstract The free energy of the initial charge separation in bacterial photosynthetic reaction centers has been modified by placing oriented Langmuir-Blodgett films of the purified protein between external electrodes in a planar capacitor and applying a field of nearly 106 V/cm. The near-infrared transient absorption changes associated with the decay of the excited state of the bacteriochlorophyll dimer and the initial charge separation was measured with 300 fs time resolution with and without applied field. The surprisingly small field induced rate changes of the oriented systems compared to unoriented systems suggest that modulation of the energy gap between excited bacteriochlorophyll dimer and the charge separated state with bacteriochlorophyll monomer reduced is the principal influence of electric field on rate. The field induced quantum yield failure observed at longer timescales appears to be associated with modulation of the bacteriopheophytin to bacteriochlorophyll dimer charge recombination.

Fast photovoltage measurements in photosynthesis. I. Theory and data evaluation

AbstractFast photovoltage measurements are a valuable tool for the study of trapping of excitation energy, kinetics of charge separation, and relative distances of intermediary acceptors in the photosynthetic reaction center. Recent advances of both, the technique and the data analysis have not been described before. Here, in a first article of this sequence, we describe a theoretical model of the primary photosynthetic reactions and the computer analysis of transient photovoltage as well as of fluorescence data. The model assumes free energy migration between photosynthetic units and takes into account specific effects of high energy excitation like the acceleration of the trapping kinetics, the quenching of the excited state by oxidized reaction centers, and the nonlinear process of exciton‐exciton annihilation. Furthermore, the finite duration of the picosecond flashes is considered. The approximations involved in this model are discussed with respect to different functional antenna organizations. In a second article of this sequence we describe the experimental set‐up, calibration procedures and the application of the present theory to experimental data. © 1995 John Wiley & Sons, Inc.

Fast photovoltage measurements in photosynthesis. II. Experimental methods

AbstractFast photovoltage measurements in the pico‐ and nanosecond time range represent a valuable tool for the study of excitation energy trapping, kinetics of charge separation, and the location of intermediary acceptors in the photosynthetic reaction center. However, data recording and data analysis are complex. In a preceding article (Wulf and Trissl, Biospectroscopy, 1 (1995), pp. xx–xx) we have described the theoretical fundamentals of data analysis. Here we describe the experimental set‐up, calibration procedures, and strategies for the determination of the parameters involved. Limitations of the method will be discussed. © 1995 John Wiley & Sons, Inc.

Effect of the electric field on the electron transfer processes in symmetrical dimeric systems

The kinetics of photo-induced charge separation resulting from reversible outer-sphere electron transfer processes in symmetrical one-dimensional dimeric systems, such as bianthryl, perturbed by an external electric field, in liquid solutions are investigated. The theoretical approach is based on Zusman-type kinetic equations, and applies to adiabatic electron transfer processes. Model calculations demonstrating the response of the steady state decay of the three-surface system to the external electric field perturbation as a function of the dielectric relaxation rate of the solvent, reorganization energy and the free energy are presented.

Energy transfer and charge separation kinetics in photosystem I. 2. Picosecond fluorescence study of various PS I particles and light-harvesting complex isolated from higher plants

Two different Triton X-100 isolated photosystem (PS) I particle preparations as well as isolated light-harvesting complex (LHC) I from spinach have been studied by steady state and time-resolved fluorescence with a time resolution of about 5 ps. The dependence of the fluorescence kinetics and spectra on two parameters was investigated in detail: firstly, the solubilizing detergent used in the measurements was varied between Triton X-100, octylglucoside and SB12 (zwitterionic detergent). Secondly, the PS I particle size and type were varied, studying what is believed on the basis of literature data to be PS I-native and PS I core, respectively. All of these changes had pronounced effects on the kinetics. By global analysis procedures, we generally find four and in some cases five lifetime components in all data sets. Our results indicate that all PS I preparations studied, including the PS I-core complex lacking LHC I, are heterogeneous, displaying at least a bimodal distribution in antenna size. We attribute this size distribution to the action of Triton X-100 used in the isolation procedure. The fastest lifetime component in the range of 6–14 ps, depending on antenna size, appears as a rise term at wavelengths above 710 nm in all particles and is attributed to an energy transfer in the antenna system from the main pool of short wavelength absorbing pigments (F690) to pool(s) of long wavelength absorbing and fluorescing pigments (F720 and/or F735). The observation of such an antenna equilibration component at room temperature is crucial for the understanding of the excitation kinetics. Furthermore, for the sample that is believed to be “native” PS I, we found two lifetime components of 31–49 ps (depending on the detergent) and 130 ps. We attribute both of them to an overall charge separation, but originating from different particle types in the sample. We thus propose a fundamental antenna size heterogeneity. The shorter time most probably corresponds to intact PS I cores, whereas only the longer one originates from “native” PS I with antenna size of about 200 Chl. The data suggest that the excitation kinetics in all of these PS I particles is close to the trap-limit.

Primary processes and structure of the Photosystem II reaction center: II. Low-temperature picosecond fluorescence kinetics of a D 1-D 2-cyt- b-559 reaction center complex isolated by short Triton exposure

The fluorescence kinetics of a D 1-D 2-cyt- b-559 reaction center complex isolated by short Triton-exposure has been measured with picosecond resolution as a function of temperature below 150 K. The data were analyzed by combined global analysis of data measured with different time resolutions and are presented as decay-associated fluorescence spectra (DAS). Emphasis is given to the resolution and assignment of the fast components below 100 ps. Six lifetime components were generally necessary for a good fit of the data over a long time-range. The shortest lifetime of 13–24 ps (depending on temperature) is attributed to an energy transfer from (accessory) Chlorophyll to P680, presumably via pheophytin. A component in the range of 40–70 ps is attributed to energy transfer from pheophytin to P680. These components can only be properly resolved and assigned from measurements at temperatures below about 40 K. An ultrashort component of 1–6 ps, which had been resolved in our previous measurements (Biochim. Biophys. Acta (1991) 1060, 237–244) was not resolved in the particles studied here. We propose that the absence of the ultrashort component in this study is attributable to the larger chlorophyll content of the present short-term Triton-exposed D 1-D 2-cyt- b-559 preparation as compared to the previous long-term Triton-exposed RC particles. In the particles studied here the effective charge separation kinetics in the short time-range is rate-limited by relatively slow energy transfer components (lifetimes from 13 to 24 ps) upon preferential excitation of the accessory chlorophylls. In the long time-range a multicomponent charge recombination kinetics giving rise to four fluorescence lifetimes in the range of 1.56–49.7 ns are resolved. All these components show basically identical DAS with maxima near 682 nm.

Energy transfer and charge separation kinetics in photosystem I: Part 1: Picosecond transient absorption and fluorescence study of cyanobacterial photosystem I particles

The energy transfer and charge separation kinetics of a photosystem I (PS I) core particle of an antenna size of 100 chlorophyll/P700 has been studied by combined fluorescence and transient absorption kinetics with picosecond resolution. This is the first combined picosecond study of transient absorption and fluorescence carried out on a PS I particle and the results are consistent with each other. The data were analyzed by both global lifetime and global target analysis procedures. In fluorescence major lifetime components were found to be 12 and 36 ps. The shorter-lived one shows a negative amplitude at long wavelengths and is attributed to an energy transfer process between pigments in the main antenna Chl pool and a small long-wavelength Chl pool emitting around 720 nm whereas the longer-lived component is assigned to the overall charge separation lifetime. The lifetimes resolved in transient absorption are 7-8 ps, 33 ps, and [unk]1 ns. The shortest-lived one is assigned to energy transfer between the same pigment pools as observed also in fluorescence kinetics, the middle component of 33 ps to the overall charge separation, and the long-lived component to the lifetime of the oxidized primary donor P700(+). The transient absorption data indicate an even faster, but kinetically unresolved energy transfer component in the main Chl pool with a lifetime <3 ps. Several kinetic models were tested on both the fluorescence and the picosecond absorption data by global target analysis procedures. A model where the long-wave pigments are spatially and kinetically connected with the reaction center P700 is favored over a model where P700 is connected more closely with the main Chl pool. Our data show that the charge separation kinetics in these PS I particles is essentially trap limited. The relevance of our data with respect to other time-resolved studies on PS I core particles is discussed, in particular with respect to the nature and function of the long-wave pigments. From the transient absorption data we do not see any evidence for the occurrence of a reduced Chl primary electron acceptor, but we also can not exclude that possibility, provided that reoxidation of that acceptor should occur within a time <40 ps.

Intramolecular charge separation and recombination in non-polar environments via long-distance electron transfer through saturated hydrocarbon barriers

Time-resolved microwave conductivity and fluorescence spectroscopy techniques have been used to monitor the kinetics of charge separation and recombination following photo-excitation of donor-spacer-acceptor (DSA) molecules in which the spacer is a rigid saturated hydrocarbon bridge of length varying from 4.6 to 13.5 Å. The solvents used were all completely non-polar saturated hydrocarbons with relative dielectric constants varying from 1.8 to 2.3. The lifetimes of the highly dipolar, charge-separated states formed increase initially with increasing length of the spacer but eventually decrease for distances longer than approximately 9 Å. At that point the lifetime becomes sensitive to the dielectric constant of the medium and the temperature which was varied between 175 and 375 K. At the transition distance delayed donor fluorescence is observed. The results are explained in terms of the decrease in the Coulomb energy with increasing distance which raises the energy level of the charge separated state and eventually brings it close to the energy level of the locally excited donor (LED) state. Under these conditions charge recombination occurs preferentially via the LED state by thermally activated back electron transfer. The enegetics underlying this change in recombination mechanism are discussed.

Electric field effect on the picosecond fluorescence of Photosystem II and its relation to the energetics and kinetics of primary charge separation

Across the thylakoid membrane of spinach chloroplasts a diffusion potential of defined magnitude can be created by rapid changes in the KCl concentration. The resulting electric field leads to an increase in the yield of photosystem II (PS II) fluorescence for positive membrane voltages — positive in the inner thylakoid space — and a decrease for negative membrane voltages (Dau and Sauer, 1991, BBA 1098, 49–60). We have how studied this phenomenon by measurement of picosecond fluorescence decay. Based on the kinetic PS II model of Schatz et al. (1988, Biophys. J. 54, 397–405), the fluorescence decays were interpreted in terms of the rate constant of primary charge separation (formation of reaction center cation radical and pheophytin anion radical) primary charge recombination (recombination of primary biradical to chlorophyll singlet state) and secondary charge separation (reduction of primary quinone acceptor). For increasingly positive thylakoid voltages, the results are indicative of a relatively small but significant decrease in the rate constant of primary charge separation (by about 8% per + 100 mV thylakoid voltage) and a much larger increase (by about 50% per + 100 mV) of the rate constant of primary charge recombination. Nevertheless, due to the high sensitivity of the PS II fluorescence yield to changes in the rate constant of primary charge separation, the field-induced increase of fluorescence yield results mainly from the decreased rate constant of primary charge separation, and to a smaller extent (about one third of the increase) from the increased rate constant of charge recombination. The free energy difference of the primary radical pair was found to change by 17 meV per 100 mV thylakoid voltage. The relation between the rate constant of primary charge separation and the free energy difference appears to be linear within the accessible range of free energy changes (−15 meV to +22 meV); the rate constant of primary charge separation increases with increasingly negative free energy differences by 6% per 10 meV. This free energy dependence is compared with model calculations for a sequential two-step and for a super-exchange model of the primary PS II charge separation.

Electric field effect on the picosecond fluorescence of Photosystem II and its relation to the energetics and kinetics of primary charge separation

Across the thylakoid membrane of spinach chloroplasts a diffusion potential of defined magnitude can be created by rapid changes in the KCl concentration. The resulting electric field leads to an increase in the yield of photosystem II (PS II) fluorescence for positive membrane voltages — positive in the inner thylakoid space — and a decrease for negative membrane voltages (Dau and Sauer, 1991, BBA 1098, 49–60). We have how studied this phenomenon by measurement of picosecond fluorescence decay. Based on the kinetic PS II model of Schatz et al. (1988, Biophys. J. 54, 397–405), the fluorescence decays were interpreted in terms of the rate constant of primary charge separation (formation of reaction center cation radical and pheophytin anion radical) primary charge recombination (recombination of primary biradical to chlorophyll singlet state) and secondary charge separation (reduction of primary quinone acceptor). For increasingly positive thylakoid voltages, the results are indicative of a relatively small but significant decrease in the rate constant of primary charge separation (by about 8% per + 100 mV thylakoid voltage) and a much larger increase (by about 50% per + 100 mV) of the rate constant of primary charge recombination. Nevertheless, due to the high sensitivity of the PS II fluorescence yield to changes in the rate constant of primary charge separation, the field-induced increase of fluorescence yield results mainly from the decreased rate constant of primary charge separation, and to a smaller extent (about one third of the increase) from the increased rate constant of charge recombination. The free energy difference of the primary radical pair was found to change by 17 meV per 100 mV thylakoid voltage. The relation between the rate constant of primary charge separation and the free energy difference appears to be linear within the accessible range of free energy changes (−15 meV to +22 meV); the rate constant of primary charge separation increases with increasingly negative free energy differences by 6% per 10 meV. This free energy dependence is compared with model calculations for a sequential two-step and for a super-exchange model of the primary PS II charge separation.