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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

Max-Planck-Institut für Bioanorganische Chemie, ^* Stiftstr. 34-36, D-45470 Mülheim a.d. Ruhr, Germany

Correspondence: Address reprint requests to Alfred R. Holzwarth, Max-Planck-Institut für Bioanorganische Chemie, Stiftstr. 34-36, D-45470 Mülheim a.d. Ruhr, Germany. Tel.: 49-208-306-3571; Fax: 49-208-306-3951; E-mail: holzwarth{at}mpi-muelheim.mpg.de.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS APPENDIX 1 APPENDIX 2 ACKNOWLEDGEMENTS REFERENCES

 
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 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS APPENDIX 1 APPENDIX 2 ACKNOWLEDGEMENTS REFERENCES

 
Photosystem I (PSI) is one of the two reaction centers which, by way of two consecutive photodriven electron transfer steps within the well-known Z-scheme, transport electrons from water to NADP⁺. The recent x-ray structure analysis of cyanobacterial PSI with 2.5 Å resolution (Jordan et al., 2001) in principle opens the possibility for elucidating detailed structure/function relationships for the early events in PSI and should allow us to critically examine existing ideas about the present mechanistic models. The cyanobacterial PSI contains 127 co-factors in total, from which 96 are chlorophylls (Chls). However, as recent simulations have shown, the exact spectral properties of the reaction center pigments and their excited states, the nature of the primary donor state, the energy trapping lifetimes, and the rate of the first electron transfer step in the reaction center (RC) have a profound influence on the outcome of such simulations of the ultrafast energy trapping kinetics (Byrdin et al., 2002; Sener et al., 2002; Damjanovic et al., 2002). For this reason it is important to know those parameters exactly, in advance, to arrive at valid conclusions for the primary steps in detailed calculations based on the structural parameters. Only six Chls make up the reaction center proper, where the electron transfer steps are located (for a review, see Chitnis, 2001). The high ratio of the number of antennae to RC Chls characterizes the difficulties in observing directly the electron transfer processes in the RC, without disturbance by the antenna processes. Due to the presence of the large antennae, the rise and decay of the primary radical pair state and the associated spectral changes have actually never been directly resolved in open PSI RCs but have only been deduced from indirect experiments—i.e., by comparing the kinetics with oxidized and reduced P700 (for reviews, see Brettel and Leibl, 2001; Melkozernov, 2001).

Energy trapping and charge separation kinetics in various PSI particles has been the subject of a large number of ultrafast kinetic studies (see recent reviews by Holzwarth et al., 1998; Karapetyan et al., 1999a; Brettel and Leibl, 2001; Gobets and van Grondelle, 2001; Melkozernov, 2001). The kinetics of isolated Chlamydomonas reinhardtii PSI particles has been studied by several groups using ultrafast transient absorption and fluorescence (Owens et al., 1989; Chan et al., 1989; Du et al., 1993; Hastings et al., 1995a; Melkozernov et al., 1997, 1998, 2000b; Gibasiewicz et al., 2001). At present, agreement seems to exist about the interpretation of the various kinetic components. Ultrafast components in the subpicosecond range and a component in the 2–4-ps range were ascribed to energy equilibration in the antenna and between antenna and RC core (Gibasiewicz et al., 2001), whereas a 20–25 ps-component is ascribed to energy trapping by charge separation in the reaction center (Gibasiewicz et al., 2001). Thus energy equilibration is assumed to be complete after a few picoseconds and longer-lived components are ascribed to charge separation processes.

A very similar situation exists for cyanobacterial PSI particles for which a larger number of studies have been performed. For PSI from Synechocystis and Synechococcus elongatus, sub-ps and a few ps components (2–5 ps) have also been observed in various studies (Holzwarth et al., 1993; Turconi et al., 1993; Hastings et al., 1994a,b, 1995a,b; Turconi et al., 1996; Palsson et al., 1998; Gobets et al., 1998b; Savikhin et al., 1999, 2000; Melkozernov et al., 2000a). However, the ps components were interpreted in cyanobacterial PSI as energy transfer equilibration with special red pigments, i.e., antenna Chls with absorptions significantly longer than the 700-nm absorption of the RC (Karapetyan et al., 1999a; Gobets and van Grondelle, 2001; Melkozernov, 2001), rather than energy transfer only within the core antenna and to the RC. A recent combined femtosecond fluorescence upconversion and fluorescence streak camera study characterized in detail the ultrafast antenna energy transfer steps for Synechococcus PSI which occur mostly in the sub-ps range (Kennis et al., 2001; Gobets et al., 2001a). The presence of a species-dependent, significant amount of red pigments complicates the kinetic picture for cyanobacterial PSI, and the results are not always directly comparable to those of higher plants and green algae.

The energy trapping components for Synechocystis and S. elongatus PSI were found to be in the 25 and 35-ps range, respectively. The overall trapping kinetics for Spirulina platensis PSI is the slowest, at 40–50 ps, due to the presence of a particularly large amount of the red pigments in this cyanobacterial PSI complex (Karapetyan et al., 1997, 1998, 1999a,b; Karapetyan, 1998; Gobets and van Grondelle, 2001; Gobets et al., 2001b).

To make the comparison and discussion easier we have summarized in Scheme 1 the different kinetic models presently discussed in the literature for the energy trapping and early electron transfer processes in PSI. Note that some simplifications have been made in these schemes to generalize the models and focus on the most essential processes. Scheme 1 A shows the essentially trap-limited kinetic scheme proposed by Melkozernov et al. for both C. reinhardtii and cyanobacterial PSI (Gibasiewicz et al., 2001; Melkozernov, 2001). (We note that Melkozernov and co-workers did not present a detailed kinetic scheme in their articles; therefore, to make the different models comparable, we have translated the interpretations of Melkozernov et al.'s work into a kinetic scheme. The lifetime for charge separation in Scheme 1 A is an effective lifetime and does not represent an intrinsic lifetime of charge separation.) Scheme 1 B shows a model proposed by Savikhin and co-workers earlier (Savikhin et al., 2000) and Scheme 1 C shows a recently revised interpretation (Savikhin et al., 2001). Scheme 1 D shows the model proposed by Gobets and co-workers for cyanobacterial PSI (Gobets and van Grondelle, 2001; Gobets et al., 2001b). Note that in the latter model we have added the secondary electron transfer step which is missing in their model because the data were based on fluorescence kinetics only. In all the models shown in Scheme 1 the energy trapping and formation of the first radical pair occurs with a lifetime of 20–25 ps. This is also the case for Scheme 1 C if the antenna is excited despite the fast intrinsic charge separation step of 1.3 ps. Models 1 A and 1 B are based on both transient absorption and fluorescence kinetic data. Model 1 C is based on transient absorption data alone.

SCHEME 1  Summary of the presently discussed models in the literature for the energy trapping and early electron transfer steps in PSI RCs. (A) The essentially trap-limited kinetic scheme proposed by Melkozernov and co-workers for both C. reinhardtii and cyanobacterial PSI (Gibasiewicz et al., 2001; Melkozernov, 2001). (B) Transfer-to-trap-limited scheme proposed by Savikhin et al. (2000); (C) revised interpretation by Savikhin et al. (2001); and (D) transfer-to-trap-limited model (simplified) as proposed by Gobets and co-workers for cyanobacterial PSI (Gobets and van Grondelle, 2001; Gobets et al., 2001b). Note that in the latter, the antenna-to-RC transfer of 20 ps (energy trapping) is further slowed down by the red pigments.

View larger version (61K): [in this window] [in a new window]   FIGURE 1  Three-dimensional plot of the femtosecond transient absorption kinetics measured on two different timescales and resolutions (see Materials and Methods for details) for PSI core particles from C. reinhardtii at room temperature. (A) 670-nm excitation with 430 pJ/pulse, and (B) 700-nm excitation with 760 pJ/pulse. Note that the A scale is reversed (negative is up) for better visibility of the surface.

The energy transfer and charge separation processes in PSI can be described by a system of coupled differential equations within so-called compartment models (see, e.g., Holzwarth et al., 1987, 1998; Holzwarth, 1996). Even changing only one rate constant in the system, as is certainly done when going from open (P700 reduced) to closed (P700 oxidized) RCs, would have an effect on several, if not all observed lifetimes, albeit to various extents. Although the qualitative insight of forming double difference spectra between kinetics of two different states may be quite useful, such procedures can potentially lead to spurious signals and transients (partly due to the difference formation of kinetics with slightly changed lifetimes) that may lead to erroneous interpretations. It would thus be more desirable to solve the kinetic scheme and the associated transient spectra directly from the transient absorption data. Such kinetic modeling is performed rarely due to the complexities of the kinetic models and/or due to the uncertainties in the data. The insight gained from such modeling is, however, very valuable, and should be free from the problems related to forming difference spectra between transient spectra of different states or of different delay times, as discussed above.

Another problem that needs to be addressed in detail concerns the question of the nature of the primary electron donor and/or the exact redox state of the first intermediate. It is generally assumed that what is usually called P700⁺ is already formed in the first electron transfer step (all the interpretations of ultrafast data cited above assume such a model; for a review, see Brettel and Leibl, 2001) due to the oxidation of one of the central Chls of the RC—i.e., P_A or P_B. We note, however, that so far no solid experimental evidence exists to support this assignment. The nature of P700⁺ has typically been measured and tested only at times much later than the formation and decay of the primary radical pair. A recent electron paramagnetic resonance study showed that the electron hole is most likely located on the Chl P_B of the RC, with a partial delocalization on P_A (Käss et al., 2001). In principle, however, other initial electron transfer steps and sequences are possible and not excluded by the present data. Thus it may be possible that P700⁺ forms only in a secondary electron transfer step. Such a scheme was, for example, demonstrated recently for Photosystem II reaction centers (Prokhorenko and Holzwarth, 2000). In contrast to general assumptions that the primary electron donor should be the pseudodimer P_D1/P_D2 (Zouni et al., 2001), it was shown that, at least at low temperatures, the primary electron donor is the accessory Chl. A similar electron transfer process was observed earlier in bacterial RCs (van Brederode et al., 1999; van Brederode and van Grondelle, 1999), although it plays only a minor role due to the energetically high-lying excited state of the accessory BChl. Similar schemes cannot be excluded at present for PSI either.

The purpose of this work is twofold: 1), to critically re-examine the current interpretations regarding the energy trapping times and mechanism of charge separation; and 2), to test the current models for the secondary electron transfer steps. To this end, we performed ultrafast transient absorption spectroscopy with low excitation intensity and high signal-to-noise (S/N) ratio on open PSI RC core complexes from C. reinhardtii, and then analyzed these kinetic data using kinetic compartment modeling to arrive both at a kinetic scheme and, at the same time, obtain the corresponding transient spectra of the intermediates. Only if both pieces of information, the kinetics and the associated intermediate spectra, yield meaningful results can we be reasonably certain about the validity of the kinetic description of the underlying processes. These data strongly suggest that none of the currently discussed models for the energy trapping and the early electron transfer processes in PSI complexes correctly describes the experimental kinetics. A revised kinetic scheme that describes the early processes up to 100 ps is presented.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS APPENDIX 1 APPENDIX 2 ACKNOWLEDGEMENTS REFERENCES

 
Biochemical preparation and measurement conditions
Cells of C. reinhardtii CC2696 were grown and the PSI complexes prepared as described in Witt et al. (2002). The cells were harvested and thylakoids prepared as in Krabben et al. (2000). Isolation of the PSI complex was performed according to the method of Hippler et al. (1997), with slight modifications. The thylakoid membranes were resuspended in H₂O to a Chl concentration of 0.2 mg/ml and were then solubilized once with 0.9% (w/v) n-dodecyl ß-d-maltoside (DM) for 20 min at 4°C under stirring. After addition of NaCl to a final concentration of 50 mM, stirring was continued for an additional 10 min before centrifugation. The supernatant was loaded onto a sucrose density gradient and centrifuged for 16 h at 170,000 g. The lowest band was collected to obtain the PSI particles, diluted with 5 mM Tricine-NaOH (pH 7.5), 0.02% DM, and 100 mM NaCl, and concentrated with an Amicon ultrafiltration cell (Millipore, Bedford, MA). Chl concentrations were determined according to the method of Porra et al. (1989).

For the measurements the PSI complexes were diluted to an OD₆₇₆ = 0.71/mm in 5 mM Tricine-NaOH (pH 7.5), 0.02% DM, 100 mM NaCl, 40 mM Na ascorbate, and 50 µM phenoxy-methosulfate as redox mediator. Integrity of the sample was checked by absorption spectroscopy before and after the measurements. No changes in the spectrum were observed. The measurements were performed at room temperature (22°C).

Femtosecond measurements and analysis
Transient spectra in the femtosecond-to-nanosecond time range have been measured with open (reduced) P700 on these PSI particles at room temperature. The sample was contained in a 1-mm pathlength rotating cuvette which was also shifted sideways. Thus the cycle time to the same volume was 1 min, which allowed enough time for reopening of the RCs after a turnover. The fs-transient absorption kinetics were measured as described in Croce et al. (2001) using a Ti-sapphire regenerative amplifier system (Quantronix, model 4810, East Setauket, NY) pumping an optical parametric amplifier (Topas, Light Conversion, Vilnius, Lithuania) for excitation and a homebuilt photodiode array detector system. In short, the pulse width was 60 fs with a spectral width (full-width at half-maximum) of 8–9 nm. The excitation intensities in the 120–130-µm diameter spot were such that <0.3 photons were absorbed per PSI particle per shot at a 3-kHz repetition rate.

The primary data analysis has been performed by lifetime distribution analysis and is represented as lifetime density maps, as described previously (Croce et al., 2001). For the analysis the data from several measurements, recorded in different time ranges of 5 ps and 300 ps and with different resolutions per point of 13 fs and 0.5 ps, were combined and analyzed globally to calculate a single lifetime density map (Croce et al., 2001). This also involves correction for the minor chirp in the white-light continuum and deconvolution with the excitation pulse. In addition the original spectral resolution in the raw data of 0.5 nm/point (see Fig. 1) was limited to 3 nm by binning several camera pixels, which results in a higher S/N ratio. In summary the lifetime density plots represent the amplitudes of the lifetime components in a quasi-continuous lifetime range. Thus the kinetics is described as

which is replaced for analysis by a discrete sum of exponentials,

where n is a large number (typically 70–100; see Eq. 1 in Croce et al., 2001, for more details). The individual (fixed) lifetimes _i of this distribution themselves have no physical meaning but serve only as a suitable basis function set for the description of the total kinetics. The maxima and shoulders of these lifetime distributions A_i() represent the prevailing physical lifetimes of the system (Holzwarth, 1996). White-yellow spots in the maps represent positive amplitudes A_i and reflect either decay of an absorption or the rise of a bleaching/stimulated emission. Blue-to-black spots represent negative amplitudes and reflect either decay of a bleaching/stimulated emission or the rise of an absorption. The orange background represents the zero amplitude level.

View larger version (26K): [in this window] [in a new window]   FIGURE 2  Transient absorption difference spectra for PSI particles after correction for chirp and deconvolution with the excitation pulse (see the original data in Fig. 1). (A) 670-nm excitation, and (B) 700-nm excitation.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS APPENDIX 1 APPENDIX 2 ACKNOWLEDGEMENTS REFERENCES

 
Experimental kinetics
Transient absorption spectra have been measured for 670 nm (only antenna excitation) and 700 nm (primarily RC excitation) excitation wavelength for open (P700 reduced) PSI particles from C. reinhardtii CC2696 on the timescale of tens of femtoseconds to hundreds of picoseconds at room temperature. The excitation intensity was low enough to primarily cause single excitations per PSI particle. In fact our excitation intensity was slightly smaller than that used recently for characterization of exciton migration in C. reinhardtii PSI (Gibasiewicz et al., 2001). Detailed calculations show that our excitation conditions on average provide <0.3 excitations per PSI particle. Taking into account the Poissonian distribution, only 10% of all excited particles receive a second excitation (for 670-nm excitation; less for 700-nm excitation). Thus our excitation conditions for 670 nm are located close to the onset of exciton annihilation. When decreasing the excitation intensity by a factor of two we did not observe any changes in the kinetics that would significantly change the results. However, our primary aim in this work is the elucidation of the energy trapping and electron transfer processes and not of the precise, very early, antenna equilibration processes. Thus the excitation conditions provide a good compromise between linearity and high S/N ratio, which is important for the resolution of the complex kinetics at longer times.

The original transient kinetics of C. reinhardtii PSI cores are shown in Fig. 1 A for 670-nm and Fig. 1 B for 700-nm excitation for two different timescales. The decay of the initial antenna bleaching after excitation and the formation of the P700⁺ difference spectrum is directly visible. The absorption difference spectra at various delay times are shown in Fig. 2 A, for 670-nm excitation, and Fig. 2 B, for 700-nm excitation. They are calculated from the lifetime density maps of the transient kinetics (Fig. 3) for Fig. 3 A, 670-nm, and Fig. 3 B, 700-nm excitation wavelengths. Inspection of Figs. 1 and 2 indicates that the antenna and antenna/RC core equilibration processes are essentially finished on a timescale of 2 ps. The difference spectra at later times show the overall trapping of excitation and the formation/decay of various radical pair states. More detailed analysis of Fig. 2, A and B, reveals that after excitation ultrafast spectral evolution takes 100 fs. After that time the initially very narrow bleaching band has broadened and the characteristic stimulated emission (SE) sidebands for the vibronic transitions of excited antenna Chls 730–740 nm has developed. For 670-nm excitation the spectrum has been substantially red-shifted at 1-ps delay time, with some minor further development of the spectrum up to 2 ps. At that time the antenna excited-state equilibration seems to be completed. This can be deduced from the absence of further red-shift in the absorption bleaching maximum at longer times (see Fig. 2 A). For 700 nm the initial narrow bleaching band is located at 699 nm and widens up to 100 fs, together with the development of the characteristic SE side band at 745 nm. We believe that this ultrafast time process characterizes the equilibration among the exciton states in the RC core that is directly excited primarily at 700 nm. The RC core comprises at least the six RC Chls and perhaps one or two of the relatively tightly coupled connecting Chls (see the structure of Synechococcus PSI (Jordan et al., 2001)). Most of the spectral development of the excited state distribution is finished already at 1-ps delay in this case, again with some further minor spectral development up to 2–4 ps. Thus, as for 670-nm excitation, the excited state equilibration seems to be complete at this time, as judged from the absence of further blue-shift in the absorption bleaching maximum at longer delay times. This provides a first indication that processes on a timescale longer than 3 ps should reflect the energy trapping by charge separation and secondary electron transfer processes. The lifetime density maps in Fig. 3, A and B, present in a condensed form the total kinetics in the system. The exact three-dimensional data sets from which these maps are reproduced are used in the detailed kinetic modeling below.

View larger version (50K): [in this window] [in a new window]   FIGURE 3  Lifetime density maps (see Materials and Methods for details) of the transient absorption decays of PSI particles as obtained from the data shown in Fig. 1. (A) 670-nm excitation, and (B) 700-nm excitation.

View larger version (22K): [in this window] [in a new window]   FIGURE 4  Results of kinetic modeling. Simplest model with one antenna pool, the RC pool, and two RPs. (A) SADS for 670-nm excitation; (B) SADS for 700-nm excitation; (C) kinetic scheme with optimized rate constants (in units of ns-1) for the model and resulting lifetimes (bottom); and (D) time dependence of relative concentrations of the intermediates in the model for 670-nm excitation (the initial excitation is always 1, corresponding to 100% initial excited state at t = 0). Also given in this figure is the ratio of excitations in the RC to the main antenna pool Ant1. This allows us to judge the timescale of antenna/RC* equilibration. Note that the first RP rises with the dominant lifetime component of 8.5 ps. [General comment for Figs. 4–10: The same type of presentation will be used for the different models in Figs. 5–10 as well. Therefore, please refer to this figure caption for details, while the other captions are provided in short notation. Ant1 denotes the main antenna pool, Ant0 denotes the short wavelength antenna pool, RC* the excited reaction center, and Core* (only used in Fig. 9) an additional exciton state of the RC proper. In general the rate constants (in units of ns-1) in the models have been rounded to the nearest decade, except for the rate constants <50 ns-1. The error in the rate constants is typically in the range of 10% for most rate constants. For the secondary electron transfer rates it is closer to 5%. In some cases the error may be > or < than 10%, which will be indicated in the text specifically. For 670-nm excitation an additional component of 1.4 ns for the disconnected LHC 1 was taken into account (the corresponding SADS for LHC 1 is shown only in Fig. 4 A and is omitted in the other SADS for clarity of presentation). No LHC 1 was taken into account due to negligible excitation for 700-nm excitation experiments (for detailsm see text).]

View larger version (24K): [in this window] [in a new window]   FIGURE 10  Full kinetic scheme including the effects of charge recombination from the first RP. Two antenna pools, the RC, and three RPs are taken into account. (A) SADS for 670-nm excitation; (B) kinetic scheme with rate constants and resulting lifetimes (bottom); (C) time dependence of the intermediates; and (D) amplitude matrix Aij for the different lifetimes. The time dependence of concentrations Cj(t) of each intermediate follows the equation where Aij represents the amplitude matrix and i are the lifetimes. The excitation conditions are provided in the excitation vector denoted Exc. The radical pair rises with a dominant lifetime of 7 ps. See Note in Fig. 4, legend, for further details.

View larger version (31K): [in this window] [in a new window]   FIGURE 5  Kinetic model with a hypothetical scheme where charge separation occurs with a lifetime of 20 ps (energy trapping and rise of first RP). This scheme corresponds closely to most current models in the literature for the early processes in PSI from both C. reinhardtii and most cyanobacteria (for details see Results and Discussion). (A) SADS for 670-nm excitation. (B) Kinetic scheme with rate constants (in units of ns-1) and resulting lifetimes. (C–E) Comparison of experimental transient absorption kinetics at 742 nm (solid line) with the fitted curve from the model (dashed line). The three parts show the comparison for different models: (C) for the hypothetical model of Fig. 5 (this model fits the data very poorly, see text); (D) for the model shown in Fig. 4; and (E) for the model shown in Fig. 7. See Note in Fig. 4, legend, for further details.

View larger version (17K): [in this window] [in a new window]   FIGURE 9  Modified scheme (for 700-nm excitation only) where the ultrafast RC equilibration component of 150 fs has also been taken into account. The outer antenna pool Ant0 is omitted, since essentially no excitation is returning to that pool upon 700-nm excitation. (A) SADS for 700-nm excitation; (B) Kinetic scheme with rate constants and resulting lifetimes (bottom); and (C) time dependence of concentrations of intermediates. The first RP rises with a dominant lifetime of 7 ps. See Note in Fig. 4, legend, for further details.

The main difference between 670-nm and 700-nm excitation is the opposite amplitudes of the transient components for the antenna equilibration. For 670-nm excitation there occurs a bleaching decay (negative amplitude) 675 nm in the 300–800-fs and the 2–4-ps components, whereas the amplitude of these DADS is positive (rise of a bleaching) at 690 nm. In contrast, for 700-nm excitation the 1-ps component has a negative amplitude at 700 nm (decay of a bleaching) and a positive amplitude (rise of a bleaching) at 675 nm. These reversed amplitudes in the two sets of data simply reflect the antenna equilibrations occurring from different starting distributions, i.e., uphill energy transfer from the pigments excited at 700 nm, as already discussed for the transient absorption spectra at different delay times for Fig. 2, A and B. Also the 100–200-fs component reflects decay of bleaching at 700 nm and rise of bleaching at 665–680 nm, indicating uphill energy equilibration. The absence of the 2–4-ps antenna equilibration component for long-wavelength excitation has also been noted by Gibasiewicz et al. (2001), and their transient absorption data on the short timescale are in fact quite similar to the ones reported here.

The difference spectra at long delay times (300 ps, see Fig. 2, A and B) should reflect the final radical pair state on this timescale (the P700⁺ state) and are thus expected to be identical for the two excitations (this is the same component as the so-called nondecaying, i.e., ND, component in the work of other authors; see, e.g., Gibasiewicz et al., 2001). Surprisingly at first glance these final spectra are quite different for the two excitation wavelengths (see Figs. 1 and 2). The same observation has been made by Gibasiewicz et al. (2001) in their transient spectra of the ND component. Inspection of Fig. 3 A in the long lifetime range and comparison to Fig. 3 B indicates that there is a specific difference in the two (ND) lifetime components; i.e., for 670-nm excitation, there occurs an additional 1–2-ns lifetime component showing a bleaching band centered at 675 nm which is absent in Fig. 3 B. We have performed steady-state fluorescence experiments on these samples (data not shown) that indicate the presence of a substantial amount of energetically detached light-harvesting complex (LHC I), which emits at 740 nm at 77 K. This is responsible for the 1–2-ns component contribution to the long-lived (ND component) bleaching spectrum and thus for the distortion in the 670-nm excitation ND component. It is possible (and necessary) to include this component in the kinetic modeling. It will be demonstrated below that taking into account this energetically detached antenna in the kinetic model completely removes this difference in the long-lived transient spectra of the final radical pair for the two excitation wavelengths.

For 700-nm excitation no contribution of the uncoupled antenna is present due to the negligible absorption of LHC I at this wavelength in our samples. Thus in this experiment the long-lived (ND) transient spectrum should reflect completely the undistorted P700⁺–P700 difference spectrum. The main bleaching maximum of this component is located at 697 nm, and the second minor band at 680 nm. The positions of these maxima and the overall shape of this spectrum (ratio of 1/0.3 of the two main bands) are very close to the P700⁺ absorption difference spectrum measured on the ms-to-s timescale (see the ms-spectrum shown in Gibasiewicz et al., 2001, for a comparison), and thus clearly reflects the P700⁺ difference spectrum without any significant distortions. In contrast to Gibasiewicz et al. we thus believe that any relaxations of the protein after the radical pair formation have only a minor influence on the shape of this spectrum. A possible protein relaxation step might perhaps be assigned to the very weak 200-ps component, which slightly changes the shape of this difference spectrum on the 200-ps timescale. The most likely reason for the difference of the long-lived (ND) spectrum in our case and that reported in Gibasiewicz et al. (for long wavelength excitation) is the presence of a higher amount of detached LHC I in their samples, such that excitation at 700 nm still partially excited that complex in their experiments. We have tried to eliminate as much of this complex as possible in the isolation procedure, although it does not seem to be possible to remove it entirely without resorting to harsher methods which are prone to remove some of the minor polypeptides as well. We estimate 20–25% excitation contribution for LHC I at 670 nm (see below) in our sample. At 700 nm the absorption contribution of such an amount of LHC I to the total sample absorption is expected to be essentially negligible, and our data in fact show that no LHC I component is required to describe the 700-nm experiments. In the following kinetic models we will thus only take into account the excited LHC I for 670-nm excitation and not for 700-nm excitation.

It will be important for the later discussion and interpretations that, entirely independent of any detailed kinetic modeling, it is possible to conclude from the data discussed so far that excited state equilibration is essentially complete after 3 ps. (This conclusion is also in good agreement with the data of Gibasiewicz et al., 2001.) Furthermore it follows from these and other data in the literature about excited Chl a difference spectra, that over almost the complete detected wavelength range the processes of energy transfer/equilibration and charge separation/electron transfer show spectral contributions and changes. There exists, however, one wavelength region in our data where there is essentially no contribution from either antenna bleaching (GB) nor excited state absorption (ESA). This is the region of 745–760 nm. Rather, in that region there occurs a weak but characteristic absorption of radical pair states (Nuijs et al., 1986, 1987; van Gorkom and Nuijs, 1987). Thus, it should be possible in that wavelength region to observe and detect, without even resorting to any kinetic models, the time course of the development of the primary radical pair state(s). In that wavelength region we observe in our data a distinct component corresponding to a lifetime of 6–9 ps which has not been resolved in previous work. This component appears both in the 670-nm and in the 700-nm excitation experiments. It has a negative amplitude and thus represents the rise of an absorption. We believe that the only possible explanation for this component is the development of the primary radical pair state, since it cannot be explained by an energy transfer process which would have to show a positive amplitude due to the rise of a bleaching of a long-wavelength SE signal (see Figs. 2 and 3). Inspection of Fig. 2, A and B, shows that the absorption increase in the time range of 10–30 ps at 755–760 nm is highest and that it decays to approximately one-half that value up to 300 ps. Fig. 3 A also shows that the 40–45-ps component has a spectrum quite similar to that of the 6–9-ps component, but with opposite amplitude (positive, reflecting the decay of an absorption). This absorption decrease can thus only reflect a secondary electron transfer step. The most important conclusion from these findings is, however, that the primary radical pair formation does occur with a lifetime of 6–9 ps. This is in contrast to the present interpretation that the energy trapping by charge separation has a 20–25-ps lifetime in C. reinhardtii PSI (Melkozernov et al., 1997, 1998; Gibasiewicz et al., 2001), quite similar to the energy trapping lifetime (20–25-ps lifetime) of Synechocystis PSI (Turconi et al., 1996; Gobets et al., 1998b, 2001b; Savikhin et al., 1999, 2000; Melkozernov et al., 2000a; Gobets and van Grondelle, 2001). If the energy trapping in C. reinhardtii PSI indeed occurs with a lifetime of 6–9 ps this would drastically change the models for energy transfer, trapping, and electron transfer in this RC complex. Although these qualitative observations provide very important and fundamental information for the understanding of the nature and kinetics of the underlying processes, we do not consider this a final proof. Rather this conclusion should be supported by a kinetic model analysis where both the rate constant scheme as well as the corresponding species-associated intermediate difference spectra (SADS) should be in agreement with the conclusions drawn. This is done in the following part.

Kinetic modeling
We now proceed with kinetic modeling of the transient absorption data presented above. C. reinhardtii PSI does not seem to contain any red Chls in its core antenna according to the literature data but seems to contain the red pigments in the peripheral LHC-I complexes (Melkozernov, 2001) which are not attached in our preparation. In the PSI structure of Synechococcus (Jordan et al., 2001) the RC Chls are quite separated from all the antenna Chls (the P700 Chls are separated by 20–30 Å from all other Chls), except for the two so-called connecting Chls, which are closer to the RC Chls. It has been reported, however, that these connecting Chls, contrary to earlier belief, do not play a major role in the energy equilibration between antenna and RC (Byrdin et al., 2002). It thus seems reasonable to expect that the RC complex, comprising at least the six RC Chls, but possibly including the two connecting Chls, represents an entity wherein excitation equilibration occurs quite rapidly, i.e., on the timescale of 100–200 fs (see, e.g., Gibasiewicz et al., 2002). This is even more likely due to the fact that the six RC Chls are fairly strongly coupled excitonically (Beddard, 1998a,b). A similar situation occurs in the PSII RC and it is well known that most of the exciton equilibration among the states in the RC proper (as measured, e.g., in the D1–D2 RC complex) occurs in 100 fs, and that only a minor part of the equilibration takes up to 400 fs (Merry et al., 1996; Müller et al., 1996a,b; Prokhorenko and Holzwarth, 2000). A similarly fast equilibration is likely to occur among the exciton states in the PSI RC, as proposed already by Gibasiewicz et al. (2001, 2002), although the exact kinetics of the ultrafast equilibration process was not fully resolved there. This view is in fact well-supported by the data in Fig. 2 B, where the RC is excited directly. If this model is correct, the first electron transfer step would most likely occur from an almost-completely equilibrated RC core, even if we assume that the (intrinsic) rate of the first electron transfer step is very fast, e.g., 2 ps^-1. If this is the case, the fast electron transfer step would most likely not be observable with any substantial amplitude in a transient absorption experiment of intact PSI. Rather the observable rate would be slower by a factor of up to 6–8, reflecting the distribution of the excitation over 6–8 Chls in the RC, depending on which Chls should be included in the rapid equilibration process and charge separation from an equilibrated RC core. If correct, the conclusion from this situation would be that we should be able to observe a state (intermediate) in transient absorption (and most likely also in fluorescence) that reflects the absorption difference of the excited but equilibrated RC core proper. This situation would be rather different from the assumptions made in previous models where it was assumed that either 1), the excited RC does not attain any appreciable population due to slow energy transfer from the antenna and faster charge separation (Gobets et al., 1998a, 2001b; Gobets and van Grondelle, 2001; see also this article, Scheme 1 D); or 2), the other popular model, where only P700 (reflecting essentially only the special Chl pair P_A P_B) is excited and observable in the kinetics (Savikhin et al., 1999, 2000, 2001) (see Scheme 1, B and C). We will test all of these various possibilities in our kinetic modeling. Surprisingly Melkozernov and co-workers (Melkozernov et al., 1997; Gibasiewicz et al., 2001) did not resolve a RC component, although in their model the equilibration of the antenna with the RC is faster than the trapping by charge separation.

We start our kinetic modeling without any assumptions by just taking into account the situation that the model needs to describe at least the four dominant experimentally observed lifetime components (see Fig. 3), which means that there must be at least four different species or intermediates present. In more extended models, five or six components should be present, reflecting the additional 1–2 lifetime components present in the data. The simplest possible model, ignoring for the time-being the energy equilibration processes on a timescale <500 fs, should include at least one antenna pool, the excited RC complex, and two radical pair states. The initial excitation conditions should be such that for 670-nm excitation the antenna is almost exclusively excited (plus the detached LHC I complex which contributes an additional, but unconnected lifetime component), whereas for 700-nm excitation the RC core is excited to 70–90% and the core antenna only to a small percentage.

The method used for kinetic modeling is described in the Materials and Methods section. Here only one point needs to be mentioned in addition. Our kinetic modeling does not only aim at a good kinetic description of the data in terms of lifetimes, although such a solution is in fact always possible given enough lifetime components and/or intermediates (in a first-order rate equation model, the number of species is always identical with the number of lifetimes in the system). In every case at least one trivial solution (a sequential model with or without back-reactions) exists. In reality many different kinetic models will satisfy the conditions of merely a good kinetic fitting in terms of lifetimes. Thus, without any further criteria to judge the different formal solutions we cannot differentiate the good from the poor models. These additional criteria required consist in 1), getting the rate constants of the model in a physically meaningful range; 2), obtaining a reasonable probability of initial excitation according to the absorption cross-sections of the different pigment pools; and 3), the most important of these additional criteria, the shape and amplitude of the resulting intermediate spectra (SADS). Kinetic modeling without calculating these intermediate spectra is almost meaningless, since there will always exist a mathematically optimal fitting solution as long as the rate constant scheme yields the correct lifetimes present in the data (Holzwarth, 1995, 1996). Thus, when developing kinetic models, "solutions" will often be obtained where the SADS are clearly meaningless. Such cases can be discarded easily. However, it also happens that some aspects of the spectra appear to be quite reasonable whereas others may be questionable. It is therefore important to further define the criteria by which resulting SADS should be judged in our particular case at hand. The difficulty lies in the problem that these criteria cannot be implemented easily in a quantitative manner into a kinetic fitting program. For this reason we have developed the semiautomatic fitting and kinetic modeling program described above, which allows, at every stage, visual inspection of all important parameters. Even though the criteria for the SADS cannot be defined in such a rigorous quantitative manner as the criteria for a good kinetic fit (like, e.g., the usual ²-values; see Holzwarth, 1995, 1996), we nevertheless need to define, as clearly as possible, the criteria we used here for judging the SADS, which are given in the following: a difference spectrum of a Chl excited state (or a spectrally broadened inhomogeneous pool of Chls) should typically have only one bleaching maximum, a small SE side band in the range 720–740 nm (appearing as a small bleaching band), and an ESA band starting at wavelengths 20–30 nm to the blue from the bleaching maximum. The SE side band is particularly revealing and important. The SADS of radical pairs can have several bleaching maxima but they should have a characteristic small absorption increase above 720 nm (Nuijs et al., 1986, 1987; Holzwarth et al., 1993). Furthermore, the amplitudes of the excited state difference spectra should be fairly similar as long as they reflect mostly nonexcitonically coupled states (or pools of such pigments). Thus, the SADS of antenna pools should have similar amplitudes, whereas the RC^* SADS may perhaps have somewhat larger amplitudes due to exciton delocalization which could increase the extinction coefficient as compared to merely monomeric Chls. The reason for this is that the area under the bleaching curves (neglecting excited state absorption) must be proportional to the oscillator strength of the underlying transitions (Holzwarth, 1995, 1996). For the radical pair spectra no such criteria for the amplitudes can be formulated.

Fig. 4 shows the data for the minimal kinetic model as discussed above. For 670-nm excitation (Fig. 4 A) the additional LHC I antenna with a lifetime in the 1.4-ns range was taken into account as a separate component to describe the contribution of this complex to the long-lived bleaching spectrum (called the nondecaying or ND component by other authors; see, e.g., Melkozernov, 2001), which would otherwise distort the resulting RP spectrum. This antenna has no contribution in the models for 700-nm excitation. As shown in Fig. 4 B, it is possible to describe the experiments for the two excitations within the same kinetic model, the same rate constants, and, within the error limits, the same SADS. The only difference between the two sets of spectra is the slightly larger amplitude and the somewhat narrower width of the RC^* difference spectrum for 670-nm excitation as compared to 700-nm excitation. Reasons for these small differences will become clear in the further discussion below. Here we simply note that this scheme represents the simplest possible compartment model that describes the main features of the kinetics but still does not reflect all important details of the kinetics. The main reason for these discrepancies can, however, be assigned to the fact that for 700-nm excitation the main part of the excited state equilibration occurs with a lifetime of 100–200 fs, which is not reflected in this kinetic model at all. Also the 400–500-fs energy equilibration component is not reflected properly. Instead, the overall energy equilibration is described by an average 800-fs component. Fig. 4 C shows the kinetic scheme and the resulting associated rate constants together with the lifetimes of this model. We note that such a uniform model for drastically different excitation conditions has not been achieved so far for any PSI particle according to our knowledge. The resulting SADS satisfy all the criteria provided above. The antenna SADS and the RC^* SADS show the expected SE bands at long wavelength and have only one bleaching band. The two radical pair SADS show an absorption increase above 740 nm. The most important result of this simple model is, however, the fact that the first radical pair shows a spectrum with a strong bleaching band at 680 nm, in addition to a second bleaching maximum at 694 nm. These are the features that have been deduced indirectly previously for the spectrum of the first radical pair, but have never been resolved directly with open RCs in PSI particles. (Note that the RP difference spectra of C. reinhardtii are blue-shifted by 5–7 nm versus those from cyanobacteria; Melkozernov et al., 1997, 2000a,b; Savikhin et al., 2001.) The 680-nm bleaching disappears mostly in the second electron transfer step, resulting in a spectrum for the final radical pair that is essentially identical to the long timescale P700⁺ difference spectrum, as already mentioned above. Thus all the SADS look very reasonable. Furthermore, this kinetic model resolves, for the first time, the excited states spectrum of the presumably equilibrated RC with a bleaching maximum at 689 nm. Such a feature has neither been resolved in transient absorption nor fluorescence kinetics before.

View larger version (17K): [in this window] [in a new window]   FIGURE 7  Kinetic scheme with one antenna pool, the RC, and three RPs. (A) SADS for 670-nm excitation; (B) SADS for 700-nm excitation; and (C) kinetic scheme with rate constants and resulting lifetimes (bottom). The energy trapping and rise of the first RP occur with a dominant lifetime of 7.7 ps. See Note in Fig. 4, legend, for further details.

View larger version (17K): [in this window] [in a new window]   FIGURE 6  Extended kinetic model with two antenna pools, the RC, and two RPs for 670-nm excitation. (A) SADS; (B) kinetic scheme with rate constants and resulting lifetimes (bottom); and (C) time dependence of concentrations of intermediates. The energy trapping and rise of the first RP occurs with a dominant lifetime of 8.9 ps. See Note in Fig. 4, legend, for further details.

Reducing the rate constant for charge separation (as done deliberately in the model of Fig. 5) has, however, several adverse consequences: first, the antenna SADS now shows substantial excited state absorption above 710 nm and no SE band; i.e., the antenna SADS now has some characteristics of a radical pair spectrum. Even more drastic is the consequence on the SADS of the first radical pair, wherein we entirely lose the expected strong bleaching band at 680 nm. In addition, this model yields quite a poor fit to the experimental kinetics at most wavelengths. We show here only the data in the 740-nm range, which is fitted very poorly by the model with the slower trapping time as compared to the model shown in Fig. 4, due to the lack of a 6–9-ps component (see Fig. 5, C–E). Note, however, that despite the much better fit, the agreement between experiment and fit is not yet perfect for our simple model either. In any case we have to discard the slow trapping scheme on various grounds and conclude that even in the simplest model that can be developed to explain the main features of the present data we get an energy trapping and charge separation with a lifetime in the range of 6–9 ps, i.e., by a factor of three faster than assumed so far by other authors.

More refined kinetic models
The model shown in Fig. 4 is in various ways too simple and needs extension, although it is well-suited to describe the main features of the kinetics and yield good SADS. First it describes only four lifetimes (including the ND component), whereas the kinetic data (Figs. 1–3) indicate the presence of six major lifetime components, not even including the 100–200-fs component(s). Thus the simple scheme of Fig. 4 averages several kinetic components. The additional components would include at least one more energy equilibration component, thus allowing us to separate the sub-ps and the 1.5–2-ps energy transfer components. One additional lifetime component is required to better describe the lifetime range from 10–45 ps, which shows a very broad distribution 680–700 nm and some complexities which are not described by a single 25-ps component (see Figs. 3 and 4). It is easy to include an additional antenna equilibration component as shown in Fig. 6. The two antenna components Ant₀ (short wavelength antenna pool) and Ant₁ (main core antenna pool) have peak bleaching wavelengths of 678 and 682 nm in their SADS, representing a slightly blue-shifted smaller antenna pool and the major core antenna, respectively. This model extension substantially improves the description of the fast energy equilibration processes, which are now described with two lifetimes of 600 fs and 1.9 ps (see kinetic scheme in Fig. 6, B and C). The spectral shapes of the SADS look very reasonable, with smooth band shapes, SE in the long wavelength range for all the excited states, absorption increases for the RPs on the long wavelength side, and ESA in the short wavelength region for the excited states. The amplitude of the RC^* SADS is somewhat larger than for the antennae, reflecting probably some increased oscillator strength due to exciton coupling in the RC. The shapes of the radical pair spectra are not affected significantly as compared to those resulting from the simpler model in Fig. 4. This was expected since we have already discussed above that energy transfer and electron transfer/trapping processes occur on different timescales in this PSI particle. The SADS for 700-nm excitation are not shown for this model, inasmuch as the outer antenna Ant₀ does not receive any appreciable population upon 700-nm excitation (for a more detailed analysis of the 700-nm experiment, see Fig. 9, below).

The energy trapping by charge separation in this model occurs with a lifetime of 8.9 ps, quite similar to the one in the model of Fig. 4. (Note again that this lifetime is not the inverse intrinsic electron transfer rate and also not the inverse apparent charge separation rate from the RC, which is actually 400 ns^-1.) In Fig. 6 C the time dependencies of the various intermediates are shown. A 4:1 excitation probability for 670-nm excitation for the two antenna pools best describes the data. The RC^* state reaches a maximal concentration of 25%, whereas the first radical pair reaches a maximal concentration of nearly 60%. The populations in the inner antenna Ant₁ and the RC^* are equilibrated within 5 ps, i.e., in a time shorter than the effective energy trapping lifetime of 8.9 ps. This points to a kinetics which is considerably more trap-limited than diffusion-limited or transfer-to-trap-limited. In this model the overall trapping lifetime is strongly dependent on the electron transfer rate constant. The model shows that the energy equilibration processes within the antenna on the one hand and between antenna and RC on the other hand can be described very well in a relatively simple way with two antenna pools in this PSI particle. Again the RC^* SADS has its maximum at 689 nm, only 8 nm longer than the main antenna pool. Although this model describes the energy transfer processes (occurring upon 670-nm excitation) very well, there is still some deviation between experimental and model kinetics for lifetimes in the 10–45-ps range. This will be improved in the next step.

Before going to more complex models it is interesting to compare the excited state equilibration times with the apparent lifetime for energy trapping (6–9 ps). This can be done if we set the rate constant for charge separation in the scheme of Fig. 6 B to zero without changing the other rate constants. The scheme then gives the kinetics that would be observed if by some means the electron transfer could be stopped entirely without any other changes. The data show that the core antenna and the RC^* equilibrate with the longest component lifetime of 2 ps. This is the actual slow equilibration time between antenna and RC. However, in the intact system with open RC the RC^* population is disturbed by the ongoing charge separation and thus the equilibration effectively takes a little longer time.

More complex electron transfer models
In keeping with the main aim of this article we will now try to improve the description of the longer-lived electron transfer processes rather than the energy transfer processes. In order not to complicate the kinetic model too much we will in the following first ignore again the details of the antenna processes, as shown in Fig. 6, and return to a one antenna pool description. This is valid as we have already shown above that the spectra and kinetics related to the components above 3 ps are not influenced significantly by the details of the antenna energy transfer processes. At the end we will introduce again the antenna equilibration and provide a full kinetic model. However, for the time being, things can be kept simpler by just focusing on the radical pair kinetics.

The data in Fig. 3 indicating the qualitative assignment of the effective charge separation (equal to energy trapping) step to a lifetime <10 ps, supported by the models shown in Figs. 4– 6, require the inclusion of a further radical pair intermediate to better describe the lifetime range from 10–45 ps which contains more components than described by the models presented so far. With only two radical pair intermediates the broad kinetic feature in Fig. 3, A and B, which spans the lifetime range from 10 to 40 ps, cannot be described exactly. Note that the inclusion of any back electron transfer reactions in the kinetic scheme, as may be necessary eventually (see below), would not increase the number of lifetime components, but would simply change some lifetimes in the model—which would, in turn, require some changes in the other rates of the kinetic model to arrive again at the correct experimental lifetimes.

Fig. 7 shows the SADS (Fig. 7, A and B) and the kinetic scheme that best describe the data for both excitation wavelengths with one