Accumulated Photon Echo Studies on Bacterial Photosynthetic

electron-phonon coupling, and the Huang-Rhys factor is determined to be about S ) 1.7. The longer decay components are attributed to charge transfer f...
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6786

J. Phys. Chem. B 1997, 101, 6786-6790

Accumulated Photon Echo Studies on Bacterial Photosynthetic Reaction Centers: Charge-Transfer Rate Distribution and Electron-Phonon Coupling P. Schellenberg,† R. J. W. Louwe, S. Shochat, P. Gast, and T. J. Aartsma* Department of Biophysics, UniVersity of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands ReceiVed: April 30, 1997; In Final Form: June 3, 1997X

The dephasing kinetics of the excitation in the P-870 band of Rb. sphaeroides R26 and the mutant Rb. sphaeroides (M)Y210W was measured by accumulated photon echo experiments. The photon echo signal shows an extremely fast component and a nonexponential longer decay. The fast component is attributed to electron-phonon coupling, and the Huang-Rhys factor is determined to be about S ) 1.7. The longer decay components are attributed to charge transfer from the primary donor. The multiple decay components can be explained by assuming dispersive kinetics due to configurational and energetic disorder. The results are compared with those of incoherent pump-probe and hole-burning experiments.

1. Introduction

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The determination of the structure of the reaction centers (RCs) from purple bacteria to atomic resolution by X-ray crystallography1,2 has been a highly stimulating factor in the research on these pigment-protein complexes, especially in unraveling the mechanism of electron transfer. The threedimensional structure of the bacterial RCs shows approximately a C2-symmetric arrangement of the cofactors in these systems. The primary electron donor in the RC was unambiguously identified as a bacteriochlorophyll (BChl) a dimer, forming the special pair P. The strong interaction between the two BChls and the interactions with the protein contribute to a large red shift of the absorption band relative to that of monomeric BChl. The absorbance of P has a maximum around 870 nm at room temperature (the P-870 band). Upon optical excitation of the special pair, an electron is transferred from P down the active branch of cofactors, those which belong to the so-called L-subunit of the protein complex. The first step, the transfer from P to the bacteriopheophytin ΦL, occurs within about 3 ps at ambient temperature and accelerates to 1.2 ps at cryogenic temperature.3 This indicates that no thermal activation energy is required for this process. The role of the accessory BChl a molecule located between the primary donor and ΦL is still a matter of debate. It is usually assumed that it is involved either in a superexchange mechanism4 or as an intermediate in a two-step sequential electron transfer.5,6 In a subsequent process, the electron is transferred from ΦL to the quinones QA and QB to stabilize the charge separation7-9 on the time scale of tens of milliseconds. Suitable methods for studying the excited state dynamics of the pigments involved in electron transfer in the RC are incoherent pump-probe and photon echo experiments in the time domain and hole-burning spectroscopy in the frequency domain. Incoherent pump-probe and photon echo experiments provide the lifetime of the excited state T1 and the total dephasing time T2, respectively. The frequency domain equivalent of the total dephasing time is the homogeneous line width Γh, which can be obtained from hole-burning spectroscopy. The relationship between these observables is expressed in the equation * Corresponding author. E-mail: [email protected]. † Present address: Department of Biophysics, University of Ulm, D-89081 Ulm, Germany. X Abstract published in AdVance ACS Abstracts, July 15, 1997.

S1089-5647(97)01457-0 CCC: $14.00

)

1 1 1 1 ) + πT2 π 2T1 T2*

Γh )

(1)

where T2* is the pure dephasing time. Note that the dephasing of an excited state is a much narrower criterion than the population decay. For example, energy transfer between nearly degenerate states would lead to dephasing, but not to a change in transmission and would therefore not be observed in incoherent pump-probe experiments. Elastic phonon scattering or interaction with specific low-frequency modes characteristic for amorphous solids also leads to dephasing without de-excitation. Similar considerations apply to reversible electron or energy transfer between chromophores, as for example in the parking-state model10,11 for photosynthetic RCs. Therefore, photon echo methods open special opportunities for elucidating the structure-function-dynamics relationships in chromoproteins12 and in photosynthetic systems in particular. The information gained from photon echo experiments is the time domain equivalent of that obtained from hole-burning spectroscopy. In particular, the decay of the photon echo signal in stimulated and accumulated photon echo experiments is just the Fourier transform of the spectral features observed in the frequency domain. In the frequency domain, the hole-burned spectrum usually consists of a relatively sharp zero-phonon line and a broad phonon side band. The phonon side band gives rise to an ultrafast decay in the time domain, whereas the zerophonon line width can be correlated with much longer decay components.13-16 The strength of the electron-phonon coupling can also be determined by means of photon echo measurements. Under the condition of a sufficient time resolution (or equivalently, a sufficiently broad pulse to cover the whole phonon side wing), the Debye-Waller factor R and Huang-Rhys factor S of the electronic transition can be calculated from the amplitude ratio of the slow decay component AZPL to that of the total decay AΣ at zero delay (t ) 0):15

[

]

AZPL(t ) 0)

R)

AΣ(t ) 0)

1/2

) e-S

(2)

The Huang-Rhys factor S corresponds to the most probable number of phonons from a single mode involved in the transition. To achieve a high time resolution, it is not necessary to have a pulse duration on a femtosecond time scale, since the time © 1997 American Chemical Society

Bacterial Photosynthetic Reaction Centers resolution in photon echo experiments is determined by the correlation time of the optical electric field. Thus one can use non-transform limited laser pulses, a method which is commonly referred to as incoherent or stochastic photon echo generation.14,15,17 Transient absorption and time-resolved fluorescence measurements10,11,18 on RCs of purple bacteria with subpicosecond time resolution with excitation in the P-870 band reveal a nonexponential population decay of the excited state, which has been discussed in terms of a parking state model10,11 and of a dispersion of transfer rates.19,20 Hole-burning experiments21-23 show that narrow holes can be burned in the red wing of the dimer absorption band of photosynthetic purple bacteria. This is in contradiction to earlier accumulated photon echo and hole-burning experiments by Meech et al.24,25 who observed only ultrafast dephasing which was attributed to relaxation to an intradimer charge transfer state. In this article we will present additional results of accumulated photon echo (APE) measurements on purple bacterial reaction centers. The photon echo decay in Rb. sphaeroides R26 is characterized by multiple time constants. A very fast decay component is attributed to vibronic relaxation due to excitation into the phonon sideband manifold, while the slower components reflect electron transfer. The results are discussed in relation to those of previous photon echo measurements,24,25 incoherent pump-probe,3,10,11,19,26 and hole-burning experiments.21,23 We will also present results on the mutant M(Y)210W of Rb. sphaeroides, in which electron transfer occurs at a much lower rate.27 In this mutant, the M210 tyrosine residue in the vicinity of the accessory BChl a in the active branch has been replaced by tryptophane. 2. Experimental Section Reaction centers from Rb. sphaeroides R26 and (M)Y210W are prepared as previously described.27 The reaction centers are dissolved in a buffer and detergent solution (0.025% LDAO, 10 mM Tris-HCl buffer, 1 mM EDTA) at pH 8. Glycerol is added (66 vol %) prior to cooling in order to achieve a clear glass at low temperature. The concentration is adjusted to an optical density of about 0.3 at the measuring wavelength under the experimental conditions. The sample is cooled down to 1.4 K in a liquid helium bath cryostat. The APE technique is a variation of three-pulse stimulated photon echo generation.28,29 By the APE method, advantage is taken of a long-lived bottleneck state in the excitation-relaxation cycle of a lightabsorbing molecule. The echo is generated by recovering the phase information accumulated in the ground state population over many pulse pairs. Because of this accumulation effect, it is possible to get a very high S/N ratio using low-energy pulses, while background rejection can be achieved by heterodyne detection using a double modulation technique. Details of the experimental setup are described in ref 29. The picosecond laser pulse train is generated with a single jet dye laser that is synchronously pumped by the frequencydoubled output of an actively mode-locked Nd:YAG laser. The double modulation is achieved by using the transmitted beam of an opto-acoustic modulator (OAM) operated at 8 MHz and a mechanical chopper at 250 Hz to modulate the pump and probe beam, respectively. In bacterial RCs, one can either use the charge-separated state P+ QA- or the triplet state, 3P, of the special pair as the bottleneck state. The bottleneck state used for accumulation has to decay completely during a dark phase of a chopper cycle to achieve maximal signal. Comparing the lifetimes of the triplet state and the charge-separated state (about 1 and 40 ms at cryogenic temperatures, respectively), it is clear that the use of the former state is preferable because higher

J. Phys. Chem. B, Vol. 101, No. 34, 1997 6787

Figure 1. APE signal at 670 nm of chlorophyll a in alcohol glass at 100 K as a function of the time delay between excitation pulses. The solid lines represent fits to a sech2 function. Open circles: data obtained with transform-limited pulses, the fitted curve has a width (fwhm) of 2.2 ps. Filled circles: data obtained with stochastic excitation pulses; the fit in this case has a width (fwhm) of 110 fs.

modulation frequencies can be applied in the heterodyne detection scheme. Efficient population of the triplet state can be obtained by inhibition of electron transfer to the secondary acceptor QA. This is achieved by illuminating the sample during freezing in the presence of ascorbate as an agent rereducing P+, by which QA is accumulated in the reduced state.9 To obtain an increased time resolution, some photon echo experiments are performed using stochastic excitation.14,15,17 By removing the wavelength-selective element from the dye-laser cavity, the spectral width of the laser pulses can be increased up to approximately 10 nm, depending slightly on the dye or dye mixture. The wavelength of the laser can then be adjusted by changing the relative concentration of two spectrally adjacent laser dyes. For measurements on the P-870 absorption band of bacterial reaction centers we used a mixture of LDS867 and LDS925 dissolved in ethylene glycol/propylene carbonate. Special care has to be taken to prevent an unequal dispersion of the two beams by optical components in the light path, which may lead to reduced time resolution and artificial oscillations in the observed signal.30 Therefore, optically identical components were used in both beam paths. This included the OAM, although the one in the probe beam was not active. The time resolution of the experiment was verified by measuring the accumulated photon echo decay of chlorophyll a in an alcohol-glass matrix at 670 nm and at a temperature of approximately 100 K. The results obtained with transformlimited and with stochastic excitation pulses are shown in Figure 1. At 100 K the time constant of dephasing is much shorter than the electric field correlation time of a pair of excitation pulses, and therefore the results in Figure 1 are representative for the time resolution that can be obtained. In both cases, the instrument response function is symmetric around zero delay and can be accurately fitted to a sech2 function, as shown by the solid lines in Figure 1. In the case of stochastic, spectrally broad excitation, the time resolution was determined to be about 100 fs, although the actual pulse duration is on the order of several picoseconds. Given the value of 110 fs for the fwhm of the data in Figure 1, in combination with the spectral bandwidth in this case of about 7 nm, the time bandwidth product is found to be 0.5 which is close to the theoretical value of 0.32 assuming sech2 pulses.31 We assume that this result also applies to excitation of RCs at wavelengths around 900 nm.

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Figure 2. APE decay at 915 nm of isolated reaction centers of Rhodobacter sphaeroides R26 at 1.5 K. The dashed line is obtained by expanding the vertical scale by a factor of 10.

Figure 3. APE decay at 910 nm of isolated reaction centers of the (M)Y210W mutant of Rhodobacter sphaeroides R26 at 1.5 K. The inset shows the region around zero delay at an expanded scale.

In the case of transform-limited pulses, the instrument response function shown in Figure 1 is in excellent agreement with the autocorrelation function determined with the autocorrelator. The width (fwhm) of the instrument response function is typically 2.0-2.5 ps, which corresponds to a transform-limited spectral bandwidth of about 5 cm-1. 3. Results Figure 2 shows the echo decay signal of Rb. sphaeroides R26 in a stochastic APE experiment with excitation at 908 nm and a bandwidth of about 10 nm. A very large signal is observed around t ) 0, as well as a signal decaying on a picosecond scale with a relatively small amplitude. The latter is enlarged in the inset of Figure 2 by a factor of 10 for clarity. The ratio of the amplitude of the slowly decaying signal to that of the total amplitude is 1:30. The slowly decaying signal can be fitted to a biexponential decay function with time constants of 1 and 4 ps and relative contributions of 80% and 20%, respectively. The fast decaying component is attributed to rapid vibronic relaxation resulting from excitation into the phonon sideband of the P-870 transition. The slowly decaying signal is associated with optical dephasing of the zero-phonon excited state. We will refer to the latter as the zero-phonon decay, by analogy to the zero-phonon transitions. A similar decay pattern is also observed in stochastic APE measurements of the (M)Y210W mutant of Rb. sphaeroides, shown in Figure 3. However, the zero-phonon decay is much longer, yielding time constants of 18 and 120 ps. The decay curves show a dip which is clearly visible at t < 1 ps in particular in the (M)Y210W mutant (see inset of Figure 3). This

Schellenberg et al.

Figure 4. Representative APE decay curves, at different wavelengths of excitation, of isolated reaction centers of the (M)Y210W mutant of Rhodobacter sphaeroides R26 at 1.5 K.

Figure 5. Solid circles: wavelength dependence of the relative contribution R (right-hand scale) of the phonon sideband to the total APE intensity at zero delay, of RCs of the (M)Y210W mutant at 1.5 K. The dashed line through the data points is a fit to a Gaussian distribution function (dash-dotted line) of zero-phonon transition frequencies. The solid line is the absorption spectrum of the (M)Y210W RC at 4 K.

feature is presumably due to a quantum beat between the decay of the zero-phonon state and that of the phonon sideband.17,32 To determine the wavelength dependence of the echo decay, Fourier-transform limited pulses were used in order to achieve a better spectral resolution. Some typical results obtained with the (M)Y210W mutant are shown shown in Figure 4. In the case of this mutant it is relatively easy to distinguish between the zero-phonon and the phonon sideband contributions because of the longer lifetime of P*. Results for native RCs of Rb. sphaeroides R26 (not shown) are very similar. Although the time constants for the decay components are independent of wavelength, their relative amplitudes change significantly in native as well as in mutant RCs. The wavelength dependence of the amplitude ratio of the fast and the slow decay is shown in Figure 5, where the decay amplitudes have been normalized to the amplitude of the fast component. This ratio increases toward the blue part of the spectrum. In the maximum of the inhomogeneously broadened absorption band of P-870, the zerophonon contribution to the photon echo signal has vanished completely. From the wavelength dependence of the amplitude ratio, a quantitative estimate can be obtained for the distribution of zero-phonon lines in the absorption band P-870. Assuming a Gaussian distribution, the central wavelength is found to be 910 nm for Rb. sphaeroides R26 and 913 nm for Rb. sphaeroides (M)Y210W. The width of the distribution in both cases is about 6 nm (fwhm).

Bacterial Photosynthetic Reaction Centers

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4. Discussion There are two striking features in APE experiments on RCs of Rb. sphaeroides and its mutant (M)Y210W. The first is that the zero-phonon decay of the APE signal is clearly nonexponential and can only be fitted well by a sum of at least two exponentials. The second feature is the ultrafast decay at zero delay, which dominates the APE signal and whose relative amplitude is wavelength dependent. In the blue part of the P-870 band it is the only component which is observed. In the following, these two observations are explained in terms of the electron transfer kinetics of the RC and the electron-phonon coupling. The results will be compared with those of hole-burning and transient absorption experiments. 4.1. Electron Transfer Kinetics. As discussed in the Introduction, the total dephasing measured in accumulated photon echo experiments is determined by contributions of the population decay as well as the pure dephasing of the excited state. The time scale of pure dephasing is usually on the order of nanoseconds or longer at sufficiently low temperatures. Taking into account the conditions of the experiments reported here, pure dephasing can be neglected in the present discussion: T1 , T2*. Consequently, we assume that the zero-phonon decay of the APE is determined by the lifetime of P*, i.e., by the rate of primary electron transfer. The optical dephasing of the excited state of P in bacterial RCs is clearly nonexponential. One possibility to analyze our experimental data is by using a biexponential function to describe the multiple time constants we observed in the zerophonon decay of the APE. Such a biexponential decay has been observed in transient absorption33 and in time-resolved fluorescence10,11,18 measurements, and now also in APE experiments. Hamm et al.11 describe several electron transfer models which may give rise to a multiexponential decay of P*. One of these is the so-called parking-state model, where it is assumed that electron transfer also takes place along the inactive branch of the pigments in the M subunit of the RC, although eventually all the charge is transferred to the active branch in the L subunit. Within this model, however, we would expect a monoexponential decay in photon echo experiments, because the rate of dephasing would be determined by the sum of the rates of electron transfer to the active and the inactive branch. This is clearly in contradiction with our experimental observations. Therefore, the biexponential decay cannot be attributed to the parking-state mechanism. Similar arguments apply to the biexponential photon echo decay in RCs of the (M)Y210W mutant, with fitted time constants of about 20 and 120 ps. It is more likely then that the nonexponential character of the photon echo decay must be attributed to a distribution of rate constants caused by the intrinsic disorder of the protein environment. A distribution of rate constants is observed in various kinds of experiments on disordered systems like glasses and proteins.34-36 The implications of disorder and dispersive kinetics for primary electron transfer in photosynthetic reaction centers have been discussed by Small et al.37,38 A decay with a distribution of rate constants can usually be represented well by a stretched exponential function φ(t, T):

φ(t, T) ) exp{-(k(T)t)β}

(3)

where k(T) is the temperature-dependent rate. β is commonly referred to as the dispersion parameter and is a measure for the width of the distribution (0 < β e 1). Note that if β ) 1, the function φ(t, T) reduces to a simple exponential law with a single rate constant. For the photon echo decays (obtained with stochastic excitation) of Rb. sphaeroides R26 and M(Y)210W reaction centers

we find that k ) (1.9 ps)-1 and k ) (45 ps)-1, respectively, while β ) 0.65 in both types of RCs. The value for β is comparable to typical values observed in amorphous solids such as glasses and proteins.35,36 The APE decay curves may be compared with the excited state decay measured in pump-probe measurements. In terms of a biexponential fit, we find that the long decay component of 120 ps in the photon echo decay of the (M)Y210W mutant deviates significantly from that found in incoherent pump-probe measurements (300 ps at 4.2 K).39 This observation may indicate that there is some spectral diffusion on the time scale of the experiment which is determined by the lifetime of the triplet bottleneck state.40 Another possibility is that a parking state is involved at this relatively long time scale, in addition to a dispersion of transfer rates. Recently, Heller et al.41 observed electron transfer to the inactive branch in a double mutant of Rb. capsulatus, presumably due to an increase of the reduction potential of BChlL. Such a process may also occur in R26 and (M)Y210W RCs but will only play a role if electron transfer along the active branch is slowed down sufficiently. Heller et al.41 estimated a time constant of 100 ps for electron transfer to the inactive branch in the double mutant of Rb. capsulatus, similar to the long time constant observed in the accumulated photon echo decay. The homogeneous line width obtained in hole-burning spectroscopy on Rb. sphaeroides R2621 corresponds to the dephasing component with a time constant of τ = 1 ps in our experiments. Assuming a distribution of rate constants, this zero-phonon line width seems to correspond to the short-time limit of the dephasing time distribution. The longer time constants of this distribution may be difficult to detect in hole burning, considering the relative amplitudes of the different decay components in the case of native RCs and a possible interference with power broadening. It may also be that the hole-burning efficiency varies within the distribution of RCs, favoring the formation of holes corresponding to the shorter time constants. 4.2. Electron-Phonon Coupling. The observed fastdecaying signal around zero delay is the Fourier transform of the phonon side wing in the frequency domain. Therefore, the amplitude of the photon echo decay around t ) 0 is a measure for the strength of electron-phonon coupling. Using eq 2 we calculate that the Huang-Rhys factor S is equal to 1.7 (assuming that the homogeneous line width of the vibronic transitions are comparable to or narrower than the bandwidth of the laser). This is in reasonable agreement with the value of 2.1 as determined in hole-burning investigations.21 The strong electron-phonon coupling gives rise to a large phonon sideband which dominates the blue part of the spectrum. This is also apparent in Figure 5. The zero-phonon line envelope is located in the red edge of the spectrum with a width of 2σ ) 12 nm. The maximum of this envelope is located at λmax ) 910 nm and at λmax ) 913 nm for Rb. sphaeroides R26 and the mutant (M)Y210W, respectively. In earlier experiments, Meech et al.24,25 observed very broad holes in hole burning, and an ultrafast decay in accumulated photon echo experiments. They interpreted this ultrafast dephasing in terms of a strong coupling of the excited singlet state to a charge transfer state of the primary donor.24,25 However, given the wavelengths of excitation in these early experiments, we conclude from our results that the ultrafast decay component is associated with vibronic relaxation following excitation into the phonon side-band. Nevertheless, the observed strong electron-phonon coupling implies a large shift of the nuclear equilibrium positions of the host atoms with respect to the chromophore upon excitation and is often observed

6790 J. Phys. Chem. B, Vol. 101, No. 34, 1997 in connection with a charge redistribution in the chromophore. Such a charge redistribution gives rise to a significant change of the equilibrium configuration of the site of the chromophore, resulting in a low Frank-Condon-factor for the 0-0 transition and a relatively enhanced contribution of the phonon sideband. Support for a large charge transfer character was also obtained from Stark experiments on RCs of Rb. sphaeroides and Rps. Viridis.42 In the case of the primary donor, an additional vibronic contribution to the spectrum may come from the intermolecular vibrations within the special pair. These so-called marker modes of the special pair are observed as a specific structure in the phonon sideband of hole-burning spectra21,43 and in Raman spectroscopy44 as well as in stimulated fluorescence measurements with femtosecond resolution.45 The Huang-Rhys factor of these marker modes was estimated to be on the order of 1.21,43 Although these modes may give rise to oscillatory features in a photon echo decay, they are not observed in our experiments. Most likely, they are disguised by the ultrafast decay of the contribution of the whole phonon sideband to the photon echo signal. The charge redistribution can be seen as a precursor to the charge separation along the chain of the chromophores. It is reasonable to assume that the charge within the special pair is aligned in such a way that the electron density is larger to the side of the active wing. This could imply that the environment around the special pair preconditions the electron transfer going along this direction. 5. Summary APE experiments on isolated reaction centers of Rb. sphaeroides R26 and the (M)Y210W mutant show a strong nonexponential behavior of the dephasing of the special pair at 1.5 K, which is determined by the primary step of electron transfer. This nonexponential behavior is attributed to the inherent disorder of the protein environment. The decay patterns of the photon echo signal in R26 and (M)Y210W RCs are comparable with those found in hole-burning and incoherent pump-probe experiments. The previously discussed possibility of a parking state as the origin of the nonexponential kinetics in transient absorption experiements can be excluded on the basis of the accumulated photon results. The mutant (M)Y210W shows a relatively long electron transfer time, the longest one found so far for bacterial reaction centers with a single mutation. Form the APE decays we conclude that the electron-phonon coupling of the special pair is very strong in isolated bacterial RCs. This is primarily due to a large charge transfer character of the excitation of the special pair. The absorption profile of the special pair dimer is heavily influenced by this strong electron phonon coupling. In fact, the inhomogeneous distribution of zero phonon lines is limited to the red wing of the P-870 band. Acknowledgment. We acknowledge the support of S. Jansen for the preparation of the reaction centers. This investigation was supported by the Life Science Foundation (SLW) which is subsidized by the Netherlands Organization for Scientific Research (NWO). P.S. thanks the European Community for a grant from the Human Capital and Mobility Program, contract No. ERBCHBGCT930361. References and Notes (1) Deisenhofer, J.; Epp, O.; Miki, R.; Huber, R.; Michel, H. Nature 1985, 318, 618. (2) Allen, J. P.; Feher, G.; Yeates, T. O.; Komiya, H.; Rees, D. C. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5730.

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