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J. Phys. Chem. B 1997, 101, 10560-10567
Temperature Dependence of Excitation Transfer in LH2 of Rhodobacter sphaeroides To˜ nu Pullerits,* Susan Hess, Jennifer L. Herek, and Villy Sundstro1 m Department of Chemical Physics, Lund UniVersity, Box 124, 22100 Lund, Sweden ReceiVed: June 27, 1997; In Final Form: October 13, 1997X
Using two-color pump-probe femtosecond spectroscopy, the temperature dependence of the energy transfer rate within the peripheral light-harvesting antenna (LH2) of the photosynthetic bacterium Rhodobacter sphaeroides has been measured. The energy transfer time from B800 to B850 is determined to be 0.7, 1.2, and 1.5 ps at 300, 77, and 4.2 K, respectively. These data, combined with earlier results, have been analyzed with regard to the crystal structure and spectroscopic properties of the purple bacterial LH2 complex. We conclude that the transfer within B800 occurs mainly via the incoherent Fo¨rster hopping mechanism. For B800 to B850 transfer, estimates based on the Fo¨rster formula yield considerably slower transfer times than experimentally observed, suggesting that an additional mechanism may be involved in enhancing the transfer rate. We suggest two possibilities: transfer via the upper excitonic component of B850 band and/or transfer mediated by a carotenoid molecule.
1. Introduction Photosynthetic purple bacteria are among the simplest photosynthetic organisms. During the last few decades, the primary photophysical and photochemical processes in these bacteria have been subject to numerous studies employing a variety of biochemical and spectroscopic techniques.1,2 Recently, the atomic structure of the peripheral antenna (LH2) of purple bacteria Rhodopseudomonas acidophila3 and Rhodospirillum molischianum4 was resolved. Such availability of highresolution structure, in conjunction with selectively engineered mutants5 and ultrafast laser sources capable of spanning the spectral region of the LH2 absorption bands, has made these photosynthetic systems one of the most popular objects in current photosynthesis research, which may eventually lead to a major breakthrough in understanding the primary steps in photosynthesis (for a recent review see 6). The homology of the LH2 protein sequences from different purple bacteria suggests that their structures are very similar.3,7,8 LH2 of Rhodobacter sphaeroides, one of the best studied purple bacteria, has two distinct absorption bands, B800 and B850, termed according to the characteristic Qy absorption wavelength of the bacteriochlorophyll (Bchl) pigments. Steady-state fluorescence polarization studies by Kramer and co-workers indicated a fast excitation transfer among B800 molecules.9 It is remarkable that in this early work many of the basic structural features of the LH2 antenna were correctly predicted. Femtosecond pump-probe measurements later revealed that at room temperature the B800 transient absorption anisotropy decays with a time constant of 0.8-1.6 ps, interpreted as a consequence of excitation transfer within the inhomogeneously broadened B800 band.10 In addition, a ∼300 fs isotropic decay component was observed and related to possible vibrational relaxation and/ or excitation transfer. The authors proposed that the fast isotropic and slower anisotropy decays might reflect the same excitation transfer process among molecules with near parallel transition dipole moments. A fast component of isotropic decay in B800 was also observed at 77 K.11 To rule out the possibility of intramolecular contributions to the fast decay, Bchl a molecules in solution and in low-temperature glasses were studied.11 Furthermore, anisotropy decay measurements and X
Abstract published in AdVance ACS Abstracts, December 1, 1997.
S1089-5647(97)02095-6 CCC: $14.00
corresponding computer simulations indicated that excitation transfer from molecules in the high-energy wing of the inhomogeneous distribution of B800 is slowed at low temperature because of the weak electron-phonon coupling.11 Monshouwer et al.12 did not observe any distinctive fast component of the B800 decay, most likely because the pulses they used had about the same duration as the fast decay time observed by Hess et al.11 Instead, they observed a gradual increase of the decay time as the pump-probe wavelength was scanned over the B800 absorption band from the blue to the red, which they interpreted as a manifestation of excitation transfer inside of B800. In a more recent work, Monshower and van Grondelle13 found a 400 fs rising component at the red part of the B800 spectrum after exciting at the blue edge of the band, giving a more direct evidence of the excitation transfer. Also, the variation of the hole width over the B800 band observed in spectral hole-burning experiments was interpreted as a consequence of excitation transfer among B800 Bchl molecules.14 Contrary to these lowtemperature studies, room temperature three-pulse photon echo experiments indicate no significant B800 f B800 transfer.15 However, in more recent work based on fluorescence upconversion measurements and Fo¨rster calculations, Fleming and coworkers conclude that energy transfer within the B800 band at room temperature probably takes place but on a slower time scale (∼ 1 ps)16 than what is reported for 77 K.11 Early time-resolved experiments found that excitation transfer from B800 to B850 in Rb. sphaeroides at room temperature occurs on a picosecond time scale.17,18 In subsequent work performed with sub-picosecond resolution, Shreve et al.19 estimated the transfer step to be 0.7 ps at room temperature. This time constant has recently been verified a number of times with femtosecond time-resolution.10,15,16 Spectral hole burning experiments from two groups provide results in mutual agreement suggesting that the B800 f B850 transfer slows down to 2.4 ps at liquid helium temperatures.20,21 Time-domain studies at 77 K have demonstrated the same trend. However, the reported time constants for B800 f B850 excitation transfer at 77 K vary from ∼2.5 ps (obtained in early experiments with relatively limited time resolution10) to 1.8 11 and 1.2 ps.12 It was also reported that this time constant appears to increase with increased intensity of the excitation laser,12 which may account for the longer time constants observed in some earlier 77 K studies. In a series of mutants of Rb. sphaeroides where © 1997 American Chemical Society
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the B850 band was progressively blue-shifted, it was found that the B800 f B850 transfer time at 77 K decreases as the separation between the two bands increases, i.e., with decreasing spectral overlap.22 This work suggested that this transfer step occurs via the Fo¨rster incoherent hopping mechanism. The conclusion was further supported by structural data3 which show that the distances from B800 molecules to the nearest B850 molecules are about 18 Å, leading to a relatively weak coupling between these molecules. In an interesting set of experiments, Wu et al.23 observed that the hole width in B800 of Rps. acidophila depends on the excitation wavelength, a trend similar to that reported earlier by De Caro et al.14 In addition, they found that increasing the pressure changes the dependence of the hole width on the wavelength.23 The wavelength of the cutoff between the narrow and wide holes appears to be correlated with the pressure shift of the B850 band relative to the B800 band, suggesting that an upper excitonic component of B850 is involved in the dynamics and responsible for the broadening of the holes. If this proves to be the case, it would be the first observation of the upper excitonic component of B850. In this article, we report femtosecond two-color transient absorption measurements of the LH2 antenna in Rb. sphaeroides. The experiments were performed at room temperature, 77 K, and 4.2 K. Various mechanisms behind the dynamics and temperature dependencies are discussed. The article is organized as follows: A brief overview of our experimental setup is presented in section 2. In section 3, the experimental results are summarized, and in section 4 we discuss results of simulations as compared to the experimental findings, as well as various open questions. Conclusions are drawn in section 5. 2. Materials and Methods The experiments employed a femtosecond spectrometer with 100-120 fs pulses generated by an amplified Ti:Sa laser system. The initial infrared 100 fs, 82 MHz pulses from a mode-locked Ti:Sa oscillator were amplified in a Ti:Sa regenerative amplifier from a few nanojoules to approximately 300 µJ at 5 kHz repetition rate. Amplified 795 nm pulses were split in two parts, one to excite the B800 band of the LH2 complex and the other for generation of white probe light in a 1 mm path length water cell. On order to minimize excitation annihilation and sample degradation, the excitation intensity was kept below 1.5 × 1014 photons cm-2 pulse-1, and no significant intensity dependence of the measured kinetics was observed by varying the excitation intensity by a factor of 10. To compensate for the intensity fluctuations of the probe light, a dual beam detection system based on a three-photodiode arrangement was used.24 To select the analyzing wavelength, reference and probe beams were focused on to the slit of a single-grating monochromator (resolution ∼ 4 nm); both beams were detected by near infrared photodiodes. Sample preparations have been described elsewhere.25 In order to obtain an optical quality glass at 4.2 K, the sample was diluted with glycerol and Tris buffer at pH 8 in the ratio 66% glycerol and 33% buffer solution, such that the linear absorbance was ∼0.5 at 850 nm. The measured transient absorption kinetics were analyzed as a sum of exponentials convoluted with the apparatus response function, using the Spectra Solve software. 3. Experimental Results Figure 1A shows the kinetics obtained at different temperatures by using pump and probe wavelengths 795 and 800 nm, respectively. We have selected this experimental configuration
Figure 1. (A) Two-color transient absorption signals in chromatophores of Rb. sphaeroides at three different temperatures. Pump and probe wavelengths are 795 and 800 nm, respectively. (B) 4 K transient absorption signal with pump and probe wavelengths at 800 and 840 nm. Fitting parameters for the kinetic curves are given in Table 1.
since, in one-color experiments, different four-wave mixing signals may give rise to fast kinetic components during the period of pump-probe overlap which are not related to the energy transfer. While coherence interactions are also present in two-color experiments, their effects are minimal. The signal at all temperatures consists of initial ground-state bleaching (BL) and stimulated emission (SE) of B800 which is followed by the excited-state absorption signal of B850. The kinetics were fitted as a sum of three exponentials, including a minor very fast (200-300 fs) component and a dominant, temperaturedependent component (0.7-1.5 ps). The accuracy of the estimation of the fastest component is not very high, and we were not able to observe any evidence of an associated temperature dependence. To obtain a good fit, a much slower (30 ps) component with negative amplitude (due to the excitedstate absorption of the B850 band) was added. On the basis of our earlier anisotropy studies,10,11 we assign the fast component to energy equilibration within the inhomogeneously broadened B800 band. The slower component corresponds to energy transfer from B800 to B850. The fitting results are shown in
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Pullerits et al.
TABLE 1: Results of Best Fits to the Kinetic Data Presented in Figure 1 temp (K)
probe wavelength (nm)
T1 ps (A1) (30%
T2 ps (A2) (15%
T3 ps (A3) (20%
response function (fs)
293 77 4 4
800 800 800 840
0.3 (1) 0.3 (1) 0.4 (1)
0.7 (4) 1.2 (6) 1.5 (6) 1.3 (-1)
28 (-0.5) 36 (-0.2) 31 (-0.2) ∞ (1)
80 120 120 120
more detail in Table 1. We can see that the excitation transfer time from B800 to B850 increases from ∼0.7 ps at room temperature to ∼1.5 ps at 4.2 K. Confirmation of the 4.2 K transfer time is shown in the Figure 1B, in which pump and probe wavelengths of 800 and 840 nm were employed. Here, the kinetics are fitted to a single-exponential rise (τ ∼1.3 (0.2 ps), directly reflecting the energy transfer from B800 to B850. Fitting the experimental decay curves to a sum of three exponentials is not a trivial task as significantly different combinations of lifetimes may yield a good fit. Furthermore, in our low-temperature studies, the pulses are passing through a number of cryostat windows which broadens the pulses slightly; how much exactly, we do not know. In addition, LH2 samples are known to suffer from irreversible photo damages which alter the kinetics if the excitation intensities are too strong.12,26 Special care was taken to minimize these problems. The fitting results reported in Table 1 represent an average of fits to several curves measured with the same conditions; the error estimate is based on the statistical behavior of the lifetime in the fitting. To account for possible pulse broadening in the cryostat, we have varied the pulse lengths used in the fitting procedure. The uncertainty in the pulse width does not allow us to make any conclusions about a possible rising component at low temperature. The error limits of the fastest lifetime also reflect this uncertainty (longer lifetimes do not depend on the pulse length). Experiments were carried out with very low excitation intensities, and test traces were always taken with about three times lower and two times higher intensities. No intensity dependence of the kinetics was observed. 4. Simulations and Discussion Fo1 rster Transfer. The transfer rate between two states can be expressed via the Fermi golden rule. In our case the initial state is D*A, where D* refers to an electronically excited donor and A to the electronic ground state of the acceptor. Analogously, the final state is DA*. As was first shown by Fo¨rster, this rate can be related to the experimentally observable fluorescence spectrum of the donor and the absorption spectrum of the acceptor.27 Rather than use absolute values of the extinction coefficient and the natural lifetime (as is done in the most common formulations of the Fo¨rster equation), we have chosen to represent the transfer rate via an overlap integral of normalized absorption and fluorescence spectra and the interaction between electronic states:
kji ) 1.18V2
∫Fi(ν) Aj(ν) dν ) 1.18V2Θ
(1)
where k has units of ps-1, V is the dipole-dipole interaction in cm-1, and Θ is the overlap integral between the donor fluorescence and acceptor absorption spectra for which the intensity (area) has been normalized to 1 on the cm-1 scale. It was shown by Dexter28 that similar expressions could also be derived for electron exchange and multipole interactions. Recently, it was pointed out that short-range excitation transfer rates may be influenced by “through-configuration” interactions,29,30 which in some cases may be even stronger than the
above-mentioned “standard” Dexter terms. In the current analysis, we will concentrate on Fo¨rster transfer only, as described by eq 1. Spectral Overlap. Homogeneous spectra in general consist of a purely electronic transition (zero phonon line (ZPL)) and transitions by which vibrations and/or lattice phonons are created or destroyed (phonon wing (PW), vibronic lines).31 The shape of the spectra depends on the coupling of the phonons and vibrations with the electronic transition, on phonon and vibronic frequencies, and on temperature. At low temperature, ZPLs and vibronic lines are sharp and can be observed via fluorescence line narrowing32 or spectral hole-burning33 techniques. At higher temperatures, the ZPL disappears and the overall spectrum attains a Gaussian shape.34 To evaluate the overlap integral, we have used a spectral simulation procedure which makes use of the linear harmonic Franck-Condon approximation.34,35 In this approach, the corresponding homogeneous absorption and fluorescence spectra are mirror images, symmetric with respect to the ZPL position. The input parameters of the simulation are the vibrational frequencies, the one-phonon profile, and the corresponding Huang-Rhys factors (S). These parameters can be obtained from hole-burning21 or selectively excited fluorescence experiments.36,37 Reddy et al.21 have observed a number of satellite holes of the B850 band at 280, 340, 560, 750, and 920 cm-1. They argue that the 280 cm-1 hole is perturbed by the antihole of B850 and therefore the real frequency is closer to 200 cm-1, in agreement with fluorescence measurements.36 In addition, they estimate that the Franck-Condon factors of the 750 and 920 cm-1 modes are about 0.05. It is worth noting that in a similar work on photosystem 1 (PS1), Gillie et al.38 observed a full set of vibrational frequencies of Chl a and estimated the corresponding Franck-Condon factors. At the same time, Peterman et al. studied selectively excited fluorescence spectra of Chl a in light-harvesting complex 2 (LHCII).37 While there is remarkable similarity between the frequencies and relative intensities of the vibronic transitions reported in these two different works, the values for the Franck-Condon factors reported by Peterman et al.37 are significantly smaller than the corresponding values in the hole-burning work.38 It is quite unlikely that such a big and systematic variance could fully originate from the different surroundings of the Chl a molecules in the two different pigment protein complexes (note that the hole-burning experiments were performed on PS1 and the fluorescence studies on LHCII). Thus, it appears to us that the Franck-Condon factors of Chl a may have been overestimated in the hole-burning study. To the best of our knowledge, the hole-burning study on LH2 is the only frequency-domain work in which the Franck-Condon factors of vibrational modes of Bchl a in antenna systems have been estimated.21 In a timeresolved study, Chachisvilis et al.39 observed coherent nuclear motions with a frequency of 120 cm-1, however they did not estimate the corresponding Franck-Condon factor. While there is no evidence that the Franck-Condon factors in ref 21 are overestimated, we consider these estimates as an upper limit of the Franck-Condon factors of Bchl a in LH2. We also carried out simulations of the overlap integral with 10 times smaller S values. Several studies21,23 have concluded that the electron-phonon coupling of Bchl a in antenna systems is weak, suggesting S values from 0.3 to 0.6. In most of the systems with essentially monomeric Bchl pigments (e.g., B800) it was found that the PW has a maximum at about 20-30 cm-1.14,21 For B820, a dimer of Bchl a,40 a much broader PW was observed with a
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TABLE 2: Frequencies of the Vibrational Modes and Corresponding Huang-Rhys Parameters Extracted from Literature21,35,36 B800b
B800 -1 a
-1 a
ω (cm )
S
ω (cm )
S
15 125 200 340 560 650 750 920
0.3 0.01 0.01 0.01 0.01 0.01 0.05 0.05
50 200 340 560 650 750 920
0.3 0.01 0.01 0.01 0.01 0.05 0.05
a ω of the first mode corresponds to µ of phonons in eq 2 which has a maximum at 2µ. b For B850 we have used here a value of µ which roughly corresponds to that of the B820 dimer.
maximum of the wing at around 110 cm-1.35 Here we point out that the B850 ring of LH2 consists of Rβ-pairs very similar to what is suggested for B820. In all studies, the PW appears to be substantially asymmetric (see, e.g., refs 14, 23, and 37). Hence, we have chosen the following shape for the one-phonon function:
{
(ν2/2µ3) exp(-ν/µ), 0 e ν φ(ν) ) 0, ν
