Photosynthesis Research 66: 225–233, 2000. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
225
Regular paper
Selective quenching of the fluorescence of core chlorophyll–protein complexes by photochemistry indicates that Photosystem II is partly diffusion limited Robert C. Jennings∗ , Gianluca Elli, Flavio M. Garlaschi, Stefano Santabarbara & Giuseppe Zucchelli Centro CNR sulla Biologia Cellulare e Molecolare delle Piante, Dipartimento di Biologia, Universit`a di Milano, via Celoria 26, 20133 Milano, Italy; ∗ Author for correspondence (e-mail:
[email protected], fax: +3902-26604399) Received 18 February 2000; accepted in revised form 5 November 2000
Abstract The spectral characteristics of fluorescence quenching by open reaction centres in isolated Photosystem II membranes were determined with very high resolution and analysed. Quenching due to photochemistry is maximal near 687 nm, minimal in the chlorophyll b emission interval and displays a distinctive structure around 670 nm. The amplitude of this ‘quenching hole’ is about 0.03 for normalised spectra. On the basis of the absorption spectra of isolated chlorophyll–protein complexes, it is shown that these quenching structures can be exactly described by assuming that photochemistry lowers the fluorescence yield of the reaction centre complex (D1/D2/cytb559) plus CP47, with quenching of the former complex being approximately double that of the latter complex. These data, which qualitatively indicate that there are kinetically limiting processes for primary photochemistry in the antenna, have been analysed by means of several different kinetic models. These models are derived from present structural knowledge of the arrangement of the chlorophyll–protein complexes in Photosystem II and incorporate the reversible charge separation characteristic of the exciton/radical pair equilibration model. In this way it is shown that Photosystem II cannot be considered to be purely trap limited and that exciton migration in the antenna imposes a diffusion limitation of about 30%, irrespective of the structural model assumed. Abbreviations: CP – chlorophyll-protein complex; D1/D2/cytb559 – reaction center complex of PS II; Fo – minimal fluorescence; Fm – maximal fluorescence; Fv – variable fluorescence (Fv = Fm − Fo ); LHCII, light harvesting complex of PS II; P680 – primary electron donor of PS II; RC – reaction centre Introduction Photosystem II of higher plants is a large, multisubunit complex, which catalyses the conversion of light energy into chemical energy via the creation of an electrochemical gradient of about 1eV associated with stable charge separation. Seven distinct chlorophyll binding proteins are distinguished, of which six have a purely antenna function (Jennings et al. 1996a) and one binds the primary electron donor, P680, as well as a number of primary electron transport cofactors. The photosystem is conventionally subdivided into two distinct parts. (1) An outer or peripheral antenna
which consists of a family of chl a/b binding proteins known as LHC IIa,b,c , CP29, CP26, CP24. LHC IIa,b,c is responsible for about 55% of total light absorption in PS II with the three minor chl a/b complexes accounting for about 16% (Jennings et al. 1993). 2) A core made up of the chl a binding proteins CP43 and CP47, having only antenna function, and the reaction centre complex (D1/D2/cytb559). The core antenna complexes account for about 25% of total light absorption and the reaction centre complex about 4% (Jennings et al. 1993, 1994, 1996b). The overall topological organisation of these subunits, in which the chl a/b binding complexes are peripheral to the
226 core complexes, initially made largely on the basis of detergent fractionation studies (Peter and Thornber 1991; Dainese et al. 1992; Jansson 1994), has in general terms been confirmed in recent years by electron microscope analysis of detergent fractionated PS II particles and scanning-tunneling microscopy of membrane bound PS II (e.g. Hankamer et al. 1997; Lukins and Oates 1998; Boekema et al. 1999; Barber et al. 1999). In these proposed structures, the minor chl a/b complexes bind most of the the major LHC II antenna subunit to the core. Concerning the core itself, while there is general consensus that the D1/D2/cytb559 complex directly binds the CP47 antenna subunit, the situation concerning CP43 is less clear with different opinions being expressed (eg Hankamer et al. 1997; Lukins and Oates 1998, Hankamer et al. 1999). The marked biochemical complexity of the pigment binding system of PS II which emerges is in sharp contrast with the energetic characteristics of the component parts. From a careful analysis of the distribution of energy levels associated with the major chlorophyll spectral pools of all complexes, the picture emerged of a PS II made up of almost isoenergetic subunits (Jennings et al. 1993, 1994, 1996b) and in which the free energy for exciton diffusion from the external antenna to the core is around –0.3kB T at room temperature. Thus, the excited state distribution over the entire pigment system is expected to be rather homogeneous and substantially thermalised before the occurrence of primary photochemical trapping, which in PS II is rather slow, with literature values for the mean trapping time (τ m ) being reported in the 280– 480 ps range (Roelofs et al. 1992; Gilmore et al. 1996; Vasilev et al. 1998). This substantially isoenergetic nature of the chl-binding complexes means that it is virtually impossible to directly analise excitation energy flow between them by picosecond time resolved techniques. In fact, in fluorescence lifetime studies, only two clearly defined decay components are usually detected and their decay associated spectra are almost identical (Roelofs et al. 1992; Dau and Sauer 1996). In addition, Dau and Sauer (1996) have detected an approximately 15 ps process which has the characteristics of an energy transfer component. These results have given rise to a PS II model in which the energy transfer processes between the antenna and P680 are thought to be extremely fast with respect the mean trapping time, τ m , which is therefore exclusively determined by the trap (exciton radical pair equilibration model; (Van Grondelle 1985; Schatz et al. 1988)). Thus, antenna processes are not usually considered in
this kind of model though several groups (Briantais et al. 1996; Gilmore et al. 1996) have suggested that energy transfer from the external antenna complexes to the core might occur within several picoseconds and be irreversible. In this latter scheme, P680 is thought to equilibrate only with the core antenna. While the exciton/radical pair equilibration model provides an excellent description of the fluorescence decay kinetics of PS II, the proposal that antenna rate processes are not relevant to the trapping rate has never been directly investigated. In the present paper, we examine this proposal by comparing steady state fluorescence measurements at open and closed reaction centres. It is demonstrated that primary photochemistry quenches the fluorescence of the reaction centre complex and to a lesser extent that of CP47, clearly implying that antenna processes are to some extent limiting. This information is incorporated into a simple, multistate, kinetic model of energy transfer in PS II, based on the exciton radical pair equilibrium model, in which the subunit structural information is also explicitely considered. Materials and methods BBY-grana were prepared from freshly harvested spinach leaves as described by Berthold et al. (1981) but omitting the last Triton-X100 treatment (Van Dorssen et al. 1987). This preparation was shown to be free of Photosystem I by mild gel electrophoresis and contained only Photosystem II of the α-type by fluorescence induction in which the kinetics were described by a single exponential (unpublished observations). The BBY recovery yield was between 25–35% with respect to the thylakoids, on a chlorophyll basis. Experiments were performed on material resuspended in 30 mm Tricine (pH 8), 10 mM NaCl, 5 mM MgCl2 , 0.2 M sucrose, at a chlorophyll concentration of 2 µg ml−1 . Fluorescence emission spectra were measured with an EG&G OMA III (model 1460) multichannel spectrometer. The resolution of this apparatus was about 0.5 nm pixel−1 . Excitation light (475 nm) was provided by a Heath monochromator plus two Corning 4–96 filters. This assured the absence of stray light above 650 nm. Fluorescence emission was filtered across a Schott OG 530 filter. Spectra were accumulated for 12 different preparations in order to have over 3·106 counts in the peak channel at Fm . Fluorescence was maintained near the Fo level by means of an extremely weak excitation beam illuminating only
227
Figure 1. Description of the Fo /Fm quenching hole in terms of the chlorophyll–protein complexes. The dotted line represents the experimental dataand the solid curve the fit determined according to Equaiton 3, with K = 0.03. Panel A: X (λ) = D1/D2/cytb559 complex; panel B: X (λ) = D1/D2/cytb559 complex × 0.41 + CP43 × 0.59; panel C: X (λ) = D1/D2/cytb559 × 0.41 + CP47 × 0.59; panel D: X (λ) = D1/D2/cytb559 × 0.18 + CP43 × 0.41 + CP47 × 0.41. The Fo /Fm spectrum and the simulated quenching hole were normalised on the basis of the subtended areas between 640 and 700 nm.
a small part of the sample, which was under continual stirring. Emission spectra at Fm were determined for the same sample after addition of DCMU (25 µM) plus 2 mM hydroxylamine. The Fm /Fo ratio was about 3.3. The D1/D2/cytb559 complex was obtained according to the method of Chapman et al. (1988) and the room temperature absorption spectrum measured as
previously described (Cattaneo et al. 1995). The absorption maximum at room temperature was close to 676 nm. Pigment analysis indicated a chl/pheophytin ratio of 3.0. The CP43 and CP47 preparation procedure and room temperature absorption spectra have been published previously (Jennings et al. 1993).
228 Results and discussion It has been previously shown that there are small but significant differences in the emission spectra of PS II at Fo with respect to Fm (Jennings et al. 1991; Dau and Sauer 1996). Maximal quenching due to photochemistry was to the red of the emission maximum, while the shorter wavelength emittors, and in particular chl b, were shown to be less quenched. A clear interpretation of these data was not forthcoming at that time. We have recently noticed, however, that the overall band shape of this fluorescence quenching structure could be approximated by assuming a low fluorescence yield of the core complexes, and in particular of the D1/D2/cytb559 complex. This prompted us to carefully re-investigate this aspect using PS IImembranes (BBY particles) as these contain only the PS II units of the α type. Data, presented in Figure 1 (dotted lines) as the normalised Fo /Fm ratio, are the result of an extensive series of measurements in which over three million counts were accumulated in the peak channel. It is clear that the Fo /Fm ratio displays a broad quenching hole with a quenching minimum near 650 nm and maximum near 687 nm. A broad structure is also apparent in the 670–680 nm region. The amplitude of the quenching hole is about 0.03 in terms of the normalised spectra. While it is clear that the most direct way of analysing the fluorescence quenching hole in terms of the chlorophyll/protein complexes would be based on the measured emission spectra of the isolated complexes, unfortunately this is not possible. Even the mild detergent treatments employed in particle preparation lead to significant perturbation of the fluorescence spectrum of most antenna complexes (e.g. Croce et al. 1996) due to the uncoupling of a small number of chlorophylls which have a high fluorescence yield. As absorption spectra are much less modified by mild detergent treatment, we have based our analysis on the chlorophyll/protein absorption spectra. That this is possibile can be seen from inspection of the Stepanov expression which connects the absorption A(ν) with the fluorescence F(ν) (Equation (1)) as modified by Ketskeméty et al. (1961) for a variable fluorescence yield 8(ν) across the absorption band. This is a reasonable approach due to the extremely rapid spectral equilibration within them (e.g. Bittner et al. 1995; Visser et al. 1996; Connelly et al. 1997; Gradinaru et al.1998), an essential condition for the thermal equilibration assumption implicit in Equation (1).
Figure 2. Absorption spectra of the BBY particles and of the core chlorophyll–protein complexes. BBY: (—); CP47: (.....); CP43: (- - -); D1D2:( –.–.). The source of these spectra are indicated in the ‘Materials and methods’.
F (ν) − hν ∝ ν 2 8(ν)e kB T A(ν)
(1)
where ν is the frequency, h is the Planck constant, kB is the Boltzmann constant and is the absolute temperature. Thus, when F(ν) is substituted by Fo (ν) and Fm (ν) it is clear that the Fo (ν)/Fm (ν) ratio spectrum can be determined from the ratio of the product of the relative absorption spectrum with its fluorescence yield (Equation (2)) and that the quenching hole (Figure 1) may be interpreted in terms of changes in the fluorescence yield across the PS II absorption band (the absorption terms Ao (ν) and Am (ν) do not change during fluorescence induction) 8o (ν) Ao (ν) Fo (ν) = × Fm (ν) 8m (ν) Am (ν)
(2)
The Fo (ν)/Fm (ν) ratio was calculated by modifying the absorption weighting of a particular complex according to Equation (3). P SI I (λ) − K × X(λ) Fo (λ) = Fm (λ) P SI I (λ)
(3)
where PS II (λ) is the absorption spectrum of PS II membranes, X (λ) is the absorption spectrum of a chlorophyll–protein complex or some linear combination of the absorption spectra of more than one chlorophyll–protein complex and K is an appropriate weighting factor which gives the intensity of the quenching hole. We emphasise that while the quench-
229 ing hole is, of course, due to changes in the fluorescence yield across the spectrum, from Equations (1–3) it is clear that this may be calculated using absorption. The measured absorption spectra used in these calculations are shown in Figure 2. The quenching minimum near 650 nm in the experimental Fo /Fm spectrum is clearly associated with chl b emission and the maximum near 687 nm to chl a emission. Due to ultrafast energy transfer and equilibration within the chlorophyll–protein complexes, as discussed above, this indicates that it the external antenna complexes binding chl b are less quenched and thus any attempt to describe the Fo /Fm quenching hole in terms of the chlorophyll–protein complexes requires the search for a decreased fluorescence yield associated with the three core complexes (D1/D2/cytb559, CP43, CP47) which bind only chl a. In Figure 1, we show the best descriptions (full lines) obtainable considering a lower fluorescence yield associated with either (A) D1/D2/cytb559, (B) D1/D2/cytb559 plus CP43, (C) D1/D2/cytb559 plus CP47 and (D) D1/D2/cytb559 plus CP43 plus CP47 according to their weighting in the core complex. It is evident from Figure 1A that while the assumption of a specific quenching on the D1/D2/cytb559 complex alone does in fact describe the major structures present in the Fo /Fm quenching spectrum, it is unsatisfactory in describing the fine detail. However, when the D1/D2/cytb559 complex and CP47 are considered together (Figure 1C), an excellent detailed description is obtained. The combination of D1/D2/cytb559 with CP43 (Figure 1B) is less satisfactory as is the description obtained with a combination of all three of the core complexes (Figure 1D). We therefore conclude that primary photochemistry leads to a lowering of the fluorescence yield of both the reaction centre complex and CP47. We can see no evidence from these data that CP43 is also quenched though to exclude this out of hand at the moment may be unwarranted as the difference in the fit quality between Figure 1C and D is not great. However, as the best description does not point to quenching of CP43 in the following analysis, we shall not consider this possibility further. It should be pointed out that biochemical evidence suggests that CP47 binds more tightly to the core complex than CP43 (Dekker et al. 1990; Barbato et al. 1992; Zheleva et al. 1998) which may correlate with the present evidence. The decrease in fluorescence yield of D1/D2/cytb559 and CP47 can be quantified on the basis of our previous estimation of the relative Boltzmann excited state
population for the PS II chlorophyll–protein complexes in the absence of photochemistry (Jennings et al. 1993, 1994, 1996b). For the reaction centre, complex this was estimated at 5% of the total PS II emission and 15.6% for CP47. Using these figures and the data presented in Figure 1C we estimate that these values decrease to 3.7% and 13.9%, respectively, at open reaction centres. Thus the photochemistry induced fluorescence quenching is considerably greater on the D1/D2/cytb559 (0.26) than on CP47 (0.11). These values should not be regarded as absolute values for PS II as they are subject to several factors which are not readily quantifiable. Thus, the exact value of the Fo /Fm , usually higher for BBY particles than for intact chloroplasts, is expected to modify the intensity of the quenching hole and hence the amount of quenching of the complexes. Furthermore, owing to the minor, inevitable, variation in particle preparation , as pointed out above, some quenching of CP43 may occur which is not resolved in these experiments. In the case of an equal quenching on both CP43 and CP47 (Figure 1D), the photochemical induced quenching is estimated as 0.11 for D1 /D2 /cytb559 and 0.06 for each of the two core antenna complexes. Thus the expected greater quenching on the RC complex with respect to the core complexes is general. These conclusions are important for kinetic modelling of PS II as they provide information on the relative rates of population and depopulation of the reaction centre complex under these conditions and indicate that energy flow into it is, to some extent, kinetically limiting. In the following, we present a model description of the Fo quenching hole in terms of a simple five state kinetic scheme which takes into account available structural data for PS II. Four of the states are chlorophyll–protein complexes or groups of chlorophyll–protein complexes, with the fifth being pheophytin (Figure 3). The justification for using single states to model chlorophyll–protein complexes is that excitation equilibration within them is about two orders of magnitude faster than the mean trapping time (τ m ) (Bittner et al. 1995; Visser et al. 1996; Connelly et al. 1997; Gradinaru et al. 1998). Detailed balance between coupled chlorophyll–protein complexes was obtained using the equilibrium, excited state population values previously reported (Jennings et al. 1993, 1994, 1996b) for all PS II chlorophyll– proteins. In agreement with the exciton radical pair equilibration model, reversible charge separation was incorporated in the model, as this well describes the main properties of fluorescence decay experiments
230
Figure 3. Five-state models used in calculations. Model A is based on a scanning-tunneling microscope study (Lukins and Oates 1998), while Model B is based on numerous electron microscope analyses (Hankamer et al. 1997; Boekema et al. 1999). Chl a/b proteins are LHC II + CP24 + CP26 + CP29 and I is the primary pheophyin electron acceptor. The equilibrium population values used were: Chl a/b proteins = 0.652; CP43 = 0.142; CP47 = 0.156; D1 /D2 cytb559 = 0.05. For further details see text and Table 1.
(Schatz et al. 1988; Roelofs et al. 1992). Primary charge separation is modelled as proceeding from the D1/D2/cytb559 complex and its macroscopic rate con-
stant (k2 = 80 ns−1 ) is based on a molecular rate for pheophytin reduction of 330 ns−1 and assuming P680 to be a chl dimer. We point out that the exact value of this process is not known with certainty with literature values falling in the 50–1000ns−1 range from time resolved fluorescence and absorption measurments mostly performed with isolated D1 /D2 /cytb559 (Wasielewski et al. 1989; Roelofs et al. 1991; Visser et al. 1995; Donovan et al. 1997; Van Brederode and Van Grondelle 1999; Trissl 2000). We have chosen a value which falls in the fast part of this distribution as this is in greater agreement with the overall rate of primary photochemistry for intact PS II when the antenna size and mean energy level is considered (Schatz et al. 1988; Jennings et al. 1996b). We point out, however, that in these model calculations, this value may vary several times without significantly modifying the conclusions. The other primary electron transfer processes are within the range usually suggested (Schatz et al. 1988; Dau 1994). In searching for an acceptable model description of PS II at open reaction centres, we have used several experimental parameters towards which the simulations converge. We wish to emphasise that as all these experimental parameters are expected or are known to display large variation, above and beyond the statistical measurement errors, we do not attempt to find exactly converging descriptions. The fit criteria used are: 1. The equilibrium excited state population (fluorescence) values (at closed traps) for the four states associated with the chlorophyll–proteins are 0.652 (chla/b proteins), 0.142 (CP43). 0.156 (CP47), 0.05 (D1/D2/cytb559) (Jennings et al. 1993, 1994, 1996b). The fluorescence yield associated with the D1/D2/cytb559 complex and CP47 is lowered by photochemistry giving rise to a quenching hole amplitude of about 3% (see above). Quenching of the reaction centre complex is taken as about 0.26 and of CP47 as about 0.11. 2. A two component fluorescence decay. There is little agreement in the literature on the exact values of the PS II fluorescence decay which range from mean decay values (τ m ; where τ m is the first central moment or centre of gravity of the fluorescence decay) near 280 ps to about 500 ps at open reaction centres (Roelofs et al. 1992; Briantais et al. 1996; Gilmore et al. 1996; Vasilev et al. 1998). The component lifetimes reported also display similar fluctuations. This variability almost certainly reflects the well known variability in Fv /Fm values
231 reported in the literature with the longer lifetimes corresponding to relatively low Fv /Fm ratios. Detailed fluorescence decay analyses of PS II have mostly been performed for PS II samples in which the τ m values are in the 400–500 ps range (Roelofs et al. 1992). In the following, we simulate decays for τ m values of this kind. In these model calculations, the three complexes of the so-called core complex are explicitly represented (Figure 3) and two different organisations for CP47 and CP43 are considered. In one case, Model A, only CP47 is directly coupled to the reaction centre complex as suggested by a recent spectroscopic study using scanning-tunneling microscopy (Lukins and Oates 1998), while in the other case, Model B, both CP43 and CP47 are directly coupled to the reaction centre, in agreement with interpretation of a large body of electron microscope data (Hankamer et al. 1997; Boekema et al. 1999). The chl a/b binding complexes of the socalled external antenna are here considered as a single state. This is acceptable as all four chl a/b complexes are almost isoenergetic (Jennings et al. 1993, 1994, 1996b) and pooling them together into a single state simply assumes rapid energy flow between them. In general terms, description of the quenching hole requires that energy transfer into the reaction centre complex be relatively slow (kinetically limiting). If these values are assumed to be kinetically nonlimiting, and of course maintaining detailed balance, the photochemistry induced perturbation is essentially absent. Thus the presence of this quenching structure, associated with the ‘core’ complexes and particularly with the reaction centre complex itself, indicates that energy flow into D1/D2/cytb559 constitutes some kind of kinetic constraint. Model A. The exact kinetic requirements for this model are basically threefold: (1) energy flow from the chl a/b proteins into CP43 needs to be non-limiting while that into CP47 is slower; (2) the rate of energy transfer between CP47 and CP43 should be relatively slow (kinetically slightly limiting) in order that the photochemical induced fluorescence quenching does not spread out to cover CP43; 3) energy flow into the D1/D2/cytb559 complex needs to be kinetically limiting though sufficiently fast to allow transfer of the quenching process to CP47. An example of this kind of solution is given in Table 1 where the rate values given for the core processes may be varied by up to approximately 20–30% without significantly modifying the description.
Table 1. Simulations with models A and B (A)
(B)
Model parameters k1 a k2 k−2 k3 k−3 k4 k−4 k5 k−5 k6 k−6
2.2 80 0.8 56 175 20 18 >40b >184b 18 74.7
2.2 80 0.8 56 175 5.6 15.9 >40b >184b 20 83
Model calculation results τ1 A1 c τ2 A2 τ3 A3 τm 8FD1/D2 d 8FCP 47 QH e
10 –4.5 252 59.5 578 43.8 457 3.4 13.9 3.3
10 –3.1 245 60.8 578 42.4 452 3.5 13.9 3.1
a Units are: k, ns−1 ; τ , ps; A, 8F and QH are given
as per cent values. b Kinetically non limiting process. c Amplitudes (A) were calculated by summing
over all chlorophyll–protein states. d 8F XX – Fluorescence yield of the subscript
chlorophyll–protein complex. e QH – quenching hole.
Model B. The exact kinetic requirements for this model are: (1) energy flow from the chl a/b proteins into CP43 needs to be non-limiting while that into CP47 is slower and slightly rate limiting; (2) energy flow into the D1/D2/cytb559 complex from both CP43 and CP47 needs to be kinetically limiting though it should be faster for transfer between CP47 and D1/D2/cytb559 than for CP43 and the RC complex. It will be noted that a characteristic common to both schemes is that energy coupling between the external antenna and CP47 is weaker than that to CP43. This conclusion finds some support from structural studies which localise CP43 in a more ‘external’ position in the ‘core’ structure, apparently in closer ‘average’ physical contact with the chl a/b complexes (e.g. Boekema et al. 1999).
232 We wish to emphasise that while the quenching hole is rather sensitive to the values of the rate processes in the core, the fluorescence decay dynamics display a lesser sensitivity. It is possible to analyse the relative importance of antenna and reaction centre dynamics on the mean trapping time (τ m ) by means of the expression τ m = τ d + τ t r (Kudzmauskas et al. 1983; Valkunas 1986) where τ d is the time of excitation diffusion within the antenna, often known as the ‘first passage time’ and τ t r is given by (P·kcs )−1 . P is the probability that P680 is excited and kcs is the rate of charge separation. For the models τ d is calculated by assigning a very high value (saturating) to k2 and k−2 = 0. The values which come out are 145 ps for Model A and 131 ps for Model B. Thus we conclude that for PS II, antenna processes impose a diffusion limiting component of about 30% on the overall trapping rate. This conclusion is independent of whether or not there is quenching on CP43 as well as CP47. The important model parameter is the amplitude of the quenching hole associated with the core complexes. Concerning the fluorescence decay itself, it is apparent that, for the present five state model, formally there are an equal number of decay components i.e. more than the usual two or three components detected experimentally. The two fastest components, which are less than 5 ps lifetime, in all cases have almost zero amplitude when summed over all states. Thus they are expected to be experimentally invisible in a time resolved fluorescence experiment. The third component, which has a decay time of about 10–12 ps with both positive and negative amplitudes, corresponds to the transfer component of Dau and Sauer (1996). It is the two slowest components which represent the commonly observed decays and it can be seen that both the lifetime values and relative amplitudes are within the range of published values. As mentioned above, the model calculations presented here should be taken in a general sense without overdue emphasis being given to the exact values of the rate processes. This is because the large experimental variability of the modelled parameters precludes any attempt at greater precision. However, notwithstanding this limitation, it is clear that energy flow into the RC complex is rather slow. This conclusion would seem at first sight to be in contradiction with the basic assumption of the exciton radical pair equilibrium model as originally proposed (Schatz et al. 1988; Roelofs et al. 1992) which is that of a purely trap limited system in which antenna processes do not
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