J. Phys. Chem. 1995,99, 13537-13544
13537
Ultrafast Initial Reaction in Bacterial Photosynthesis Revealed by Femtosecond Infrared Spectroscopy P. Ha"*
and W. Zinth
Institut f i r Medizinische Optik, Ludwig Maximilians Universitat Miinchen, Barbarastrasse 16, 80797 Miinchen, Germany Received: March 28, 1995; In Final Form: July 7, 1995@
The early temporal evolution of the transient infrared difference spectrum of bacterial reaction centers of Rhodobacter sphaeroides between 1000 and 1600 cm-' reveals a strong 200 fs component which has not yet been detected in femtosecond experiments in the visible or near infrared region. A detailed analysis of the experimental data shows that this fast component can be explained by a considerable change of the electronic structure of the primary electron donor, the special pair, during this time. A possible explanation of the reaction relates this process with an ultrafast initial intramolecular charge separation in the special pair P.
Introduction The photosynthetic reaction center is responsible for the initial storage of photon energy into chemical energy by transferring an electron through the transmembrane protein. Because of the importance of this process in the global energy balance, the reaction center has received strongly increasing attention in the last decades. The determination of the three-dimensional structure with atomic resolution by X-ray diffraction gave the first detailed insights into the microscopic function of this In the reaction center (RC) there are two potential electron transfer branches (A and B), each starting from a dimer of strongly coupled bacteriochlorophyll (BChl) molecules (PL and PM coupled to the special pair P) and continuing with a BChl monomer (BL and BM), a bacteriopheophytin (BPhe, HL and HM),and a quinone (QAand QB). Surprisingly, the electron is transferred only through one branch, the active A branche5 This observation,the '"directionality of the electron transfer", is one of the remaining questions of bacterial photosynthesis. The basic mechanism of the transfer reaction is known from time-resolved femtosecond spectroscopy in the visible and near infrared spectral region, which tests the electronic states of the chromophores. These experiments reveal that after electronic excitation of the special pair, the electron reaches BL in ~ 3 - 4 ps, HL in a second faster (in 1 ps) electron transfer step, and finally Qn in ~ 2 0 0 However, the kinetics of the decay of the electronically excited state P* is not fully understood, since recent experiments have shown that P* does not decay monoexponentially: At early delay times, an oscillating signal in the stimulated emission signal with a period of W300 fs was observed even at room temperature.8 These results are discussed in the frame of a coherent wave packet motion in the excited state. Time-resolved fluorescence measurements have demonstrated that at later delay times the decay of the excited state has to be fit with at least two time constants ( ~ 2 . 5and 7-11 P S ) . ~ . ' O Commonly, this result is explained by a distribution of energy levels of the first charge-separated state P+B-. To obtain a more detailed microscopic picture of the electron transfer reaction and in particular of the structure-function relationship, femtosecond IR spectroscopy testing the vibrational part of the spectrum was started In a previous paper,I4 we presented femtosecond IR data of RCs of Rhodo-
* To whom correspondence should be addressed. @
Abstract published in Advance ACS Abstracts, August 15, 1995.
bacter sphaeroides in the spectral range between 1000 and 1800 cm-I. Since we want to address questions raised in this paper, we have to summarize here the basic results of ref 14. It was shown that on a longer time scale (> 1 ps), the temporal evolution of the IR spectrum can be explained by the wellknown electron transfer kinetics. Time-resolved IR difference spectra were presented reflecting the intermediates states P* (recorded 1 ps after electronic excitation), P+H- (10 ps), and P+Q- (1000 ps), respectively. The spectra are well described in the high-frequency region (> 1600 cm-I). Here, the C=O bands of the chromophores and the surrounding protein side chains are found. Since the C=O vibrations are predominantly localized to one single molecular bond and since the density of fundamental modes is small in this spectral region, a detailed assignment of those bands was possible. Therefore, most steady-~tate'~-'~ and time-resolved IR spectroscopy' ' - I 4 focused on this part of the spectrum. However, the low-frequency region (< 1600 cm-I) could not be explained adequately: It is dominated by vibrational modes of the tetrapyrrole ring system of the special pair's chlorophyll molecules. So far, no detailed band assignment of the steadystate and time-resolved IR difference spectra in this spectral region is available because of the difficulties that arise from the delocalization of these vibrational modes over extended regions of the molecules. A first assignment of resonance Raman spectra of the ground state was given in refs 20 and 21. Three striking and unexplained features are observed in the low-frequency part of the spectrum. (1) In steady-state FTIR difference spectra of P+Q-, three broad positive bands having considerable oscillator strength are found around 1550, 1480, and 1290 cm-I. From a comparison of the light-induced P+Q-/PQ difference spectrum with electrochemically induced P+/P and Q-/Q difference s p e ~ t r a ~ ~ - ' ~ , * ~ it is known that the main contribution to these absorption bands originates from oxidation of the special pair (or from a response of its nearest neighbor) and not from the reduction of the quinone. The time-resolved data revealed that the P+H-/PH spectrum has equivalent properties and that similar strong positive difference bands also occur in the P*/P difference spectrum around 1530, 1460, and 1260 cm-I. (2) Throughout the whole mid-IR region, the difference spectra of P* and of P+ are essentially positive. Consequently, the absorption cross sections of several vibrational modes are increased upon electronic excitation as well as upon oxidation.
0022-3654/95/2099-13537$09.00/0 0 1995 American Chemical Society
13538 J. Phys. Chem., Vol. 99, No. 36, 1995 This effect is seen even in FTIR spectra, when P'Q- is accumulated by steady-state illumination (see Figure 3d in ref 14). However to our knowledge, this effect was not explicitly discussed in previous steady-state experiments: Often, the zero line of the FTIR difference spectra was dropped out (see for example ref 15 and 23) or even realigned in order to obtain a difference spectrum centered around zero (see for example ref 17). This treatment was justified by the limited accuracy of the base line in a FTIR difference spectra (W. Mbtele, personal communication). However, careful steady-state FTIR spectroscopy well reflects this absorption i n ~ r e a s e . ' ~ Recent timeresolved experiments (not influenced by base line problems) in the high-frequency range (1600-2000 cm-1)11-13 showed the same effect. However, due to the strong contribution of the C=O modes, the effect is not so clear in this spectral range. (3) In certain spectral regions, a significant contribution of a very fast kinetic component with a time constant of approximately 200 fs was detected. No related feature was observed in visible or near-IR time-resolved spectroscopy. Apparently, the low-frequency part of the spectrum reflects interesting properties of the special pair P. In the present paper we will present more experimental data taken in this spectral range and give a detailed analysis of the early evolution of the IR spectra. In addition, we will show that the increase in IR absorption is related to the electronic properties of P* and P+. Materials and Methods The experimental conditions are described in detail in refs 14,24, and 25. Here only the basic properties of the experiment are presented: A pump and probe scheme was used. The sample was excited by 150 fs pulses at 865 nm with an energy density of approximately 10 pJlmm2. The absorbance change of the sample was recorded by tunable probing pulses generated by difference frequency mixing of the output of a regenerative Ti:sapphire amplifier (repetition rate 1 kHz) and that of a synchronized traveling wave dye laser in a AgGaS2 crystal. Pulses between 1000 and 2000 cm-I with a pulse duration of ~ 3 0 fs 0 and a spectral bandwidth of ca. 60 cm-' were obtained. To receive sufficient spectral resolution for spectroscopy of narrow IR transitions (which typically have a bandwidth of 1020 cm-l), the probing pulses were dispersed in a grating spectrometer after passing the sample. A spectral resolution of 5-10 cm-I was obtained. A 10-element IR detector array was used in order to cover the spectral bandwidth of the probing pulses simultaneously. The cross correlation width (fwhm 330 fs) and the delay zero point of the experimental setup were deduced from an independent experiment observing free carrier absorption in a thin silicon sample. Since free carrier absorption arises instantaneously,this method allows deconvolution of both parameters with an estimated error of *:f50 fs. The RCs from Rb. sphaeroides R26 were prepared as described in refs 14 and 26. All experiments were done at 12 "C. Coherent Processes in IR Spectroscopy In femtosecond spectroscopy with a frequency-resolved detector (like the one used in the experimental setup), one has to consider a coherent process called perturbed free induction decay. This is of special interest in femtosecond IR spectroscopy, since the dephasing time T2 = IIAvn (Av is the bandwidth of the absorption line) of a typical absorption line is longer than the time resolution of the experimental setup. The effect can be explained as follows: The IR probing pulse excites a coherent polarization in a narrow absorption line, which decays with its intrinsic dephasing time T2. The coherent polarization radiates light, the so-called free induction decay, and therefore, will be
H a " and Zinth
Figure 1. Model calculation of the perturbed free induction decay signal. It was assumed that an isolated absorption line (bandwidth AvIc = 20 cm-I) bleaches upon short 6 function shaped excitation. For probing, a Gaussian-shaped 300 fs pulse was taken. The wavenumber
axis is labeled with the difference between the detection frequency and the resonance frequency of the absorption line. recorded by the IR detector. If an excitation pulse reaches the sample after the probing pulse (negative delay times), it cannot influence the intensity of the probing pulse itself. However, it may influence the temporal and spectral properties of the free induction decay signal, whenever the strength or position of the absorption line is changed upon excitation. As a consequence, the IR-detector displays a difference signal which decays toward negative delay times with a time constant equal to T2. As a first example, consider a single absorption line which bleaches upon electronic excitation. A theoretical description of this situation is given in refs 27 and 28. A model calculation according to eq 5 of ref 27 is shown in Figure 1. The parameters are chosen for a realistic modeling of our experimental conditions: The probing pulses are assumed to be Gaussian shaped with a pulse duration of 300 fs. The center frequency of the probing pulses and that of the absorption line are identical. For simplicity, the excitation pulses (pulse duration 150 fs) are modeled by a 6 function. The electronic dephasing is assumed to be much faster than the time resolution. For the vibrational dephasing time, a value of 600 fs corresponding to a typical bandwidth of ~ 2 cm-I 0 is taken. At positive delay times, the bleach of the Lorentzian-shaped absorption line can be seen. At negative delay times, the signal is more complicated: When the detection wavelength is tuned to the center of the absorption line, an exponentially decaying signal with a time constant equal to T2 is detected. Off-resonance, the signal is modulated with the frequency difference between band center and the detection frequency. When the function of Figure 1 is integrated over frequency, oscillating and exponentially decreasing signal components cancel. In this case, the signal rise is limited only by the cross correlation function; that is, when spectral resolution is abandoned, the full temporal resolution of the experimental setup can be used. In general, the difference signal is more complicated than the one shown in Figure 1. In a molecule as complex as the RC, there are many overlapping absorption lines changing oscillator strength and/or center frequency upon excitation. In this case, the free induction decay light field of the sample is determined by the Fourier transform of the absolute absorbance spectrum of the sample in the ground state (convoluted with the Fourier transform of the spectrum of the probe pulses). As a result, the perturbed free induction decay signal is related to the absorbance change of the sample immediately after electronic excitation (the difference spectrum at delay zero). In other
Ultrafast Initial Reaction in Bacterial Photosynthesis words, the perturbed free induction decay signal recorded before delay zero can be used to receive independent information on the difference spectrum at delay zero. This approach is correct as long as one assumes that all absorbance lines can be treated independently and are not coupled by coherent interaction. (The possibility of coherence transfer is discussed in ref 28 in the case of an absorbance line which is shifted in frequency due to the electronic excitation. However, as long as the frequency shift is small compared to the spectral bandwidth of the absorption line, the effects of coherence transfer are small.) A detailed examination of this multiabsorption line situation will be given elsewhere.29 It should be kept in mind that the perturbed free induction decay signal has its origin in the spectral properties of the difference spectrum immediately after electronic excitation. It is not connected to a temporal evolution of the sample upon a chemical or relaxational reaction; that is, it does not reflect any dynamic process of the sample. Therefore, it is important to distinguish between the temporal evolution of the difference signal caused by the perturbed free induction decay effect and those kinetic components which occur upon a real reaction. In the case of infinite time resolution (6 pulses), this distinction would be straightforward: From causality one can conclude that the perturbed free induction decay signal always is observed at negative delay times, even for complex difference spectra. Therefore, all three processes, i.e. the perturbed free induction decay signal ( t < 0), the excitation process (t = 0), and resulting reactions (t > 0), are separated in time. However, in a realistic experiment with a finite time resolution, a mixture of all three processes occurs within the cross correlation time. Therefore, a global fit algorithm is required, leading to a selfconsistent adjustment of the perturbed free induction decay signal ( t < 0) to the difference signal around delay zero (t = 0) and to possible initial reactions. Such an algorithm which allows the separation of the three components was developed and will be presented in a subsequent paper.29 Here, only a brief description of the underlying idea is given: The experimental data are related to two types of difference spectra a ( w ) and bi(w): (i) The first difference spectrum a ( o ) describes the instantaneous absorbance change of the sample upon optical excitation (difference spectrum at delay zero). As shown above, this difference spectrum is directly coupled to the perturbed free induction decay effect and is essentially determined by the Fourier transform of the perturbed free induction decay signal. (ii) The difference spectra bi(w)describe the absorbance changes of the sample due to a series of subsequent reactions. Since these incoherent spectral changes bi(w) do not cause perturbed free induction decay effects, the related temporal changes can be modeled by exponential functions (convoluted with the instrumental response function). It is important for the stability of the fit procedure that the spectral fit parameters a ( w ) and bi(w)are linear parameters leading to an algorithm insensitive to initial estimates of these parameters. For each frequency position oj there is one parameter a(wj) for the delay zero spectrum and one parameter bi(w,) for each exponential function used. In addition, only one global nonlinear fit parameter, the time constant of the initial ultrafast reaction (see the Experimental Results section), is necessary since the subsequent much slower ET rates are kept fixed at the well-known values (3.8 and 240 ps, see ref 14). In principle, the initial absorbance change a ( w ) may be determined in two ways: (i) by analyzing the perturbed free induction decay effect and (ii) by back propagation of the absorbance changes bi(o)of the later reaction steps, a ( w ) = AA(t=m) - Cbi(w). The fit procedure combines both possibilities by calculating a ( w ) in a self-consistent way.
J. Phys. Chem., Vol. 99, No. 36, 1995 13539
It was tested with the help of time-resolved IR data of a large dye molecule. These experiments will also be presented in ref 29. In the literature, an altemative technique is used for ultrafast IR In these experiments, a CW IR light beam is used as probing light. After passing the sample, the probing light is upconverted in a nonlinear crystal by a short gating pulse in order to obtain time resolution. Also under these experimental conditions, coherent signals caused by dephasing effects are observed. However, in contrast to the technique used in the present paper, these signals are observed predominantly at positive delay times and may overlap with real dynamic component^.'^ Consequently, it is more difficult to distinguish between the pure coherent effect, which does not contain dynamic information, and a real kinetic component, which is caused by a reactive or relaxational process.
Experimental Results In this section we present two direct experimental pieces of evidence which reveal that a fast initial kinetic component exists and that the P* difference spectrum 1 ps after electronic excitation is considerably different from the difference spectrum immediately after electronic excitation. In a second step, we will use the fit algorithm mentioned above to deduce the pure difference spectrum of the initially ( t = ~ 0) prepared excited electronic state. A first indication for a fast reaction is found when the early temporal evolution of the IR spectrum at 1225 cm-' and at three spectral positions centered to the P* band at 1460 cm-I is inspected (see Figure 2). In all measurements, the IR absorption does not rise simultaneously with the cross correlation function of the pump and probe pulse (broken line) but with a significant delay time. The delayed absorption rise may be modeled by an exponential function with a time constant of 150-300 fs (a value of 200 fs yields the best fit). Around 1530 cm-', in the third broad positive difference band in the 1 ps spectrum, only a weaker contribution of the 200 fs kinetic component can be found. The observed time constant lies below the fwhm of the cross correlation function between the excitation pulse and probing pulse ( ~ 3 3 fs). 0 However, due to the high amplitude of this kinetic component, it can be resolved clearly as shown in Figures 2 and 3 (with a large experimental error of the determined time constant). The following observations exclude the possibility that perturbed free induction decay is responsible for the discrepancy between the measurements and the instantaneous absorption rise: (i) The perturbed free induction decay effect can only occur at negative delay times or, in the case of finite time resolution, within the duration of the cross correlation function. However, even after the end of the cross correlation function (a > 400 fs), distinct discrepancies remain between the experimental data and the model curve (broken lines in Figure 2) calculated without a 200 fs component. (ii) Since the delayed signal rise is observed in a broad spectral region, the experiments were repeated without dispersing the probing pulses. In this case, the perturbed free induction decay effect should be absent (see previous section). As an example, Figure 3a presents results measured with undispersed probe pulses (no spectrometer, center frequency 1430 cm-' , spectral bandwidth 60 cm-I). Again, this experiment shows the delayed absorbance increase. A second experiment which was calculated by integrating the data (of a single 10-channel experiment; see ref 14 for experimental details) over a spectral bandwidth of 45 cm-' yields the same time dependence for the 1458 cm-] range (Figure 3b). Both examples prove that the
Ha" and Zinth
13540 J. Phys. Chem., Vol. 99, No. 36, 1995
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Figure 2. Transient absorbance change measured with dispersed probe pulses (frequency resolution 4-7 cm-I). Measurements are shown at 1225 cm-I and at three frequency positions around a strong P* band at 1458 cm-' (center frequency) and at 1443 cm-I (low-frequency side) and 1473 cm-' (high-frequency side). All plots show a similar delay in the rise of the absorption cross section: The broken line is a model function calculated under the assumption of an instantaneous rise of the signal; the solid line is calculated assuming an additional exponential process with a time constant of 200 fs. An additional slower kinetic component has to be introduced which describes the first ET step from P* to P'B- and which is modeled by a exponential function with a time constant of 3.4 ps.
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Figure 3. (a) Transient absorbance changes at 1430 cm-' measured with undispersed probe pulses. The probe pulses had a spectral bandwidth of 60 cm-I. (b) Transient absorbance changes centered around 1458 cm-' calculated by adding all channels of one single 10element experiment (see ref 14 for experimental details). This treatment simulates an undispersed experiment with a bandwidth of 45 cm-I. Again, both experiments show a delayed increase of absorption, confirming that it is not a consequence of the perturbed free induction decay effect.
observed signal rise is not related to the perturbed free induction decay process.
Figure 4. Transient absorbance change measured with dispersed probe pulses. Part a shows the data at a probing frequency of 1502 cm-'. Here a small perturbed free induction decay signal at negative delay times is observed. However, neither in the center of the strong negative band at 1059 cm-' (c) nor in the spectral wings of this band (1043 cm-' (c) and 1075 cm-l (d) can a perturbed free induction decay signal be detected, although a pronounced signal is expected (see Figure 1) if one assumes that the intense narrow P* band (see Figure 5b) bleaches immediately upon electronic excitation. An additional 200 fs component destroys coherence and therefore suppresses the coherent perturbed free induction decay effect. Under these assumptions, the model calculation fits well the experimental data (solid lines).
Interesting information originates from a detailed analysis of the amplitudes of the fast kinetic component at 1225 and around 1460 cm-I. The instantaneous contribution to the signal rise is small (
