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Energy trapping and detrapping in the photosynthetic bacterium Rhodopseudomonas viridis: transfer-to-trap-limited dynamics
Chemical Physics Chemical Physics 194 (1995) 275-283
ELSEVIER
Energy trapping and detrapping in the photosynthetic bacterium Rhodopseudomonas viridis: transfer-to-trap-limited dynamics K6u Timpmann a,1, Arvi Freiberg b, Villy SundstriSm a,* a Department of Chemical Physics, Lund University, Box 124, S-221 O0 Lund, Sweden Institute of Physics, Estonian Academy of Sciences, Tartu, Estonia
Received 15 November 1994
Abstract The efficiency of energy back-transfer from the reaction center to the antenna in chromatophores of the photosynthetic purple bacterium Rhodopseudomonas viridis was measured at room temperature by means of picosecond time-resolved fluorescence spectroscopy. It was found that 20 + 5% of the excitation energy selectively absorbed by the reaction center pigments in the wavelength region around 830 nm is transferred back to the antenna and gives rise to antenna fluorescence. The measured yield of energy detrapping enabled calculation of the energy detrapping (10-13 ps) i and trapping (40-50 ps)-~ rates. These results confirm that in Rps. viridis, like in bacteriochlorophyil a-containing purple bacteria, the energy transfer step from antenna pigments to the reaction center is a rate limiting step in the overall energy trapping by the reaction center. We suggest that this situation is termed 'transfer-to-trap-limited' dynamics, to distinguish it from the situation where the charge separation is the rate limiting step (trap limited) or the overall energy diffusion through the antenna is limiting (diffusion limited).
1. Introduction During the past few decades the processes of energy transfer and trapping have been thoroughly investigated in various types of photosynthetic organisms (for review see Ref. [1] and references therein), and for photosynthetic purple bacteria a considerable amount of information has been ob-
Abbreviations: RC, reaction center; LH, light harvesting; BChl, bacteriochlorophyll; P, the special pair primary electron donor of the RC; B, the accessory bacteriochlorophylls of the RC; I, the primary electron acceptor of the RC; Q, the quinone secondary electron acceptor of the RC * Corresponding author. RPermanent address: Institute of Physics, Estonian Academy of Sciences, Tartu, Estonia.
tained. Two different models, the so-called traplimited and migration-limited cases have been used to describe the energy transfer and trapping. The first model is based on the assumption of fast energy exchange between antenna and reaction center (RC) pigments, the rate exceeding the rate of charge separation in the RC [2-4]. The overall energy trapping time in this model will therefore be determined by the charge separation time from the special pair to the primary acceptor. In the opposite model the energy migration between the antenna molecules and antenna and RC is thought to be slow and constituting a rate limiting step of the overall energy trapping process [5], implying that the excitation once trapped by the RC pigments has only a low probability to escape.
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In photosynthetic purple bacteria the charge separation in isolated RCs occurs in about 3 ps [6]. Singlet-singlet annihilation [7] and time-resolved absorption anisotropy measurements [8] of Rhodospirillum (Rs.) rubrum and Rhodobacter (Rb.) sphaeroides have revealed that the energy hopping time between antenna pigments is ~< 1 ps. Low temperature picosecond fluorescence [9,10] and absorption [8,11,12] measurements of purple bacteria have shown that most of the energy equilibration over the antenna pigments is complete within ~< 10 ps. Very recent sub-picosecond investigations of the energy transfer processes in membranes of Rs. rubrum at room temperature demonstrated that spectral equilibration of the excitation density within LH1 occurs on a time scale of = 300 fs [1]. From measurements of femtosecond isotropic and anisotropy decay of the various pigments of LH1 and LH2 (B800 [13,14], B850 [15], B875 [15]) it was similarly shown that energy equilibration proceeds on the few hundred femtosecond timescale and is temperature and wavelength dependent, as a result of the spectrally inhomogeneous nature of the pigments. The trapping is considerably slower than this. Picosecond absorption measurements selectively exciting the red-most antenna pigments of Rs. rubrum and Rb. sphaeroides have shown that in the temperature range 100-200 K the antenna excited state lifetime is 35-40 ps [16], which was considered to reflect excitation transfer from antenna pigments coordinating the RC to the primary donor P. At room temperature the trapping rate was estimated to be about (20 ps) -1. The opposite process to trapping, detrapping of excitation energy from the RC to the antenna, was studied through measurements of the fluorescence excitation spectrum of antenna pigments. The results indicated negligible contribution of the RC absorption bands to the excitation spectrum of antenna fluorescence [17-19]. From this observation it was concluded that the detrapping efficiency is only a few per cent. A higher detrapping efficiency, reaching a value of 15-25%, was observed by means of time-resolved spectroscopy methods for different bacterial species [20-22]. On the basis of the measured 25% efficiency of the process the detrapping time constant in Rs. rubrum was estimated to be equal to 6 - 9 ps and the trapping time 30-40 ps [21]. The results accumulated so far
thus give evidence that in photosynthetic purple bacteria the energy trapping by the RC is characterized by the model, where the energy transfer to the RC from the antenna pigments adjacent to the RC dominates the overall trapping time [1,19,21,23]. Rhodopseudomonas (Rps.) viridis, the most investigated bacteriochlorophyll b-containing purple bacterium, may serve as an exception of this picture. In spite of considerable homology between Rps. viridis and bacteriochlorophyll a-containing purple bacteria there exist some spectral and structural differences. An intriguing spectral feature of Rps. viridis is the considerably more red Qy antenna absorption maximum (1015 nm [24]) as compared to that of the RC special pair (985 nm [25]). Despite of the seeming 'up-hill' energy transfer conditions from antenna to RC pigments, the energy trapping in Rps. viridis is found to be efficient, even at low temperatures [18] and the overall trapping time was measured to be very similar to that in BChl a-containing bacteria, 40-80 ps [26-29]. The data on the detrapping efficiency in Rps. viridis are contradictory. In early work by Olson and Clayton [30] it was concluded that excitations absorbed by the RC pigments were about 70% effective to excite antenna fluorescence. At the same time the very recent work of Otte et al. [19] suggested that at room temperature the detrapping efficiency in Rps. viridis is less than 10%. To clarify the specific character of the energy transfer and trapping processes in the BChl b-containing purple bacterium Rps. viridis we have studied the energy back-transfer efficiency from the RC to the antenna pigments by means of time-resolved fluorescence spectroscopy. The measurement was based on a comparison of the excitation spectra of the main decay component of antenna fluorescence at 1040 nm upon excitation of the sample with photochemically active (open) RCs in the state PBIQ, and with photochemically inactive (closed) RCs in the state P+BIQ in the wavelength region of 810-890 nm. It is assumed that in the case of open RCs after selective excitation of B an efficient energy transfer B --* P takes place and is followed by charge separation or energy back-transfer to the antenna, while in the case of closed RCs the back-transfer does not take place because the coupling of B* to P+ and P is different. For Rps. viridis we observed at room temperature a 20 + 5% energy detrapping efficiency
K. Timpmann et al. / Chemical Physics 194 (1995) 27.5-283
from RC to antenna pigments, and from this value the detrapping rate constant of (10-13 ps) -~ and trapping rate constant of (40-50 ps)-~ were calculated. This allows us to conclude that the energy transfer step from the antenna to RC is a slow rate limiting step of the energy trapping process in Rps. viridis, qualitatively very similar to that in BChl a-containing purple bacteria.
277
1.4
A
1.2 -.~
1.0 ~
,~100 B ~ 40
~20 ~ 0 2.
Materials
and
methods
1.0-
C
concentration, mM o - 60ps o - 140 ps
Rhodopseudomonas uiridis membrane fragments (a kind gift of Professor R. van Grondelle, Amsterdam) were diluted in 20 mM Tris-HC1 buffer (pH 8) to achieve a sample with the desired optical density ( = 0 . 3 cm ~) at 830 nm. To keep the primary electron donor in the reduced, photochemically active state, 30-50 mM sodium ascorbate was added to the sample in the buffer. The time-resolved picosecond fluorescence measurements were carried out with an apparatus described elsewhere [31]. The selective excitation of RC or antenna pigments was achieved by using = 10 ps pulses generated in a sync-pumped and cavity-dumped dye laser (Styryl 9) operating in the wavelength region 810-890 nm. The repetition rate of the laser pulses was 800 kHz. To avoid the influence of excitation annihilation processes on the fluorescence kinetics the excitation intensity was kept at the level of < 10 n photons cm -2 pulse -~. The antenna emission was detected at 1040 nm (Jobin Ivon H10-IR monochromator, 10 nm bandwidth) by an infrared-sensitive micro-channel plate photomultiplier. The measured kinetics were analyzed by a multi-exponential deconvolution procedure, taking into account the real temporal response function of the system which was 60-70 ps.
3.
Results
In the experimental mode used here the high repetition rate of the excitation pulses leads to the accumulation of the RCs in the oxidized state P+BIQ if the chromatophores are in plain buffer and no artificial electron donor is added. The fluorescence decay kinetics of the sample is in this case basically single-exponential with a time constant of = 140 ps
~ - 270 ps -
o -
2re(A*20)
-~0.5 -
I
0
I
'
[
100(I 2000 time, ps
I
3000
Fig. 1. (A) The ratio of normalized antenna fluorescence amplitudes of Rps. viridis chromatophores as a function of the concentration of sodium ascorbate. (B) Different fluorescence decay constants as a function of the concentration of sodium ascorbate. Note that 2 ns component has 20 times different scaling. (C) Two fluorescence kinetics of Rps. viridis membranes. (1) Chromatophores in plain buffer (closed RC); (2) part of the RCs are opened by sodium ascorbate. Note the apparent faster component and also a long tail in later case.
(see Fig. 1), in good agreement with earlier results [28]. In a few measurements an additional 40-50 ps decay component with a relative amplitude of 1020% was also observed, as for example in Refs. [27,29]. We believe that this decay component is due to the degradation of the sample during a prolonged experiment. The decay time of the antenna excitation was found to be independent of the excitation wavelength. To realize photochemically active RCs (in the state PBIQ) the electron donor sodium ascorbate was added to the sample. In this case the fluorescence decay kinetics became more complex, consisting of at least four decay components with time constants of about 60, 140, 270 and 2000 ps (see Fig. 1). The amplitude ratios of the decay components depend on the amount of the electron donor added to the sample, as shown in Fig. 1. The two shortest decay
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components have opposite dependence on the ascorbate concentration. When the relative amplitude of the 60 ps component increases from 50% (5 mM of ascorbate added) to 80% (70 mM of ascorbate), the relative amplitude of the 140 ps decay component decreases from 35% to 5% for the same ascorbate concentrations. The results of earlier time resolved measurements on Rps. viridis available in the literature [27-29], together with the dependence of the kinetics on artificial electron donor concentration presented here, suggest that the 60 ps decay time corresponds to the trapping of excitation energy by the reaction centers with reduced primary electron donor P, while the 140 ps decay component describes the quenching of the antenna excitations by RCs with oxidized electron donor, P+. The amplitude of the 2 ns decay component has a similar dependence on the ascorbate concentration as the 60 ps component, while the amplitude of the 270 ps decay is practically independent of ascorbate concentration. At the same time, the relative amplitudes of these two decay components depend on the excitation wavelength, being about two times (the 2 ns component) and 1.3 times (270 ps component) more pronounced for 830 nm excitation as compared to 880 nm excitation. At 830 nm a significant part of the excitation light is absorbed selectively by the RC, while at 880 nm to a good approximation only the antenna absorbs. In addition, the relative amplitude of the 2 ns component diminishes with decreased excitation intensity. From these observations we conclude that although the excitation intensity is moderate in our experiments, due to the stationary sample the electron transport chain of the RC is partially saturated and this causes photoaccumulation of a small amount of reduced primary electron acceptors of the RC, Q - , giving rise to the 2 ns component [3]. The nature of the 270 ps component is more problematic, probably it is a manifestation of several different processes (for example electron transfer from I to Q with about 200 ps time constant [32]) not fully resolved in the fitting procedures of the kinetic curves. In Fig. 2 the excitation spectra of antenna fluorescence are presented for a sample with photochemically active RCs (crosses) and with photochemically inactive RCs (circles) in the wavelength region of 810-890 nm. For comparison, the corrected absorp-
1.4
A
"-~ 1.2 1.0
~
.
IQ
''''I''''l'' B
0.4
150
C). O
10o
02
50 .= 0
. . . .
800
I
. . . .
I
,
,
0
85O 90O wavelength, rtm
Fig. 2. (A) The ratio of normalized antenna fluorescence amplitudes as a function of excitation wavelength. (B) The excitation spectra of antenna fluorescence with open (crosses) and closed (circles) RCs. The solid line is the corrected absorption spectrum of Rps. viridis chromatophores (see text).
tion spectrum (see below) of Rps. viridis chromatophores (solid line) is also reproduced. The excitation spectra were measured as the amplitude of the fluorescence decay curves detected at 1040 nm upon excitation in the wavelength region of 810-890 nm. In the case of a sample with open RCs the amplitude of the 60 ps decay component was extracted and used for the determination of the excitation spectrum. Measurements were performed up to a maximum concentration of 50 mM sodium ascorbate, which allowed us to estimate the ratio of the open and closed RCs in the sample to 8 - 1 0 at this ascotbate concentration. The two excitation spectra differ significantly around 830 nm, the wavelength region of the accessory BChl absorption band. This is demonstrated more clearly with the ratio of a normalized antenna fluorescence amplitudes with open (Fops,) and closed (Fdos~d) RCs as a function of excitation wavelength, presented in Fig. 2B. In the red part of the wavelength range )t > 850 nm the ratio Fope,/Fcloscd = 1, since the absorbance of the RC pigments is negligible compared to that of the antenna pigments at these excitation wavelengths. At shorter wavelengths the ratio Fop~n/Fdosed > 1 and it has a wavelength dependence matching the shape of
K. Timpmann et al. / Chemical Physics 194 (1995) 275-283
the RC absorption band in this wavelength region (see Fig. 2). The r a t i o Fopen/Fclosed is peaking at 830 nm and its value depends on the amount of photochemically active RCs present in the sample (see Fig. 1A). These results demonstrate that there exists a measurable energy back-transfer from the RC to the antenna at room temperature. The efficiency of the energy back-transfer is determined as [21] ~l = ( A a n t / A R c ) ( Fopen/Fclose8 -
1),
279
A
+ + ++~-~ +~"~-
4-
+
+
+
+
0
,
i
,
i
[
B
1.5
(1)
where A,, t and ARC are the absorptions at the excitation wavelength of the antenna and RC pigments, respectively. In order to obtain a correct value of the degree of back-transfer we have to determine the ratio of the antenna and the RC absorptions at each excitation wavelength. Here the main problem is to eliminate the background from the absorption spectrum of the sample, caused by scattering and absorption of uncoupled pigments. In order to describe the antenna and accessory BChl absorption spectra we fitted the antenna fluorescence excitation spectrum with a sum of Gaussians, and the 800 nm absorption band of the accessory BChl's in a RC preparation was similarly fitted to another sum of Gaussians. Then, the absorption spectrum of Rps. t,iridis chromatophores was fitted with the above Gaussians plus a constant background leaving the relative contribution from RC and antenna open. In that way the ratio of the RC and antenna absorptions in the wavelength region 810-870 nm was found (see Fig. 3B), and the energy back-transfer efficiency was calculated according to Eq. (1) (Fig. 3A). The corrected absorption spectrum is presented in Fig. 2. The average value of the detrapping efficiency after excitation of the accessory bacteriochlorophylls (B) in the RC was found to be 20 _+ 5%. The variation of the calculated 77 value from the average is more pronounced in the wings of the RC absorption band, where the RC and antenna absorption ratio is but poorly determined due to the difficulties with correcting the absorption spectrum of the sample for scattering and background absorption.
4. Discussion
The data presented in Fig. 2 demonstrate the contribution of the RC pigments in the 830 nm
0.5-
0
,
80O
85O wavelength, n m
Fig. 3. (A) Efficiency of the energy back-transfer from the RC to the antenna in Rps. ciridis chromatophores as a function of excitation wavelength. (B) The ratio of the contribution of the RC and the antenna absorption as a function of wavelength.
spectral region to the excitation spectrum of antenna fluorescence. It is shown by means of femtosecond absorption measurements on isolated RCs that there exists a very efficient and fast energy transfer from the bacteriopheophytin and accessory bacteriochlorophyll monomer to the primary electron donor P [6]. Thus, the observed antenna signal is caused by the energy back-transfer from the RC dimer P to the antenna pigments upon selective excitation of the RC accessory bacteriochlorophylls in this experiment. The RC contribution to the excitation spectrum of antenna fluorescence depends on the amount of photochemically active RCs present in the sample (see Fig. 1); with = 90% of the RCs in the photochemically active state PBIQ, the amplitude of antenna fluorescence exceeds by 1.3 times that for the sample with all RCs in the closed state and for = 60% of the RCs being in the open state the ratio of antenna fluorescence amplitudes was 1.1. Taking into account the relative absorption of antenna and RC molecules in the 830 nm spectral region (Fig. 3B) and the amount of open RCs in the sample, the energy back-transfer efficiency was calculated to be 20 + 5%. This result is similar to the energy backtransfer efficiency measured with time-resolved
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K. Timpmann et al. / ChemicalPhysics 194 (1995)'275-283
spectroscopy methods for Rs. rubrum (25%) [21], for Chromatium minutissimum (10-20%) [20] and for Rhodobacter capsulatus (15%) [22]. At the same time our data differ from those obtained for Rps. viridis by means of stationary spectroscopy [18,19,30]. In an early paper by Olson and Clayton [30] the energy transfer efficiency from the RC BChl monomer B to antenna pigments was found to be at least 70%. Such a high value was obtained by assuming an exceptionally low RC absorption relative to antenna absorption in the 830 nm absorption band. If a more realistic RC/antenna absorption ratio, like that derived in the present work, is used for the evaluation of these experimental data the energy back-transfer efficiency will be 20-30%, in good agreement with our results. In the work by Kleinherenbrink et al. [18] and Otte et al. [19] the efficiency of energy transfer from the RC to the antenna was found to be very low, ~< 2% at 6 K and ~< 10% at room temperature. As our investigation of the temperature dependence of energy detrapping efficiency in Rs. rubrum show (results will be presented elsewhere [33]), the back-transfer yield will decrease with lowering of the temperature, from = 25% at room temperature to at the most a few percent at 77 K. The room temperature results of Ref. [19] differs from ours by a factor of at least two. We believe that this discrepancy might be due to different experimental conditions. In a tifne-resolved experiment there exists an obvious control of the redox state of the RCs in the sample through the measurement of the antenna excited state lifetime, while in the stationary experiment this additional control parameter is absent. As our intensity-dependent kinetic measurements on Rps. viridis and earlier results on R. rubrum [3] show, the redox state of the RCs is very sensitive to the light intensity and electron donor concentration used in the experiment. Thus, it is important to be able to fine tune the experimental conditions while at the same time monitoring the state of the reaction center. On the basis of the measured fluorescence lifetimes and the energy detrapping yield the rate constants of forward and backward energy transfer between RC and antenna can be obtained. For that purpose numerical simulations of the time dependence of the antenna and RC excitations were performed, by means of the three-component kinetic
kl k_l
k~ P BIQ
k_2
P (P~I-Q-)
Fig. 4. Simplified kinetic model of energy transfer between antenna and RC used in numerical simulations.
scheme of Fig. 4. Here k I represents the total trapping rate of antenna excitations by the RC, k ~ is the total detrapping rate from the RC to the antenna, k 2 is the charge separation rate in the RC and k 2 the charge recombination rate. A precise description of the simulation procedure was given in Ref. [21]. The simulation was performed for a range of k~ and k ~ values until agreement was obtained between calculated and measured values of the antenna fluorescence lifetime and the detrapping efficiency. As a result, the total trapping and detrapping rates in Rps. viridis were found to be k j = ( 4 5 + 5 ps)-I and k 1 = (10 _ 2 ps) -1. These rate constants were found by using the charge separation rate k 2 = (2.8 ps)measured for isolated RCs of Rps. viridis [6]. It has been shown [34] that the charge separation rate in the presence of pre-reduced quinone acceptor ( Q - ) slows down to a value of k 2 = (5.6 ps) -1. In our experiments part of the RCs were in the state PBIQ-. Taking this into account the calculated detrapping rate constant slowed down to k_ 1 = (13 + 2 ps) ~, while the trapping rate practically did not change. From the derived total trapping and detrapping rates it is possible to deduce estimates for the pairwise rates between antenna and RC molecules. The pairwise detrapping rate kt) is related to the total detrapping rate from the RC to all nearest q antenna molecules coordinating the RC by k o = k_,/q.
(2)
The data of electron microscopy and imageprocessing techniques [35,36] indicate that in Rps. viridis the reaction center is surrounded by a ring of six antenna complexes and therefore it is reasonable to assume that q = 6. In that case the pairwise detrapping rate is k D = (60-70 ps) -a. Recently, Pearlstein introduced a mono-coordinate model for the energy trapping by the RC [37]. In this model the pairwise energy detrapping rate k D would be equal to the measured overall detrapping rate k_ ~ = (10-13
K. Timpmannet al. / ChemicalPhysics 194 (1995)275-283 ps) -~. The pairwise trapping rate k T of antenna excitations by the RC is related to the detrapping rate by the ratio of the F6rster overlap integrals for the two processes ( I T for trapping and I D for detrapping, respectively),
k T = (IT/tD) k D .
(3)
The calculation of the ratio of F6rster overlap integrals is quite problematic, since authentic spectral data of RCs in intact membranes are absent. The absorption spectrum of the isolated RC primary electron donor P is peaking at 965 nm [38], far to the blue of the main antenna absorption maximum at 1015 nm. It is believed that in intact membranes the absorption maximum of RCs is shifted by at least 20 nm to the red [18,25]. By using the absorption and fluorescence spectra of the antenna [24], taking into account the RC absorption spectrum shift, and assuming a similar shift for the RC fluorescence spectrum [38], the ratio of F6rster overlap integrals was calculated to be 1y/1D = 0.85. Now the pairwise trapping rate constant becomes k v = (70-85 ps)-1 with q = 6, and k y = (12-15 ps) i in the mono-coordinate case. This result shows that the pairwise energy trapping and detrapping rates between the antenna and RC molecules are by at least one order of magnitude smaller than the subpicosecond energy hopping rate between the antenna molecules [12,13]. This finding supports the idea that the final energy transfer from the antenna to the reaction center special pair is a rate limiting step in the overall energy trapping process. Based on early one-color p u m p probe measurements at 77 K [11] on Rb. sphaeroides and R. rubrum this view was first suggested by van Grondelle et al. [39] and somewhat later substantiated by measurements of the antenna excited state lifetime (35 ps) in Rb. sphaeroides and R. rubrum at 77 K with photochemically active (P) reaction centers [40]. Trapping kinetics measured in mutants of Rb. sphaeroides with modified charge separation rate, suggested a similar picture [41]. It should be noticed that in the original notation of trap or diffusion limited dynamics [2], the present situation with a slow rate limiting step from the antenna to the reaction center should be termed as diffusion limited trapping, because the rate limiting term resides in the so-called first passage time (Eq. (4)). This may cause some confusion since the rest of the dynamics, the
281
energy equilibration within the antenna, is very fast and occurs on the timescale of a few hundred femtoseconds [1,12-15]. Therefore, we suggest that a slightly different term is used to denote this situation with a slow, rate limiting, energy transfer step from the antenna to the reaction center namely, 'transferto-trap-limited' dynamics. It is of interest to use our measured kinetic data of Rps. viridis fluorescence and energy detrapping rate constants as input parameters to the Pearlstein model for energy migration and trapping [2]. In this model the antenna is described as a regular lattice of N equivalent sites with pigment molecules occupying each lattice point, and the energy migrating as a random walk over the antenna lattice. Such a model is applicable to Rps. viridis whose antenna is believed to be practically homogeneous [28,42,43], unlike the antenna of BChl a-containing purple bacteria which have been shown to be spectrally inhomogeneous [9,44]. The model gives the antenna exciton lifetime tex as the sum of two components, the first passage time tfpt and the revisiting time trev, where tfpt = N ( q k v ) - l +
[f(U)
-q
1]kA1N
(4)
and
trev=k2'N(lT/ID)
'
(5)
Here f ( N ) is the antenna structure function and k A is the energy hopping rate between the equivalent antenna sites. As was shown in Ref. [23], the value of the second term in Eq. (4) describing the energy migration time through the antenna to the RC is not more than a few picoseconds with a hopping rate k A of at least (1 ps) ~ [23] and assuming a two-dimensional hexagonal lattice structure. This implies that Eq. (4) can be simplified by neglecting this term in comparison with the first term describing the trapping. Then, by combining Eqs. (2)-(5), the antenna exciton lifetime can be expressed as
tex=(k
', + k 2 ' ) N ( I T / I D )
'.
(6)
It is known that there are 24 antenna molecules per RC in Rps. viridis [46]. At least for BChl a-containing purple bacteria the basic building block of the LH1 antenna is believed to be a dimer of strongly coupled antenna molecules [45]. Thus, it is reasonable to assume that N = 12, and with the values of
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k_ 1 = (10 ps) -1, k 2 = (2.8 ps) -1 and I T / I D = 0.85, the antenna exciton lifetime is found to be 190 ps. This value exceeds by approx, a factor of three the measured antenna fluorescence lifetime of 60 ps. If we assume that the antenna is organized in small clusters of four pigment molecules, probably the smallest possible N consistent with the structural data of the Rps. viridis antenna system, the antenna excitation lifetime derived from Eq. (6) is tex = 95 ps in reasonable agreement with experiment. Better matching between the measured and calculated antenna fluorescence lifetimes might be achieved by assuming that the energy detrapping rate (and therefore the trapping rate) is nearly two times faster than that used above (i.e. k_l = (5.5 p s ) - l ) , or that the ratio of the F/Srster overlap integrals is significantly more in favour of the trapping process (i.e. l - r / I D = 1.3). The first possibility implies that the energy back-transfer efficiency has to be about 40%, in disagreement with the measured yield of 20%. The second possibility is realized for example if the maximum of the absorption spectrum of the RCs in the intact membrane is shifted more to the red, to 995 nm, with other spectral parameters unchanged. This possibility is supported by very recent fluorescence site selection experiments of the Rps. viridis antenna [47], which suggest that at low temperature the antenna excitations are equilibrated in the far red wing of the antenna spectrum. In order to explain the observed charge separation efficiency at low temperature [18] the P absorption band must be more red-shifted than "the previously assumed Amax = 985 nm. Naturally, there also exist other optimizing factors of the forward energy transfer from antenna to the RC.
Acknowledgements The present study was supported by The Swedish Natural Science Research Council, the International Science Foundation, the Estonian Science Foundation and the EC contract SC1"-CT92-0796. K.T. especially acknowledges the Swedish Institute and The Swedish Academy of Sciences for a visiting scientist grant. We thank Dr T. Pullerits for many stimulating discussions.
References [1] R. van Grondelle,J.P. Dekker, T. Gillbro and V. Sundstr6m, Biochim. Biophys. Acta 1187 (1994) 1. [2] R.M. Pearlstein,Photochem. Photobiol. 35 (1982) 835. [3] A.Y. Borisov, A.M. Freiberg, V.I. Godik, K.K. Rebane and K.E. Timpmann,Biochim. Biophys. Acta 807 (1985) 221. [4] M.G. Miiller, G. Drews and A.R. Holzwarth, Biochim. Biophys. Acta 1142 (1993) 49. [5] Z.G. Fetisova,A.Y. Borisov and M.V. Fok, J. Theoret. Biol. 112 (1985) 41. [6] J. Breton, J.-L. Martin, A. Migus, A. Antonetti A. Orszag, Proc. Natl. Acad. Sci. USA 83 (1986) 5121. [7] J.G.C. Bakker, R. van Grondelleand W.T.F. den Hollander, Biochim. Biophys. Acta 725 (1983) 508. [8] H. Bergstfi3m, W.H.J. Westerhuis, W. SundstriSm, R. van Grondelle, R.A. Niederman and T. Gillbro, FEBS Letters 233 (1988) 12. [9] K. Timpmann, A. Freiberg and V.I. Godik, Chem. Phys. Letters 182 (1991) 617. [10] A. Freiberg, V.I. Godik, T. Pullerits and K. Timpmann, Chem. Phys. 128 (1988) 227. [11] R. van Grondelle, H. Bergstr6m, V. SundstrSm and T. Gillbro, Biochim. Biophys. Acta 894 (1987) 313. [12] T. Pullerits, K.J. Visscher, S. Hess, V. Sundstr6m, A. Freiberg, K. Timpmannand R. van Grondelte,Biophys. J. 66 (1994) 236. [13] S. Hess, F. Feldchtein,A. Babin, I. Nurgaleev, T. Pullerits, A. Sergeev and V. Sundstr6m. Chem. Phys Letters 216 (1993) 247. [14] S. Hess, M. Chachisvilis, E. /~,kesson, T. Pullerits and V. SundstriSm,to be published. [15] M. Chachisvilis,T. Pullerits and V. SundstrSm, to be published. [16] K.J. Visscher, H. Bergstr6m, V. SundstriSm,C.N. Hunter and R. van Grondelle,Photosynth. Res. 22 (1989) 211. [17] R.T. Wang and R.K. Clayton, Photochem. Photobiol. 13 (1971) 215. [18] F.A.M. Kleinherenbrink,G. Deinum, S.C.M. Otte, A.J. Hoff and J. Amesz, Biochim. Biophys. Acta 1099 (1992) 175. [19] S.C.M. Otte, F.A.M. Kleinherenbrinkand J. Amesz, Biochim. Biophys. Acta 1143 (1993) 84. [20] I.A. Abdourakhhmanov, R.V. Danelius and A.P. Razjivin, FEBS Letters 245 (1989) 47. [21] K. Timpmann, F.G. Zhang, A. Freiberg and V. Sundstr6m, Biochim. Biophys. Acta 1183 (1993) 185. [22] W. Xiao, S. Lin, A.K.W. Taguchi and N.W. Woodbury, Biochemistry 33 (1994) 8313. [23] O.J.G. Somsen, F. van Mourik, R. van Grondelle and L. Valkunas, Biophys. J. 66 (1994) 1580. [24] R.P. Carithers and W.W. Parson. Biochim. Biophys. Acta 387 (1975) 194. [25] A.S. Holt and R.K. Clayton, Photochem. Photobiol. 4 (1965) 829. [26] H.-W. Trissl, J. Breton, J. Deprez, A. Dobek and W. Leibl, Biochim. Biophys. Acta 1015 (1990) 322.
K. Timpmann et al. / Chemical Physics 194 (1995) 27.5-283 [27] E. Bittersman, R.E. Blankenship and N. Woodbury, in: Current research on photosynthesis, Vol. II, ed. M. Baltscheffsky (Kluwer, Dordrecht, 1990) p. 169. [28] F.G. Zhang, T. Gillbro, R. van Grondelle and V. Sundstr6m, Biophys. J. 61 (1992) 694. [29] F.A.M. Kleinherenbrink, P. Cheng, J. Amesz and R.E. Blankenship, Photochem. Photobiol. 57 (1993) 13. [30] J.M. Olson and R.K. Clayton, Photochem. Photobiol. 5 (1966) 655. [31] S. Hess, K.J. Visscher, J. Ulander, T. Pullerits, M.R. Jones, C.N. Hunter and V. Sundstr6m, Biochemistry 32 (1993) 10314. [32] V. Sundstr6m, R. van Grondelle, H. Bergstr6m, E. ,~kesson, T. Gillbro, Biochim. Biophys. Acta 851 (1986) 431. [33] K. Timpmann and V. Sundstrfim, to be published. [34] M.R. Wasielewski and D.M. Tiede, FEBS Letters 204 (1986) 368. [35] K.R. Miller, Nature 300 (1982) 53. [36] W. Welte and W. Kreutz, Biochim. Biophys. Acta 692 (1982) 479. [37] R.M. Pearlstein, J. Luminescence 51 (1992) 139. [38] P.O.J. Scherer, S.F. Fischer, J.K.H. Horber and M.E. Michel-Beyerle, in: Antennas and reaction centers of photosynthetic bacteria, ed. M.E. Michel-Beyerle (Springer, Berlin, 1985) p. 131.
283
[39] R. van Grondelle, H. Bergstr5m, V. Sundstri~m, R.J. van Dorssen, M. Vos and C.N. Hunter, in: Photosynthetic lightharvesting systems, organization and Function, eds. H. Scheer and S. Schneider (de Gruyter, Berlin, 1988) p. 519. [40] H. Bergstr6m, R. van Grondelle and V. SundstrSm, FEBS Letters 250 (1989) 503. [41] L.M.P. Beekman, F. van Mourik, M.R. Jones, H.M. Visser, C.N. Hunter and R. van Grondelle, Biochemistry 33 (1994) 3143. [42] J. Breton, D.L. Farkas and W.W. Parson, Biochim. Biophys. Acta 808 (1985) 421. [43] G. Deinum, Ph.D. Thesis, Rijksuniversiteit Leiden, The Netherlands (1991). [44] F. van Mourik, R.W. Visschers and R. van Grondelle, Chem. Phys. Letters 193 (1992) 1. [45] H. Zuber and R.A. Brunisholz, in: Chlorophylls, ed. H. Scheer (CRC Press, Boca Raton, 1991) p. 627. [46] R.W. Visschers, M.C. Chang, F. van Mourik, P.S. ParkesLoach, B.A. Heller, P.A. Loach and R. van Grondelle, Biochemistry 30 (1991) 5734. [47] R. Monshouwer, R.W. Visschers, F. van Mourik, A. Freiberg and R. van Grondelle. Biochim. Biophys. Acta, submitted for publication.
ELSEVIER
Energy trapping and detrapping in the photosynthetic bacterium Rhodopseudomonas viridis: transfer-to-trap-limited dynamics K6u Timpmann a,1, Arvi Freiberg b, Villy SundstriSm a,* a Department of Chemical Physics, Lund University, Box 124, S-221 O0 Lund, Sweden Institute of Physics, Estonian Academy of Sciences, Tartu, Estonia
Received 15 November 1994
Abstract The efficiency of energy back-transfer from the reaction center to the antenna in chromatophores of the photosynthetic purple bacterium Rhodopseudomonas viridis was measured at room temperature by means of picosecond time-resolved fluorescence spectroscopy. It was found that 20 + 5% of the excitation energy selectively absorbed by the reaction center pigments in the wavelength region around 830 nm is transferred back to the antenna and gives rise to antenna fluorescence. The measured yield of energy detrapping enabled calculation of the energy detrapping (10-13 ps) i and trapping (40-50 ps)-~ rates. These results confirm that in Rps. viridis, like in bacteriochlorophyil a-containing purple bacteria, the energy transfer step from antenna pigments to the reaction center is a rate limiting step in the overall energy trapping by the reaction center. We suggest that this situation is termed 'transfer-to-trap-limited' dynamics, to distinguish it from the situation where the charge separation is the rate limiting step (trap limited) or the overall energy diffusion through the antenna is limiting (diffusion limited).
1. Introduction During the past few decades the processes of energy transfer and trapping have been thoroughly investigated in various types of photosynthetic organisms (for review see Ref. [1] and references therein), and for photosynthetic purple bacteria a considerable amount of information has been ob-
Abbreviations: RC, reaction center; LH, light harvesting; BChl, bacteriochlorophyll; P, the special pair primary electron donor of the RC; B, the accessory bacteriochlorophylls of the RC; I, the primary electron acceptor of the RC; Q, the quinone secondary electron acceptor of the RC * Corresponding author. RPermanent address: Institute of Physics, Estonian Academy of Sciences, Tartu, Estonia.
tained. Two different models, the so-called traplimited and migration-limited cases have been used to describe the energy transfer and trapping. The first model is based on the assumption of fast energy exchange between antenna and reaction center (RC) pigments, the rate exceeding the rate of charge separation in the RC [2-4]. The overall energy trapping time in this model will therefore be determined by the charge separation time from the special pair to the primary acceptor. In the opposite model the energy migration between the antenna molecules and antenna and RC is thought to be slow and constituting a rate limiting step of the overall energy trapping process [5], implying that the excitation once trapped by the RC pigments has only a low probability to escape.
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In photosynthetic purple bacteria the charge separation in isolated RCs occurs in about 3 ps [6]. Singlet-singlet annihilation [7] and time-resolved absorption anisotropy measurements [8] of Rhodospirillum (Rs.) rubrum and Rhodobacter (Rb.) sphaeroides have revealed that the energy hopping time between antenna pigments is ~< 1 ps. Low temperature picosecond fluorescence [9,10] and absorption [8,11,12] measurements of purple bacteria have shown that most of the energy equilibration over the antenna pigments is complete within ~< 10 ps. Very recent sub-picosecond investigations of the energy transfer processes in membranes of Rs. rubrum at room temperature demonstrated that spectral equilibration of the excitation density within LH1 occurs on a time scale of = 300 fs [1]. From measurements of femtosecond isotropic and anisotropy decay of the various pigments of LH1 and LH2 (B800 [13,14], B850 [15], B875 [15]) it was similarly shown that energy equilibration proceeds on the few hundred femtosecond timescale and is temperature and wavelength dependent, as a result of the spectrally inhomogeneous nature of the pigments. The trapping is considerably slower than this. Picosecond absorption measurements selectively exciting the red-most antenna pigments of Rs. rubrum and Rb. sphaeroides have shown that in the temperature range 100-200 K the antenna excited state lifetime is 35-40 ps [16], which was considered to reflect excitation transfer from antenna pigments coordinating the RC to the primary donor P. At room temperature the trapping rate was estimated to be about (20 ps) -1. The opposite process to trapping, detrapping of excitation energy from the RC to the antenna, was studied through measurements of the fluorescence excitation spectrum of antenna pigments. The results indicated negligible contribution of the RC absorption bands to the excitation spectrum of antenna fluorescence [17-19]. From this observation it was concluded that the detrapping efficiency is only a few per cent. A higher detrapping efficiency, reaching a value of 15-25%, was observed by means of time-resolved spectroscopy methods for different bacterial species [20-22]. On the basis of the measured 25% efficiency of the process the detrapping time constant in Rs. rubrum was estimated to be equal to 6 - 9 ps and the trapping time 30-40 ps [21]. The results accumulated so far
thus give evidence that in photosynthetic purple bacteria the energy trapping by the RC is characterized by the model, where the energy transfer to the RC from the antenna pigments adjacent to the RC dominates the overall trapping time [1,19,21,23]. Rhodopseudomonas (Rps.) viridis, the most investigated bacteriochlorophyll b-containing purple bacterium, may serve as an exception of this picture. In spite of considerable homology between Rps. viridis and bacteriochlorophyll a-containing purple bacteria there exist some spectral and structural differences. An intriguing spectral feature of Rps. viridis is the considerably more red Qy antenna absorption maximum (1015 nm [24]) as compared to that of the RC special pair (985 nm [25]). Despite of the seeming 'up-hill' energy transfer conditions from antenna to RC pigments, the energy trapping in Rps. viridis is found to be efficient, even at low temperatures [18] and the overall trapping time was measured to be very similar to that in BChl a-containing bacteria, 40-80 ps [26-29]. The data on the detrapping efficiency in Rps. viridis are contradictory. In early work by Olson and Clayton [30] it was concluded that excitations absorbed by the RC pigments were about 70% effective to excite antenna fluorescence. At the same time the very recent work of Otte et al. [19] suggested that at room temperature the detrapping efficiency in Rps. viridis is less than 10%. To clarify the specific character of the energy transfer and trapping processes in the BChl b-containing purple bacterium Rps. viridis we have studied the energy back-transfer efficiency from the RC to the antenna pigments by means of time-resolved fluorescence spectroscopy. The measurement was based on a comparison of the excitation spectra of the main decay component of antenna fluorescence at 1040 nm upon excitation of the sample with photochemically active (open) RCs in the state PBIQ, and with photochemically inactive (closed) RCs in the state P+BIQ in the wavelength region of 810-890 nm. It is assumed that in the case of open RCs after selective excitation of B an efficient energy transfer B --* P takes place and is followed by charge separation or energy back-transfer to the antenna, while in the case of closed RCs the back-transfer does not take place because the coupling of B* to P+ and P is different. For Rps. viridis we observed at room temperature a 20 + 5% energy detrapping efficiency
K. Timpmann et al. / Chemical Physics 194 (1995) 27.5-283
from RC to antenna pigments, and from this value the detrapping rate constant of (10-13 ps) -~ and trapping rate constant of (40-50 ps)-~ were calculated. This allows us to conclude that the energy transfer step from the antenna to RC is a slow rate limiting step of the energy trapping process in Rps. viridis, qualitatively very similar to that in BChl a-containing purple bacteria.
277
1.4
A
1.2 -.~
1.0 ~
,~100 B ~ 40
~20 ~ 0 2.
Materials
and
methods
1.0-
C
concentration, mM o - 60ps o - 140 ps
Rhodopseudomonas uiridis membrane fragments (a kind gift of Professor R. van Grondelle, Amsterdam) were diluted in 20 mM Tris-HC1 buffer (pH 8) to achieve a sample with the desired optical density ( = 0 . 3 cm ~) at 830 nm. To keep the primary electron donor in the reduced, photochemically active state, 30-50 mM sodium ascorbate was added to the sample in the buffer. The time-resolved picosecond fluorescence measurements were carried out with an apparatus described elsewhere [31]. The selective excitation of RC or antenna pigments was achieved by using = 10 ps pulses generated in a sync-pumped and cavity-dumped dye laser (Styryl 9) operating in the wavelength region 810-890 nm. The repetition rate of the laser pulses was 800 kHz. To avoid the influence of excitation annihilation processes on the fluorescence kinetics the excitation intensity was kept at the level of < 10 n photons cm -2 pulse -~. The antenna emission was detected at 1040 nm (Jobin Ivon H10-IR monochromator, 10 nm bandwidth) by an infrared-sensitive micro-channel plate photomultiplier. The measured kinetics were analyzed by a multi-exponential deconvolution procedure, taking into account the real temporal response function of the system which was 60-70 ps.
3.
Results
In the experimental mode used here the high repetition rate of the excitation pulses leads to the accumulation of the RCs in the oxidized state P+BIQ if the chromatophores are in plain buffer and no artificial electron donor is added. The fluorescence decay kinetics of the sample is in this case basically single-exponential with a time constant of = 140 ps
~ - 270 ps -
o -
2re(A*20)
-~0.5 -
I
0
I
'
[
100(I 2000 time, ps
I
3000
Fig. 1. (A) The ratio of normalized antenna fluorescence amplitudes of Rps. viridis chromatophores as a function of the concentration of sodium ascorbate. (B) Different fluorescence decay constants as a function of the concentration of sodium ascorbate. Note that 2 ns component has 20 times different scaling. (C) Two fluorescence kinetics of Rps. viridis membranes. (1) Chromatophores in plain buffer (closed RC); (2) part of the RCs are opened by sodium ascorbate. Note the apparent faster component and also a long tail in later case.
(see Fig. 1), in good agreement with earlier results [28]. In a few measurements an additional 40-50 ps decay component with a relative amplitude of 1020% was also observed, as for example in Refs. [27,29]. We believe that this decay component is due to the degradation of the sample during a prolonged experiment. The decay time of the antenna excitation was found to be independent of the excitation wavelength. To realize photochemically active RCs (in the state PBIQ) the electron donor sodium ascorbate was added to the sample. In this case the fluorescence decay kinetics became more complex, consisting of at least four decay components with time constants of about 60, 140, 270 and 2000 ps (see Fig. 1). The amplitude ratios of the decay components depend on the amount of the electron donor added to the sample, as shown in Fig. 1. The two shortest decay
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K. Timpmann et al. / Chemical Physics 194 (1995) 275-283
components have opposite dependence on the ascorbate concentration. When the relative amplitude of the 60 ps component increases from 50% (5 mM of ascorbate added) to 80% (70 mM of ascorbate), the relative amplitude of the 140 ps decay component decreases from 35% to 5% for the same ascorbate concentrations. The results of earlier time resolved measurements on Rps. viridis available in the literature [27-29], together with the dependence of the kinetics on artificial electron donor concentration presented here, suggest that the 60 ps decay time corresponds to the trapping of excitation energy by the reaction centers with reduced primary electron donor P, while the 140 ps decay component describes the quenching of the antenna excitations by RCs with oxidized electron donor, P+. The amplitude of the 2 ns decay component has a similar dependence on the ascorbate concentration as the 60 ps component, while the amplitude of the 270 ps decay is practically independent of ascorbate concentration. At the same time, the relative amplitudes of these two decay components depend on the excitation wavelength, being about two times (the 2 ns component) and 1.3 times (270 ps component) more pronounced for 830 nm excitation as compared to 880 nm excitation. At 830 nm a significant part of the excitation light is absorbed selectively by the RC, while at 880 nm to a good approximation only the antenna absorbs. In addition, the relative amplitude of the 2 ns component diminishes with decreased excitation intensity. From these observations we conclude that although the excitation intensity is moderate in our experiments, due to the stationary sample the electron transport chain of the RC is partially saturated and this causes photoaccumulation of a small amount of reduced primary electron acceptors of the RC, Q - , giving rise to the 2 ns component [3]. The nature of the 270 ps component is more problematic, probably it is a manifestation of several different processes (for example electron transfer from I to Q with about 200 ps time constant [32]) not fully resolved in the fitting procedures of the kinetic curves. In Fig. 2 the excitation spectra of antenna fluorescence are presented for a sample with photochemically active RCs (crosses) and with photochemically inactive RCs (circles) in the wavelength region of 810-890 nm. For comparison, the corrected absorp-
1.4
A
"-~ 1.2 1.0
~
.
IQ
''''I''''l'' B
0.4
150
C). O
10o
02
50 .= 0
. . . .
800
I
. . . .
I
,
,
0
85O 90O wavelength, rtm
Fig. 2. (A) The ratio of normalized antenna fluorescence amplitudes as a function of excitation wavelength. (B) The excitation spectra of antenna fluorescence with open (crosses) and closed (circles) RCs. The solid line is the corrected absorption spectrum of Rps. viridis chromatophores (see text).
tion spectrum (see below) of Rps. viridis chromatophores (solid line) is also reproduced. The excitation spectra were measured as the amplitude of the fluorescence decay curves detected at 1040 nm upon excitation in the wavelength region of 810-890 nm. In the case of a sample with open RCs the amplitude of the 60 ps decay component was extracted and used for the determination of the excitation spectrum. Measurements were performed up to a maximum concentration of 50 mM sodium ascorbate, which allowed us to estimate the ratio of the open and closed RCs in the sample to 8 - 1 0 at this ascotbate concentration. The two excitation spectra differ significantly around 830 nm, the wavelength region of the accessory BChl absorption band. This is demonstrated more clearly with the ratio of a normalized antenna fluorescence amplitudes with open (Fops,) and closed (Fdos~d) RCs as a function of excitation wavelength, presented in Fig. 2B. In the red part of the wavelength range )t > 850 nm the ratio Fope,/Fcloscd = 1, since the absorbance of the RC pigments is negligible compared to that of the antenna pigments at these excitation wavelengths. At shorter wavelengths the ratio Fop~n/Fdosed > 1 and it has a wavelength dependence matching the shape of
K. Timpmann et al. / Chemical Physics 194 (1995) 275-283
the RC absorption band in this wavelength region (see Fig. 2). The r a t i o Fopen/Fclosed is peaking at 830 nm and its value depends on the amount of photochemically active RCs present in the sample (see Fig. 1A). These results demonstrate that there exists a measurable energy back-transfer from the RC to the antenna at room temperature. The efficiency of the energy back-transfer is determined as [21] ~l = ( A a n t / A R c ) ( Fopen/Fclose8 -
1),
279
A
+ + ++~-~ +~"~-
4-
+
+
+
+
0
,
i
,
i
[
B
1.5
(1)
where A,, t and ARC are the absorptions at the excitation wavelength of the antenna and RC pigments, respectively. In order to obtain a correct value of the degree of back-transfer we have to determine the ratio of the antenna and the RC absorptions at each excitation wavelength. Here the main problem is to eliminate the background from the absorption spectrum of the sample, caused by scattering and absorption of uncoupled pigments. In order to describe the antenna and accessory BChl absorption spectra we fitted the antenna fluorescence excitation spectrum with a sum of Gaussians, and the 800 nm absorption band of the accessory BChl's in a RC preparation was similarly fitted to another sum of Gaussians. Then, the absorption spectrum of Rps. t,iridis chromatophores was fitted with the above Gaussians plus a constant background leaving the relative contribution from RC and antenna open. In that way the ratio of the RC and antenna absorptions in the wavelength region 810-870 nm was found (see Fig. 3B), and the energy back-transfer efficiency was calculated according to Eq. (1) (Fig. 3A). The corrected absorption spectrum is presented in Fig. 2. The average value of the detrapping efficiency after excitation of the accessory bacteriochlorophylls (B) in the RC was found to be 20 _+ 5%. The variation of the calculated 77 value from the average is more pronounced in the wings of the RC absorption band, where the RC and antenna absorption ratio is but poorly determined due to the difficulties with correcting the absorption spectrum of the sample for scattering and background absorption.
4. Discussion
The data presented in Fig. 2 demonstrate the contribution of the RC pigments in the 830 nm
0.5-
0
,
80O
85O wavelength, n m
Fig. 3. (A) Efficiency of the energy back-transfer from the RC to the antenna in Rps. ciridis chromatophores as a function of excitation wavelength. (B) The ratio of the contribution of the RC and the antenna absorption as a function of wavelength.
spectral region to the excitation spectrum of antenna fluorescence. It is shown by means of femtosecond absorption measurements on isolated RCs that there exists a very efficient and fast energy transfer from the bacteriopheophytin and accessory bacteriochlorophyll monomer to the primary electron donor P [6]. Thus, the observed antenna signal is caused by the energy back-transfer from the RC dimer P to the antenna pigments upon selective excitation of the RC accessory bacteriochlorophylls in this experiment. The RC contribution to the excitation spectrum of antenna fluorescence depends on the amount of photochemically active RCs present in the sample (see Fig. 1); with = 90% of the RCs in the photochemically active state PBIQ, the amplitude of antenna fluorescence exceeds by 1.3 times that for the sample with all RCs in the closed state and for = 60% of the RCs being in the open state the ratio of antenna fluorescence amplitudes was 1.1. Taking into account the relative absorption of antenna and RC molecules in the 830 nm spectral region (Fig. 3B) and the amount of open RCs in the sample, the energy back-transfer efficiency was calculated to be 20 + 5%. This result is similar to the energy backtransfer efficiency measured with time-resolved
280
K. Timpmann et al. / ChemicalPhysics 194 (1995)'275-283
spectroscopy methods for Rs. rubrum (25%) [21], for Chromatium minutissimum (10-20%) [20] and for Rhodobacter capsulatus (15%) [22]. At the same time our data differ from those obtained for Rps. viridis by means of stationary spectroscopy [18,19,30]. In an early paper by Olson and Clayton [30] the energy transfer efficiency from the RC BChl monomer B to antenna pigments was found to be at least 70%. Such a high value was obtained by assuming an exceptionally low RC absorption relative to antenna absorption in the 830 nm absorption band. If a more realistic RC/antenna absorption ratio, like that derived in the present work, is used for the evaluation of these experimental data the energy back-transfer efficiency will be 20-30%, in good agreement with our results. In the work by Kleinherenbrink et al. [18] and Otte et al. [19] the efficiency of energy transfer from the RC to the antenna was found to be very low, ~< 2% at 6 K and ~< 10% at room temperature. As our investigation of the temperature dependence of energy detrapping efficiency in Rs. rubrum show (results will be presented elsewhere [33]), the back-transfer yield will decrease with lowering of the temperature, from = 25% at room temperature to at the most a few percent at 77 K. The room temperature results of Ref. [19] differs from ours by a factor of at least two. We believe that this discrepancy might be due to different experimental conditions. In a tifne-resolved experiment there exists an obvious control of the redox state of the RCs in the sample through the measurement of the antenna excited state lifetime, while in the stationary experiment this additional control parameter is absent. As our intensity-dependent kinetic measurements on Rps. viridis and earlier results on R. rubrum [3] show, the redox state of the RCs is very sensitive to the light intensity and electron donor concentration used in the experiment. Thus, it is important to be able to fine tune the experimental conditions while at the same time monitoring the state of the reaction center. On the basis of the measured fluorescence lifetimes and the energy detrapping yield the rate constants of forward and backward energy transfer between RC and antenna can be obtained. For that purpose numerical simulations of the time dependence of the antenna and RC excitations were performed, by means of the three-component kinetic
kl k_l
k~ P BIQ
k_2
P (P~I-Q-)
Fig. 4. Simplified kinetic model of energy transfer between antenna and RC used in numerical simulations.
scheme of Fig. 4. Here k I represents the total trapping rate of antenna excitations by the RC, k ~ is the total detrapping rate from the RC to the antenna, k 2 is the charge separation rate in the RC and k 2 the charge recombination rate. A precise description of the simulation procedure was given in Ref. [21]. The simulation was performed for a range of k~ and k ~ values until agreement was obtained between calculated and measured values of the antenna fluorescence lifetime and the detrapping efficiency. As a result, the total trapping and detrapping rates in Rps. viridis were found to be k j = ( 4 5 + 5 ps)-I and k 1 = (10 _ 2 ps) -1. These rate constants were found by using the charge separation rate k 2 = (2.8 ps)measured for isolated RCs of Rps. viridis [6]. It has been shown [34] that the charge separation rate in the presence of pre-reduced quinone acceptor ( Q - ) slows down to a value of k 2 = (5.6 ps) -1. In our experiments part of the RCs were in the state PBIQ-. Taking this into account the calculated detrapping rate constant slowed down to k_ 1 = (13 + 2 ps) ~, while the trapping rate practically did not change. From the derived total trapping and detrapping rates it is possible to deduce estimates for the pairwise rates between antenna and RC molecules. The pairwise detrapping rate kt) is related to the total detrapping rate from the RC to all nearest q antenna molecules coordinating the RC by k o = k_,/q.
(2)
The data of electron microscopy and imageprocessing techniques [35,36] indicate that in Rps. viridis the reaction center is surrounded by a ring of six antenna complexes and therefore it is reasonable to assume that q = 6. In that case the pairwise detrapping rate is k D = (60-70 ps) -a. Recently, Pearlstein introduced a mono-coordinate model for the energy trapping by the RC [37]. In this model the pairwise energy detrapping rate k D would be equal to the measured overall detrapping rate k_ ~ = (10-13
K. Timpmannet al. / ChemicalPhysics 194 (1995)275-283 ps) -~. The pairwise trapping rate k T of antenna excitations by the RC is related to the detrapping rate by the ratio of the F6rster overlap integrals for the two processes ( I T for trapping and I D for detrapping, respectively),
k T = (IT/tD) k D .
(3)
The calculation of the ratio of F6rster overlap integrals is quite problematic, since authentic spectral data of RCs in intact membranes are absent. The absorption spectrum of the isolated RC primary electron donor P is peaking at 965 nm [38], far to the blue of the main antenna absorption maximum at 1015 nm. It is believed that in intact membranes the absorption maximum of RCs is shifted by at least 20 nm to the red [18,25]. By using the absorption and fluorescence spectra of the antenna [24], taking into account the RC absorption spectrum shift, and assuming a similar shift for the RC fluorescence spectrum [38], the ratio of F6rster overlap integrals was calculated to be 1y/1D = 0.85. Now the pairwise trapping rate constant becomes k v = (70-85 ps)-1 with q = 6, and k y = (12-15 ps) i in the mono-coordinate case. This result shows that the pairwise energy trapping and detrapping rates between the antenna and RC molecules are by at least one order of magnitude smaller than the subpicosecond energy hopping rate between the antenna molecules [12,13]. This finding supports the idea that the final energy transfer from the antenna to the reaction center special pair is a rate limiting step in the overall energy trapping process. Based on early one-color p u m p probe measurements at 77 K [11] on Rb. sphaeroides and R. rubrum this view was first suggested by van Grondelle et al. [39] and somewhat later substantiated by measurements of the antenna excited state lifetime (35 ps) in Rb. sphaeroides and R. rubrum at 77 K with photochemically active (P) reaction centers [40]. Trapping kinetics measured in mutants of Rb. sphaeroides with modified charge separation rate, suggested a similar picture [41]. It should be noticed that in the original notation of trap or diffusion limited dynamics [2], the present situation with a slow rate limiting step from the antenna to the reaction center should be termed as diffusion limited trapping, because the rate limiting term resides in the so-called first passage time (Eq. (4)). This may cause some confusion since the rest of the dynamics, the
281
energy equilibration within the antenna, is very fast and occurs on the timescale of a few hundred femtoseconds [1,12-15]. Therefore, we suggest that a slightly different term is used to denote this situation with a slow, rate limiting, energy transfer step from the antenna to the reaction center namely, 'transferto-trap-limited' dynamics. It is of interest to use our measured kinetic data of Rps. viridis fluorescence and energy detrapping rate constants as input parameters to the Pearlstein model for energy migration and trapping [2]. In this model the antenna is described as a regular lattice of N equivalent sites with pigment molecules occupying each lattice point, and the energy migrating as a random walk over the antenna lattice. Such a model is applicable to Rps. viridis whose antenna is believed to be practically homogeneous [28,42,43], unlike the antenna of BChl a-containing purple bacteria which have been shown to be spectrally inhomogeneous [9,44]. The model gives the antenna exciton lifetime tex as the sum of two components, the first passage time tfpt and the revisiting time trev, where tfpt = N ( q k v ) - l +
[f(U)
-q
1]kA1N
(4)
and
trev=k2'N(lT/ID)
'
(5)
Here f ( N ) is the antenna structure function and k A is the energy hopping rate between the equivalent antenna sites. As was shown in Ref. [23], the value of the second term in Eq. (4) describing the energy migration time through the antenna to the RC is not more than a few picoseconds with a hopping rate k A of at least (1 ps) ~ [23] and assuming a two-dimensional hexagonal lattice structure. This implies that Eq. (4) can be simplified by neglecting this term in comparison with the first term describing the trapping. Then, by combining Eqs. (2)-(5), the antenna exciton lifetime can be expressed as
tex=(k
', + k 2 ' ) N ( I T / I D )
'.
(6)
It is known that there are 24 antenna molecules per RC in Rps. viridis [46]. At least for BChl a-containing purple bacteria the basic building block of the LH1 antenna is believed to be a dimer of strongly coupled antenna molecules [45]. Thus, it is reasonable to assume that N = 12, and with the values of
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K. Timpmann et al. / Chemical Physics 194 (1995) 275-283
k_ 1 = (10 ps) -1, k 2 = (2.8 ps) -1 and I T / I D = 0.85, the antenna exciton lifetime is found to be 190 ps. This value exceeds by approx, a factor of three the measured antenna fluorescence lifetime of 60 ps. If we assume that the antenna is organized in small clusters of four pigment molecules, probably the smallest possible N consistent with the structural data of the Rps. viridis antenna system, the antenna excitation lifetime derived from Eq. (6) is tex = 95 ps in reasonable agreement with experiment. Better matching between the measured and calculated antenna fluorescence lifetimes might be achieved by assuming that the energy detrapping rate (and therefore the trapping rate) is nearly two times faster than that used above (i.e. k_l = (5.5 p s ) - l ) , or that the ratio of the F/Srster overlap integrals is significantly more in favour of the trapping process (i.e. l - r / I D = 1.3). The first possibility implies that the energy back-transfer efficiency has to be about 40%, in disagreement with the measured yield of 20%. The second possibility is realized for example if the maximum of the absorption spectrum of the RCs in the intact membrane is shifted more to the red, to 995 nm, with other spectral parameters unchanged. This possibility is supported by very recent fluorescence site selection experiments of the Rps. viridis antenna [47], which suggest that at low temperature the antenna excitations are equilibrated in the far red wing of the antenna spectrum. In order to explain the observed charge separation efficiency at low temperature [18] the P absorption band must be more red-shifted than "the previously assumed Amax = 985 nm. Naturally, there also exist other optimizing factors of the forward energy transfer from antenna to the RC.
Acknowledgements The present study was supported by The Swedish Natural Science Research Council, the International Science Foundation, the Estonian Science Foundation and the EC contract SC1"-CT92-0796. K.T. especially acknowledges the Swedish Institute and The Swedish Academy of Sciences for a visiting scientist grant. We thank Dr T. Pullerits for many stimulating discussions.
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