Abstract. The initial electron transfer steps in pigment modified reaction centers, where bacteriopheophytin is replaced by plant pheophytin (R26.Phe-a RCs) ...
Photosynthesis Research 55: 153–162, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.
153
Temperature dependence of the primary electron transfer reaction in pigment-modified bacterial reaction centers Heinz Huber1 , Michaela Meyer2 , Hugo Scheer2 , Wolfgang Zinth1 & Josef Wachtveitl1,∗ 1 Ludwig-Maximilians-Universität
München, Institut für Medizinische Optik, Oettingenstr. 67, 80538 München, Germany; 2 Ludwig-Maximilians-Universität München, Botanisches Institut, Menzinger Str. 67, 80638 München, Germany; ∗ Author for correspondence Received 11 September 1997; accepted in revised form 2 February 1998
Key words: electron transfer, femtosecond spectroscopy, pheophytin a, pigment modification, reaction center, temperature dependence
Abstract The initial electron transfer steps in pigment modified reaction centers, where bacteriopheophytin is replaced by plant pheophytin (R26.Phe-a RCs) have been investigated over a wide temperature range by femtosecond timeresolved spectroscopy. The experimental data obtained in the maximum of the bacteriochlorophyll anion band at 1020 nm show the existence of a high and long-lived population of the primary acceptor P+ BA − even at 10 K. The data suggest a stepwise electron transfer mechanism with BA as primary acceptor also in the low temperature domain. A detailed data analysis suggests that the pigment modification leads to a situation with almost isoenergetic primary and secondary acceptor levels, approximately 450 cm−1 below P∗ . A Gaussian distribution (with σ = 400 cm −1 ) of the 1G values has to be assumed to account for the strong dispersive character of the kinetics in this sample. Based on these assumptions, a model is presented that reproduces the observed kinetics, heterogeneity and temperature dependence. Abbreviations: BA,B – sites of monomeric BChl-a in the RC; BChl-a – bacteriochlorophyll a; BPhe-a – bacteriopheophytin a; ET – electron transfer; FWHM – full width at half maximum; HA,B – sites of BPhe-a or Phea; λpr – probing wavelength; P – primary donor; Phe-a – pheophytin a; QA,B – quinone sites; Rb. – Rhodobacter; RC(s) – reaction center(s); RT – room temperature (indices A and B refer to the position on the active and inactive branch, respectively) Introduction The reaction center (RC) is a membrane-bound pigment–protein-complex responsible for the fundamental energy conversion process in photosynthesis. For the purple bacterial species Rhodobacter (Rb.) sphaeroides the structure of the complex is known to atomic resolution in the dark (Allen et al. 1987; Chang et al. 1991; Ermler et al. 1994) and under illumination (Stowell et al. 1997). The successful X-ray analysis in combination with time resolved spectroscopy has led to a detailed understanding of primary electron transfer (ET) reactions in bacterial photosynthesis. A clear picture about the primary steps following the excita-
tion of the RC has emerged in the last few years. The sequence of fast electron transfer reactions starts from the excited primary donor P∗ , a strongly coupled pair of bacteriochlorophyll a (BChl-a) molecules. The P∗ state can be created either by direct photoexcitation of the dimer or by energy transfer from the antenna or from other photoexcited tetrapyrrole pigments within the RC (Hunter et al. 1989; Fleming and van Grondelle 1994; Jonas et al. 1996). Only one of the two symmetrically arranged pigment branches is utilized for electron transfer. In the first charge separation reaction, the monomeric BChl-a on the active branch is reduced and the intermediate P+ BA − is formed within 3.5 ps (Holzapfel et al. 1989; Dressler et al. 1991;
PIPS NO.:158944 (M) (preskap:bio2fam) v.1.15 pres816.tex; 30/06/1998; 15:16; p.1
154 Chan et al. 1991; Arlt et al. 1993; Beekmann et al. 1995). Subsequently the electron is transferred to the BPhe-a molecule HA within 0.9 ps, followed by the much slower reduction of the quinone QA by HA − in approximately 200 ps (Martin et al. 1986; Kirmaier and Holten 1987; Holzapfel et al. 1990). Since the fast decay of the P+ BA − intermediate necessarily leads to a low population density and complicates its detection, the existence of P+ BA − as the first charge separated state was questioned for a long time (Woodbury and Allen 1995). Modern biotechnology has helped to elucidate the details of primary electron transfer. Site directed mutagenesis and specific pigment exchange are powerful tools to selectively tune the energy levels of the individual intermediates. Since exchange rates of 90% or better can now be obtained experimentally (Meyer and Scheer 1995), pigment modified RCs represent a class of both structurally and functionally interesting samples. Recently, it could be shown that the introduction of plant pheophytins (Phea) into the HA and HB sites replacing the BPhe-a molecules leads to remarkable alterations in reaction dynamics (Shkuropatov and Shuvalov 1993, Schmidt et al. 1994, 1995) and quantum yield (Franken et al. 1997b). The redox-potential of Phe-a in solution is considerably more negative (170–270 mV) than that of BPhe-a (Geskes et al. 1995), suggesting an energetic proximity of the P+ BA − and the P+ HA − intermediates in the pigment exchanged Phe-a RCs. Femtosecond time resolved experiments in the anion region of the monomeric BChl-a showed that the drastic increase of the relative free energy of the P+ HA − intermediate led to a thermal repopulation of the P+ BA − state at room temperature and consequently to a higher and long-lived population of this intermediate (Schmidt et al. 1994, 1995). The direct observation of the accessory bacteriochlorophyll anion again strongly supported the sequential character of the primary ET reactions and allowed the determination of the free energy difference between the primary donor (P∗ ) and acceptor (P+ BA − ) states (1G = –450 cm−1 ). In a further study the functional groups responsible for the differences between Phe-a and BPhe-a were addressed individually and a clear relation to the spectral and the redox properties in the modified RCs could be identified (Huber et al. 1995). The temperature dependence of the Phe-a RCs provides a crucial test for the reaction model and helps to answer the question whether the dynamics at low temperature is consistent with the idea of thermal equilibration between almost isoenergetic intermediates.
In this paper, we present fs-dynamics of R26.Phea RCs over a wide temperature and spectral region, with a focus on ground state recombination, P∗ decay and BA − population density. A reaction model is presented, which consistently explains the low temperature dynamics of the pigment modified samples.
Material and methods Femtosecond transient absorption measurements were carried out as described previously (Huber et al. 1995), with the modification that the sample was in a 2 mm cuvette, mounted on a copper frame and kept in a closed-cycle helium cryostat. The setup allowed a continuous variation of the temperature from RT to 10 K. The sample temperature was measured with an accuracy of ± 1 K with a temperature sensor mounted directly on the cuvette window. The femtosecond system is based on a Ti:Sapphire laser-amplifier system operating at a central wavelength of 870 nm. The ultrashort light pulses of the Ti:sapphire oscillator were amplified in a regenerative chirped pulse amplifier pumped by a frequency doubled 20 Hz Nd-YAG laser. This set-up provided light pulses with a duration of 100 fs (FWHM). With an appropriate attenuation weak (≈ 0.3 µJ) subpicosecond excitation pulses were selected and focused into the sample to a spot size of 0.4 mm, which corresponds to an approximate excitation of 10% of the RCs at RT. A femtosecond white light pulse is generated in a 3 mm sapphire crystal and spectrally selected in a grating monochromator. Probing pulses of adjustable spectral width in the wavelength region between 600 nm and 1400 nm were properly delayed, focused into the excited sample spot (0.2 mm) and detected with Si- (for λ < 900 nm) or InGaAs-diodes (for λ > 900 nm). In all experiments, the polarization of exciting and probing pulses is parallel, the typical instrumental response function is around 200 fs. The absorbance changes were recorded as a function of delay time tD and the data were evaluated using a rate equation model described in detail elsewhere (Finkele et al. 1990; Schmidt et al. 1995). In the simulation the free energies of the intermediate states (P∗ , P+ BA − , P∗ HA − ) enter the calculation of the backward and forward rates via the principle of detailed balance. The microscopic rates and the free energies are taken as free parameters in order to reproduce the experimental observables (time constants and population densities). Based on this nonadiabatic electron transfer model, difference cross section spec-
pres816.tex; 30/06/1998; 15:16; p.2
155 the 10–20 ns time constant indicating the decay of the radical pair state P+ QA − to the ground state (Ogrodnik et al. 1988). The time resolved data of reduced R26.Phe-a RCs therefore contain no information on the P+ HA − → P+ QA − reaction. Results Steady-state spectra
Figure 1. Steady state absorption spectra of Phe-a RCs, containing plant pheophytin in the HA and HB sites. The sample was in 20 mM Tris-HCl buffer at pH 8 and contained 0.1% LDAO, 50 mM dithiothreitol and 54% (v/v) glycerol.
tra are calculated from the transient absorption data (Figure 5). Absorbance spectra (as shown in Figure 1) were obtained using the cryostat described above in a commercially available UV/VIS spectrometer (PerkinElmer). The concentration of the samples was adjusted to values between 0.5 and 1 OD for the QY P-band (temperature-dependent between 860 nm and 900 nm). Glycerol (54% v/v) was used as a cryoprotector. Preparation, isolation and pigment exchange procedure of the Phe-a RCs is described by Meyer and Scheer (1995). To prepare the samples for time resolved spectroscopy at low temperatures, the electron transfer from HA − to QA has to be blocked, since the ground state recombination from the P+ QA − state is not completed within the 50 ms intervals between the laser pulses and the accumulation of photoproducts would be unavoidable under the given experimental conditions. The method of quinone depletion is problematic in pigment modified RCs, since the procedure seems to destroy selectively R26.Phe-a RCs, therefore creating a mixture of R26.Phe-a and native RCs with relatively poor purity. The method of quinone reduction is much more reliable, thus either 20 mM sodium dithionite or 50 mM dithiothreitol was applied to the sample solution. The complete reduction of QA was checked by monitoring the amplitude of
The steady-state absorption spectra of R26.Phe-a RCs at various temperatures are shown in Figure 1. The spectrum at room temperature is in agreement with previously reported spectra (Meyer and Scheer 1995; Schmidt et al. 1995; Franken et al. 1997a). The blueshifted QY transition (at 674 nm) and the double peak in the QX region (at 509 nm and 542 nm) characteristic of plant Phe-a (Meyer and Scheer 1995) can be clearly identified in the spectrum. The small amplitude of the remaining BPhe-a absorption at 760 nm indicates the good exchange efficiency (at both HA and HB sites) of ≥ 90%. Apparently, the quinone reduction had no influence on the spectral properties of the sample: both the BPhe-a peak and the contribution of free pigment are as small as in the untreated R26.Phe-a RC sample. The P bands also remain practically unchanged. A strong temperature dependence of the spectral position is observed only in the QY P-band, which shifts from 860 nm to 900 nm upon cooling. In the 15 K spectrum a pronounced shoulder appears at 615 nm, which can be assigned to a weak vibrational (Q0−1 y ) band of the Phe-a pigment (Franken et al. 1997a). A comparison to the wild type RCs shows that the pigment exchange does not affect the spectral characteristics of the four BChl-a molecules in the RC at any temperature. Time resolved experiments The bleaching of the QY P-band and the persistence of the charge-separated state is monitored at a probing wavelength of 850 nm. In untreated native RCs, the bleaching signal remains constant throughout the entire experimentally observed time range. Figure 2 shows the data for quinone-reduced R26.1 RCs at 10 K, where the forward electron transfer from HA to QA is blocked. A constant bleaching for delay times up to several hundred picoseconds is observed. At later delay times the bleaching signal decreases and the decay can be described by two time constants of 0.3 ns (fit amplitude 90%). The main component represents the recombination of the
pres816.tex; 30/06/1998; 15:16; p.3
156
Figure 2. Transient absorption changes of native (open circles) and R26.Phe-a RCs (closed circles), probed within the QY P band (λpr = 850 nm). The best model functions for the experimental data using the time constants given in Table 1 are plotted as solid lines.
radical pair state P+ HA − (either directly or via the triplet state 3 P). The observed values and the multiphasic behaviour of the ground state recovery agrees with previously reported measurements (Schenck et al. 1982; Ogrodnik et al. 1988, 1993, 1994). For the R26.Phe-a RCs at 10 K, the ground state recombination is found to be more pronounced than in WT. As in R26.1 RCs, two time constants are needed for a good description of the dynamics, but here the amplitude of the fast component (500–700ps) is much stronger (≥ 50%) than in WT. This component is strong enough that it could effectively compete with the forward ET reaction in ‘open’ (not reduced) RCs. This could explain the significant reduction of the quantum yield of P+ QA − formation in unreduced Phe-a samples, reported both at room temperature (Schmidt et al. 1995) and at low temperature (Franken et al. 1997b). The reason for the decrease of the 850 nm signal for the reduced sample at cryogenic temperatures (Figure 2) remains to be clarified. The spectral characteristics of 3 P indicate that enhanced triplet formation can only partly account for that effect (Lous and Hoff 1986). We find that the lifetime of the charge-separated state does not strongly depend on the temperature. Only a slight acceleration of the slow component of the recombination reaction for R26.Phea RCs with increasing temperature is found (Figure 2 and Table 1).
Figure 3. Time resolved absorption data for λpr = 920 nm. The measurements were recorded up to 3000 ps to obtain a reliable asymptote at long delay times. For improved visibility of the fast dynamics, only the first parts of the curves are shown. The curves are normalized to the same maximal signal amplitude. The inset shows the temperature dependence of the ratio of the two lifetimes τ (e−1 ) and τ (e−2 ) (Bixon et al. 1995), which reflects the dispersive character of the observed kinetics.
The temperature dependent decay of the photoexcited primary donor P∗ was monitored at 920 nm and is shown in Figure 3 (normalized to the same max. signal amplitude). The transient absorbance changes are dominated by the stimulated emission of P∗ . The P∗ decay becomes faster upon cooling; the lifetime decreases from 5.1 ps at 300 K to 2.1 ps at 10 K in a monoexponential fit. The fact that the observed decay time at room temperature is somewhat longer than the previously reported values might originate from the sample reduction. Repulsive effects from the negative charge at QA could lead to a slower ET reaction. However, since the monoexponential fit function is only a poor description of the decay, three time constants are used in a more detailed analysis for a good approximation of the curve. This is in agreement with recent experimental findings that the inhomogeneous effects on the kinetics of the primary charge separation processes are quite substantial and that therefore the description of the P∗ decay with only one lifetime is inappropriate, no matter whether the fluorescence or the stimulated emission of the primary donor was analyzed (Müller et al. 1992; Hamm et al. 1993; Jia et al. 1993). The time constants for the P∗ decay in R26.Phe-a RCs, the corresponding amplitudes and the temperature dependent changes are summarized in Table 1. For a kinetic analysis of the temperature dependence of the primary reaction that includes the implications of a heterogeneous system and is independent from special fitting models, an evaluation
pres816.tex; 30/06/1998; 15:16; p.4
157 Table 1. Time constants and amplitudes used to fit the data shown in Figures 2–4 for the probing wavelengths of λpr =920 nm (1), λpr =1020 nm (2) and λpr =850 nm (3). The fit amplitudes describe the experimentally determined transient absorbance changes at the corresponding temperatures without any further scaling factor. The temperature dependence in the total amplitude is mainly caused by the changes of the Qy (P)-band, leading to a different overlap between the spectrum of the excitation pulse (centered around 870 nm) and the Qy (P)-absorption band. It is also possible to describe the 290 K transient with the values found for the unreduced R26.Phe-a sample (∗ ) as described by Schmidt et al. 1995. Using a step-wise model, the population of the first electron transfer intermediate P+ BA − can be deduced, the values are given in the last column Temperature (K)
Time constants (ps)(1) τ 1A / τ 1B / τ 1C
Amplitudes (%)(1) –a1A / –a1B / –a1C
Time constants (ps)(2) τ1 / τ2
Amplitudes (%)(2) (–a1 ) / (–a2 )
10 30 60 90 120 180 210 240 270 290
1.5 / 18 / 300 1.5 / 14 / 300 1.6 / 12 / 300 1.6 / 9.0 / 300 1.9 / 14 / 300 2.3 / 12 / 300 2.5 / 12 / 300 2.6 / 13 / 300 3.1 / 19 / 300 3.2 / 14 / 300 3.5 / 15 / 300 (∗)
15.9 / 2.5 / 0.9 16.8 / 3.3 / 0.9 15.9 / 2.6 / 0.7 14.3 / 2.6 / 0.8 16.5 / 1.9 / 0.4 14.4 / 1.7 / 0.8 13.9 / 1.8 / 0.9 12.8 / 2.4 / 1.1 13.4 / 2.6 / 1.1 11.2 / 3.7 / 1.1 11.7 / 2.9 / 0.7 (∗)
2.1 / 0.50 2.2 / 0.48 2.3 / 0.54 2.3 / 0.55 2.5 / 0.65 2.9 / 0.66 3.2 / 0.72 3.6 / 0.89 4.4 / 1.2 5.1 / 1.3
1.2 / 3.2 1.2 / 3.2 1.1 / 3.3 0.9 / 3.4 1.2 / 3.4 2.0 / 2.4 1.1 / 3.3 1.2 / 3.0 1.6 / 2.4 1.6 / 2.2
method proposed by Bixon and coworkers (Bixon et al. 1995) is chosen. This analysis involves the τ (e−1 ) and τ (e−2 ) times, where the population has decayed to 1/e and 1/e2 . Here, the ratio r = τ (e−2 ) / 2∗ τ (e−1 ) is a measure of the non-exponentiality of the observed reaction (r = 1 for a monoexponential decay). The temperature dependence of r is shown as inset in Figure 3. It can be clearly seen that the values for r change significantly with T. The curve exhibits a U-shaped form with a pronounced minimum (r ≈ 1.2) between 100 K and 200 K; the nonexponential character of the decay curve is most evident at 10 K (r ≈ 2) and 290 K (r ≈ 1.8 ). The consequences for the electron transfer parameters and the distrubution of the 1G values of the states involved are discussed below. It has been previously shown for R26.Phe-a RCs at room temperature that the transient absorption signal in the maximum of the BChl-a anion band, i.e. at λpr = 1020 nm, can only be explained by a high and longlived P+ BA − population (Schmidt et al. 1994, 1995). Since the P+ BA − state can be observed without interfering ground state absorption at this probing wavelength, the temperature dependent extent to which this intermediate is populated can be directly analyzed here. In this wavelength range predominantly P∗ decay and ET from P+ BA − to P+ HA − contribute to the signal. Since the size of the P∗ signal is rather weak, a
Time constants (ps)(3) τ 3A / τ 3B
Amplitude ratio a3A /a3B (3)
[P+ BA − ] (%)
500 / 20000 n.d. n.d. 740 / 20000 540 / 14000 750 / 10000 740 / 10000 650 / 10000 730 / 10000 600 / 10000
1.1 n.d. n.d. 1.2 1.9 2.2 1.7 1.5 2.4 1.6
51 50 47 50 50 51 44 46 45 44
monoexponential decay with τ 1 was assumed in the fit functions. The comparison with native RCs (Figure 4, open symbols) clearly shows that in R26.Phe-a RCs an increased and long-lived P+ BA − population can be observed throughout the entire temperature range investigated. Apart from the time constant τ 1 (or τ 1A , τ 1B , τ 1C ) known from the experiments at λpr = 920 nm, a dominant faster component is necessary to describe the rise of the transients correctly. This time constant decreases from 1.3 ps at 300 K to 0.5 ps at 10 K. The 1.3 ps component is similar to the values reported earlier (Schmidt et al. 1994, 1995) and represents the decay of the P+ BA − state. The acceleration of this reaction at lower temperatures is an effect also observed in WT RCs (Lauterwasser et al. 1991). In order to explain the high P+ BA − population at low temperatures, an energetic distribution of the intermediate states (P∗ , P+ BA − , P+ HA − ) has to be assumed, if P+ HA − is below P+ BA − . A second explanation would be provided by the assumption that P+ HA − is isoenergetic or even slightly above P+ BA − . Again, the reduction of the quinone QA − could influence 1G (P+ HA − ) in this way. The spectral characteristics of the two fast kinetic components for 10 K and for 290 K are shown in Figure 5 (upper part). The amplitudes of the τ 1 component (2.1 ps or 5.1 ps) decrease with increas-
pres816.tex; 30/06/1998; 15:16; p.5
158
Figure 5. Amplitude spectra of the two fast kinetic components (upper part) and calculated spectra of the difference cross sections for P∗ and P+ BA − states (lower part) for R26.Phe-a RCs at 10 K and 290 K. The spectra were calculated from the amplitude spectra using the sequential model described in the text. Free energy differences used in the calculations were 1G(P+ BA − ) = –450 cm−1 , 1G(P+ HA − ) = –630 cm−1 for 290 K and 1G(P+ BA − ) = –450 cm−1 , 1G(P+ HA − ) = –460 cm−1 for 10 K. Figure 4. Time resolved absorption data for native (open circles) and R26.Phe-a RCs (filled circles), probed in the center of the BA − band (λpr = 1020 nm) at various temperatures. The estimated population of the P+ BA − state and the temperature dependent changes of the population density are given in Table 1.
ing probing wavelength, whereas the amplitudes of the fast τ 2 component (0.5 ps or 1.3 ps) are maximal around 1020 nm. This finding agrees well with the maximum of the BA − -absorption at 1016 nm for native RCs (Arlt et al. 1993).
Discussion The spectral characteristics of the BChl-a absorption bands observed in the steady-state spectra of R26.Phea RCs at various temperatures show, that the BChl-a pigments are not affected by the pigment exchange procedure. Also, the recently performed X-ray structure analysis of R26.Phe-a RCs (Meyer 1997) demon-
strates that the pigment exchange at the HA/B sites leaves the P and BA area practically unchanged. This suggests that the free energy of the P+ BA − intermediate also is not drastically altered. This is supported by the time-resolved experiments at low temperatures, which clearly confirm the arrangement of the energy level of P+ BA − below P∗ . Therefore, the temperature dependence of the P∗ decay can be analyzed in detail within the framework of non-adiabatic electron transfer theory, using the quantum-mechanically correct expression (Jortner 1976). For the kinetic modelling, the τ (e−1 ) and τ (e−2 ) lifetimes have been used, since this method seems to be suitable to describe dispersive kinetics (Bixon et al. 1995). For example, a modelling of the temperature dependence of the τ (e−1 ) lifetime is possible for the temperature range between 10 K and 200 K considering the change of the Franck– Condon-factor with temperature (Jortner 1976). The optimal model function is obtained with the following
pres816.tex; 30/06/1998; 15:16; p.6
159
Figure 6. Dependence of the calculated P+ HA − spectrum on the free energy difference between P∗ and P+ HA − . The optimal value is 1G(P+ HA − ) = 460 cm−1 for 10 K and 1G(P+ HA − ) = 630 cm−1 for 290 K. The P∗ and the P+ BA − spectra (dotted and dashed lines) are nearly unaffected by the variation of 1G(P+ HA − ). For the ‘sharp’ 1G(P+ HA − ) values used in this calculation, a realistic P+ HA − spectrum is obtained only within a small range of 1G(P+ HA − ). For distributed 1G values (e.g. σ = 400 cm−1 ) the P+ HA − spectrum is less sensitive to changes in 1G(P+ HA − ).
ET parameters: h¯ ω = 150 cm−1 , λ = 600 cm−1 , V = 19 cm−1 . At higher temperatures a systematic deviation of the rates from the model function is observed (not shown). Interestingly, the same deviation above 200 K was found for different ET reactions within the RC (Frolov et al. 1996) and attributed to increased mobility (‘thawing’) of the protein in this temperature
range. For a correct modelling, this effect can be accounted for by introducing a temperature dependence for the reorganization energy λ. This approach was also successfully chosen to describe the ground state recombination reaction in Rps. viridis RC (Ortega et al. 1996).
pres816.tex; 30/06/1998; 15:16; p.7
160 The kinetic heterogeneity can be analyzed by plotting the ratio r = τ (e−2 ) / 2∗ τ (e−1 ) as a function of temperature (inset Figure 3). It is found that the P∗ decay in R26.Phe-a RCs is much more dispersive than in native RCs, where r ≤ 1.2 for all temperatures. It is evident from the pronounced U-shaped form of the curve that the two lifetimes τ (e−1 ) and τ (e−2 ) differ in their temperature dependence, pointing to a different physical nature of the two P∗ components. The high kinetic heterogeneity at low temperatures (r = 2) can be explained if the lower energetic level of P∗ at low temperatures (red shift of the QY P-band in the steady state spectra) and the distribution of the free energies of all intermediates is taken into account. A Gaussian distribution of 1G with σ = 400 cm−1 was established for the radical pair state P+ HA − (Ogrodnik et al. 1994) and is assumed for P∗ and P+ BA − as well. The lack of thermal excitation for ET from low lying P∗ levels could lead to a long tail in the decay kinetics and enhance heterogeneity. These arguments apply so far to both native and R26.Phe-a RCs. For R26.Phe-a RCs an additional effect becomes important: the low lying P∗ levels can be effectively repopulated from high lying acceptor states (P+ BA − and P+ HA − ) even at low temperatures. An independent confirmation can be obtained from fluorescence up-conversion measurements showing long time tails in R26.Phe-a RCs at 20 K (Hartl 1995). On the other hand, the large value of r in R26.Phe-a RCs at higher temperatures can be explained by thermal repopulation of P∗ from the acceptor states. The amount of repopulation depends on the population density of P+ BA − , which is strongly increased in the case of the pigment exchanged sample. This additional P∗ population is absent in native RCs. A stepwise reaction model was used to calculate the difference cross section 1σi (λpr ) = σi (λpr ) − σ0 (λpr ) (where σ 0 (λpr ) corresponds to the cross section of the unexcited RC at a certain probing wavelength) of the intermediate i from the measured signal amplitudes of all kinetic components (Figure 5b). The details of this procedure are described elsewhere (Finkele et al. 1995); the free energy difference 1G(P+ BA − ) = 450 cm−1 is the recently estimated value (Nagarajan et al. 1993; Schmidt et al. 1995); a rate equation system is used, where ground state recombination reactions are allowed from all intermediates and backward and forward rates are coupled by the principle of detailed balance. The values of 1σ for P∗ are negative below 1050 nm and positive for longer wavelengths. This reflects the two (emission and absorption) contributions to the P∗ sig-
nal in this spectral range. The stimulated emission which is centered around 920 nm decreases with λ in the spectral range shown in Figure 5. Above λ = 1050 nm excited state absorption of P∗ leads to positive values of 1σ (Wynne et al. 1996). The calculated P∗ spectrum therefore reflects the expected properties of the excited primary donor. As described before, the spectrum of the intermediate P+ BA − at room temperature has a pronounced peak at 1020 nm with a shoulder towards shorter wavelengths (Schmidt et al. 1995), which can be clearly assigned to BChl-a anion radical absorption (Fajer et al. 1975). Also, at 10 K the P+ BA − spectrum is dominated by the BA − absorption; the narrower width of this band at low temperatures is clearly visible. Figure 6 shows the dependence of the calculated difference cross section 1σ (λ) on the energetic position of the intermediate P+ HA − , 1G(P+ HA − ). It becomes evident that the calculated difference cross sections of P+ HA − depend critically on the value of 1G(P+ HA − ), whereas the P∗ and P+ BA − spectra are nearly unaffected by a variation of this parameter (Figure 6). Since there is no HA − absorption in this spectral region (Fajer et al. 1975), an unstructured, weak positive absorption due to P+ is expected between 1000 and 1100 nm for the state P+ HA − . For 290 K a reasonable P+ HA − spectrum is obtained for 1G(P+ HA − ) = 630 cm−1 . This value agrees well with the value found for untreated R26.Phe-a RCs (Schmidt et al. 1994, 1995), but smaller values, as suggested in a different study (Shkuropatov and Shuvalov 1993) lead to very unrealistic 1σ curves. For the measurements at 10 K, the simulation suggests a value around 1G(P+ HA − ) = 460 cm−1 . Again, it does not seem reasonable here to place the energy level of P+ HA − above that of P+ BA − . The most plausible reaction model at low temperatures is obtained if the two acceptor states are located isoenergetically approximately 450 cm−1 below P∗ and if distributed free energies (with σ = 400 cm−1 ) of the intermediate states are assumed (Figure 7). The results suggest a temperature dependence of 1G(P+ HA − ) leading to a decrease of the free energy difference between P∗ and P+ HA − at low temperatures. This assumption has been used successfully before to describe the temperature dependence of R-26 RCs (Woodbury and Allen 1995). We cannot rule out that there is also a small temperature dependence of 1G(P+ BA − ), but a correct simulation of the data is possible without this assumption.
pres816.tex; 30/06/1998; 15:16; p.8
161 Chan CK, DiMagno TJ, Chen LQX, Norris JR and Fleming GR (1991) Mechanism of the initial charge separation in bacterial photosynthetic reaction centers. Proc Natl Acad Sci USA 88: 11202–11206 Chang C-H, El-Kabbani O, Tiede D, Norris J and Schiffer M (1991) Structure of the membrane bound protein photosynthetic reaction center from Rhodobacter sphaeroides. Biochemistry 30: 5352– 5360 Dressler K, Umlauf E, Schmidt S, Hamm P, Zinth W, Buchanan S and Michel H (1991) Detailed studies of the subpicosecond kinetics in the primary electron transfer of reaction centers of Rhodopseudomonas viridis. Chem Phys Lett 183: 270–276 Ermler U, Fritsch G, Buchanan SK and Michel H (1994) Structure of the photosynthetic reaction centre from Rhodobacter sphaeroides at 2.65 A resolution; cofactors and protein-cofactor interactions. Structure 2: 925–936 Fajer J, Brune DC, Davies MS, Forman A and Spaulding LD (1975) Primary charge separation in bacterial photosynthesis: Oxidized chlorophylls and reduced pheophytin. Proc Natl Acad Sci USA 72: 4956–4960 Finkele U, Dressler K, Lauterwasser C and Zinth W (1990) Analysis of transient absorption data from reaction centers of purple bacteria. In: Michel-Beyerle ME (ed) Reaction Centers of Photosynthetic Bacteria, pp 127–134. Springer, Berlin, Germany Fleming GR and van Grondelle R (1994) The primary steps of photosynthesis. Phys Today 47: 48–55. Franken EM, Shkuropatov AY, Francke C, Neerken S, Gast P, Shuvalov VA, Hoff AJ and Aartsma TJ (1997a) Reaction centers of Rhodobacter sphaeroides R-26 with selective replacement of bacteriopheophytin a by pheophytin a. I. Characterization of steady-state absorbance and circular dichroism, and the P+ QA − state. Biochim Biophys Acta 1319: 242–250 Franken EM, Shkuropatov AY, Francke C, Neerken S, Gast P, Shuvalov VA, Hoff AJ and Aartsma TJ (1997b) Reaction centers of Rhodobacter sphaeroides R-26 with selective replacement of bacteriopheophytin a by pheophytin a. II. Temperature dependence of the quantum yield of P+ QA − and 3 P formation. Biochim Biophys Acta 1321: 1–9 Frolov EN, Goldanski VI, Birk A and Parak F (1996) The influence of electrostatic interactions and intramolecular dynamics on electron transfer from the cytochrome subunit to the cationradical of the bacteriochlorophyll dimer in raction centers from Rps. viridis. Eur Biopyhs J 24: 433–438 Geskes C, Meyer M, Fischer M Scheer H and Heinze J (1995) Electrochemical investigation of modified photosynthetic pigments. J Phys Chem 99: 17669–17672 Hamm P, Gray KA, Oesterhelt D, Feick R, Scheer H and Zinth W (1993) Subpicosecond emission studies of bacterial RCs. Biochim Biophys Acta 1142: 99–105 Hartl I (1995) Sub-Picosekunden Emissionsmessungen zur Primärreaktion der bakteriellen Photosynthese. Diploma thesis, Institut für Medizinische Optik, Ludwig-Maximilians-University, Munich Holzapfel W, Finkele U, Kaiser W, Oesterhelt D, Scheer H, Stilz HU and Zinth W (1989) Observation of a bacteriochlorophyll anion radical during the primary charge separation in a RC. Chem Phys Lett 160: 1–7 Holzapfel W, Finkele U, Kaiser W, Oesterhelt D, Scheer H, Stilz HU and Zinth W(1990) Initial electron-transfer in the RC from Rb. sphaeroides. Proc Natl Acad Sci USA 87: 5168–5172 Huber H, Meyer M, Nägele T, Hartl I, Scheer H, Zinth W, and Wachtveitl J (1995) Primary photosynthesis in reaction centers containing four different types of electron acceptors at site HA . Chem Phys 197: 297–305
Figure 7. Schematic reaction model for the primary reactions in R26.Phe-a RCs. The forward and backward rates are termed γ ij and γ ji , respectively, γ i0 are the rates for internal conversion and recombination reactions, a Gaussian distribution of the energy levels is assumed.
In conclusion, we have shown the involvement of BA − in the primary charge separation reaction of R26.Phe-a RCs at low temperatures and presented a model that is able to reproduce the complex dynamic properties, the measured amplitudes of the kinetic components in the entire investigated spectral range and the temperature dependence of the pigment modified RC. The stepwise electron transfer is obviously the significant reaction mechanism independent of the temperature domain.
References Allen JP, Feher G, Yeates TO, Komiya H and Rees DC (1987) Structure of the reaction center from Rhodobacter sphaeroides R-26: the cofactors. Proc Natl Acad Sci USA 84: 5730–5734 Arlt T, Schmidt S, Kaiser W, Lauterwasser C, Meyer M, Scheer H and Zinth W (1993) The accessory bacteriochlorophyll: A real electron carrier in primary photosynthesis. Proc Natl Acad Sci USA 90: 11757–11761 Beekmann L, Jones MR, van Stokkum I and van Grondelle R (1995) Wavelength dependence of the stimulated emission decay of membrane bound Rb. sphaeroides reactions centers and observation of a Bchl-a anion involved in electron transfer. In: Mathis P (ed) Photosynthesis: From Light to Biosphere, Vol I, pp 495–498. Kluwer Academic Publishers, Dordrecht, the Netherlands Bixon M, Jortner J and Michel-Beyerle ME (1995) A kinetic analysis of primary charge separation in bacterial photosynthesis. Energy gaps and static heterogeneity. Chem Phys 197: 389–404
pres816.tex; 30/06/1998; 15:16; p.9
162 Hunter CN, van Grondelle R and Olsen JD (1989) Photosynthetic antenna proteins: 100 ps before photochemistry starts. Trends Biochem Sci 14: 72–75 Jia Y, DiMagno TJ, Chan CK, Wang Z, Du M, Hanson DK, Schiffer M, Norris JR, Fleming GR and Popov MS (1993) Primary charge separation in mutant RCs of Rb. capsulatus. J Phys Chem 97: 13180–13191 Jonas DM, Lang MJ, Nagasawa Y, Joo T and Fleming GR (1996) Pump-probe polarisization anisotropy study of femtosecond energy transfer within the photosynthetic reaction center of Rb. sphaeroides R26. J Phys Chem 100: 12660–12673 Jortner J (1976) Temperature dependent activation energy for electron transfer between biological moelcules. J Chem Phys 64: 4860–4867 Kirmaier C and Holten D (1987) Primary photochemistry of reaction centers from the photosynthetic purple bacteria. Photosynth Res 13: 225–260 Lauterwasser C, Finkele U, Scheer H and Zinth W (1991) Temperature dependence of the primary electron transfer in photosynthetic reaction centers from Rb. sphaeroides. Chem Phys Lett 183: 471–477 Lous EJ and Hoff AJ (1986) Triplet-minus-singlet absorbance difference spectra of reaction centers of Rhodopseudomonas sphaeroides R-26 in the temperature range 24–290 K measured by Magneto-Optical-Difference Spectroscopy (MODS). Photosynth Res 9: 89–101 Martin JL, Breton J, Hoff AJ, Migus A and Antonetti A (1986) Femtosecond spectroscopy of electron transfer in the RC of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26: Direct electron transfer from the dimeric bacteriochlorophyll primary donor to the bacteriopheophytin acceptor with a time constant of 2.8 ± 0.2 psec. Proc Natl Acad Sci USA 83: 957–961 Meyer M and Scheer H (1995) Reaction centers of Rb. sphaeroides R26 containing C-3 acetyl and vinyl (bacterio)pheophytins at sites HA/B . Photosynth Res 44: 55–65 Meyer M (1997) Pigment-modified reaction centers of Rb. sphaeroides R26.1. Thesis, University of Munich Müller MG, Griebenow K and Holzwart AR (1992) Primary processes in isolated bacterial reaction centers from Rhodobacter sphaeroides studied by picosecond fluorescence kinetics. Chem Phys Lett 199: 465–469 Nagarajan V, Parson WW, Davis D and Schenck CC (1993) Kinetics and free energy gaps of electron-transfer reactions in Rb. sphaeroides RCs. Biochemistry 32: 12324–12336 Ogrodnik A, Volk M and Michel-Beyerle ME (1988) On the energetics of states 1 P∗ , 3 P∗ and P+ H− in reaction centers of Rb. sphaeroides. In: Breton J and Verméglio A (eds) The Photosynthetic Bacterial Reaction Center – Structure and Dynamics, pp 177–184. Plenum Press, New York, USA
Ogrodnik A, Keupp W, Aumeier G and Michel-Beyerle ME (1993) Different recombination dynamics of P+ HA − in delayed emission and absorption measurments reveal an inhomogenity of radical pair energies in photosyntetic reaction centers. J Phys Chem 20: 1–25 Ogrodnik A, Keupp W, Volk M, Aumeier G and Michel-Beyerle ME (1994) Inhomogeneity of radical pair energies in photosynthetic reaction centers revealed by differences in recombination dynamics of P+ HA − when detected in delayed emission and in absorption. J Phys Chem 98: 3432–3439 Ortega JM, Mathis P, Williams JC and Allen JP (1996) Temperature dependence of the reorganisation energy for charge recombination in the reaction center from Rb. sphaeroides. Biochemistry 35: 3354–3361 Schenck CC, Blankenship RE and Parson WW (1982) Radical pair decay kinetics, triplet yields and delayed fluorescence from bacterial reaction centers. Biochim Biophys Acta 680: 44–59 Schmidt S, Arlt T, Hamm P, Huber H, Nägele T, Wachtveitl J, Meyer M, Scheer H and Zinth W (1994) Energetics of the primary electron transfer reaction revealed by ultrafast spectroscopy on modified bacterial RCs. Chem Phys Lett 223: 116–120 Schmidt S, Arlt T, Hamm P, Huber H, Nägele T, Wachtveitl J, Zinth W, Meyer M and Scheer H (1995) Primary electrontransfer dynamics in modified bacterial reaction centers containing pheophytin-a instead of bacteriopheophytin-a. Spectrochim Acta 51A: 1565–1578 Shkuropatov AY and Shuvalov VA (1993) Electron transfer in pheophytin-a modified reaction centers from Rhodobacter sphaeroides R-26. FEBS Lett 322: 168 Stowell MHB, McPhillips TM, Rees DC, Soltis SM, Abresch E and Feher G (1997) Light induced structural changes in photosynthetic reaction center: implications for mechanism of electron-proton transfer. Science 276: 812–816 Woodbury NW and Allen JP (1995) The pathway, kinetics and thermodynamics of electron transfer in wild type and mutant reaction centers of purple nonsulphur bacteria. In: Blankenship RE, Madigan MT and Bauer CE (eds) Anoxygenic Photosynthetic Bacteria, pp 527–557. Kluwer Academic Publishers, Dordrecht, the Netherlands Wynne K, Haran G, Reid GD, Moser CC, Dutton PL and Hochstrasser RM (1996) Femtosecond infrared spectroscopy of low-lying excited states in reaction centers of Rb. sphaeroides. J Phys Chem 100: 5140–5148
pres816.tex; 30/06/1998; 15:16; p.10
