Excitation Wavelength Dependence of Bacterial Reaction Center

Antonetti, A.; Orszag, A. In Ultrafast Phenomena V; Fleming, G. R., Siegman, A. E., ..... Jonathan A. Jackson, Su Lin, Aileen K. W. Taguchi, JoAnn...
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J. Phys. Chem. 1996, 100, 14228-14235

Excitation Wavelength Dependence of Bacterial Reaction Center Photochemistry. 2. Low-Temperature Measurements and Spectroscopy of Charge Separation Jeffrey M. Peloquin,† Su Lin, Aileen K. W. Taguchi, and Neal W. Woodbury* Department of Chemistry and Biochemistry and the Center for the Study of Early EVents in Photosynthesis, Arizona State UniVersity, Tempe, Arizona 85287-1604 ReceiVed: April 3, 1996; In Final Form: May 30, 1996X

Excitation with spectrally narrow (5 nm), temporally short duration (200 fs) laser pulses at a variety of wavelengths between 848 and 903 nm results in substantial excitation wavelength dependent differences in the evolution of the bacterial reaction center absorbance spectrum both before and after charge separation occurs. The transient holes in the initial electron donor band showed a more resolved vibrational band structure at 20 K, when compared to those of earlier room temperature transient hole-burning experiments (Peloquin, J. M.; Lin, S.; Taguchi, A. K. W.; Woodbury, N. W. J. Phys. Chem. 1995, 99, 1349). The dominant vibrational band observed is at 120 cm-1, in agreement with dynamic measurements of coherent oscillations on this time scale (Vos, M. H.; Rappaport, F.; Lambry, J.-C.; Breton, J.; Martin, J.-L. Nature 1993, 363, 320). At both room temperature and low temperature, there is a distribution of P to P* transition energies due to a distribution of the protein conformations in the ground state. At 20 K, one can also see a distribution of P* to P stimulated emission energies. As might be expected, the barriers to conformational interconversion are more easily crossed at room temperature, resulting in a smaller difference between the mean transition energies of the photoselectable subpopulations on the picosecond time scale relative to those at low temperature. At 20 K, much of this conformational interconversion is apparently lost when exciting near the 0-0 transition wavelength of P. More conformational interconversion appears to take place at low temperature when higher energy excitation is used, implying that P* is vibrationally hot for at least hundreds of femtoseconds following excitation on the blue side of its QY band. Of particular interest is the insensitivity of the overall charge separation kinetics to the selection of ground state conformational subpopulations by different excitation wavelengths. There appears to be very little coupling between the nuclear motion that defines the ground state transition distribution and the charge separation reaction itself. Given the apparently slow rate of vibrational relaxation of some of the excited state modes most strongly coupled to the P to P* transition, the lack of excitation wavelength dependence of the charge separation rate also suggests that these modes are not strongly coupled to the charge separation reaction. What the ground state (and possibly excited state) conformational distributions and interconversions do affect is the kinetic complexity of the absorbance changes in the 800 nm region. Specifically, the extent of involvement of fast, multiexponential decay components in this region depends strongly on the wavelength of excitation.

Introduction The photosynthetic reaction center is responsible for the conversion of light energy into a metastable form by transmembrane charge separation. R-26 reaction centers from Rhodobacter sphaeroides consist of three protein subunits (L, M, and H) and nine cofactors. The cofactors are arranged symmetrically about a pseudo-C2 axis which extends from a closely coupled pair of bacteriochlorophylls (P) on the periplasmic side of the reaction center to two monomer bacteriochlorophylls (BA and BB) to either side of P, then to two bacteriopheophytins (HA and HB) toward the middle of the transmembrane complex, and finally to two quinones (QA and QB) on the cytoplasmic side. Between the two quinones is a nonheme iron atom.1-4 An electron is transferred from the excited singlet state of P, appearing on HA with an overall time constant of about 3 ps. BA plays a critical role in this transfer, but has only a small population at any time during the electron transfer due to kinetic, thermodynamic, or quantum mechanical limitations. The * Author to whom correspondence should be addressed. E-mail: [email protected]. † Present address: Department of Chemistry, University of California at Davis, Davis, CA 95616. X Abstract published in AdVance ACS Abstracts, August 1, 1996.

S0022-3654(96)01016-7 CCC: $12.00

electron is then transferred to QA with an overall exponential time constant of about 200 ps and finally from QA to QB in about 200 µs.2,5-10 There is very little population of chargeseparated states involving B-side cofactors during this reaction in wild type reaction centers (for recent results on this topic, see refs 11-13). While it is the chlorins and quinones that serve as the redox active cofactors, the surrounding amino acid residues provide the environment in which the electron transfer reactions take place. Two aspects of the reactions that are influenced by the protein environment are the midpoint potentials of the cofactors (reviewed in ref 14) and the spectral transition energies of the cofactors (reviewed in ref 10). The large number of degrees of freedom inherent in a protein suggests that a distribution of nuclear conformations will be present in the ground state population. This conformational distribution will lead to a corresponding distribution in the transition energies and midpoint potentials of individual cofactors. This in turn leads to a distribution in the free energies of individual intermediate states in the reaction. Because the equilibrium conformation of the ground state may be different from that of the excited or chargeseparated states of the reaction center, it may also be that the state energies will be time-dependent, as the protein environment responds to state changes.15,16 © 1996 American Chemical Society

Excitation Wavelength Dependence of Reaction Centers Conformational subpopulations have been used to explain the nonexponential charge separation kinetics from P* to P+HA-.17-21 This suggests that there are two or more conformations of the protein, each with different rates of electron transfer. A roughly 150 cm-1 distribution of P f P* transition energies has been seen at 4.2 K in Rhodopseudomonas Viridis and Rb. sphaeroides by low-temperature hole burning.22,23 However, theoretical consideration of dispersive kinetics using information from holeburning measurements has suggested that the nonsingle exponential decay of P* is not due to intrinsic structural disorder in the reaction center.24 One way to explore the effects of the distribution in ground state and excited state conformational subpopulations on the electron transfer kinetics is to selectively excite subpopulations of reaction centers with narrow spectral bandwidth pulses and then observe the spectral evolution during charge separation. Previous transient hole-burning experiments at room temperature using 60 cm-1 bandwidth excitation pulses have shown that the rate of loss of P* (and therefore presumably the rate of charge separation) was essentially independent of the excitation wavelength, suggesting that the conformational changes between subpopulations selected by different excitation wavelengths are not coupled strongly to the electron transfer reaction.25 However, both the spectrum of the ground state bleaching of P and the absorbance spectrum of P* are dependent on the choice of excitation wavelength immediately following excitation. The dependence of the stimulated emission spectrum on the wavelength of excitation diminished almost completely within about 1 ps, but the excitation wavelength dependence of the ground state bleaching remained for at least 5 ps following excitation. These results confirm that a distribution of P f P* transition energies exists at room temperature and that both the P and P* states contribute to the initial distribution. However, at room temperature the effect of photoselection on the excited state spectrum is short-lived (less than 1 ps) when compared to overall charge separation rate (about 3 ps). In this report, transient hole burning and the effect of photoselecting reaction center populations is explored further at 20 K, where many of the interconversion processes between reaction center conformations should be slowed down dramatically or halted. In this way, one can test whether the conformational changes that are coupled to the ground state transition of P are also coupled to the subsequent charge separation reaction. Materials and Methods Reaction centers from Rb. sphaeroides strain R-26 were prepared essentially as described previously.26 Transient absorbance measurements performed at room temperature were done as previously described,25 and low-temperature measurements of transient absorbance were performed with quinones reduced in a glycerol glass, as described in Woodbury et al.16 Results Room Temperature Absorbance Changes. Previously, we have reported the R-26 reaction center absorption changes in the 830-950 nm region at room temperature, showing that both the early- and long-time transient spectra depend on the position of excitation within the P band.25 Figures 1 and 2 show room temperature absorbance change measurements in the 725-835 nm region exciting at 840 and 893 nm, respectively. Excitation wavelength dependent spectral changes are also observed in this region. The most pronounced difference between 840 and 893 nm excitation is during the first 0.25 ps. Short wavelength excitation at 840 nm in this time range results in an immediate

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Figure 1. Time-resolved absorbance change spectra of R-26 reaction centers at times t < 0.75 ps (panel A) and t > 0.75 ps (panel B) after excitation with 55 cm-1 wide (FWHM) 840 nm excitation pulses at room temperature. These spectra represent single time slices from a surface of absorption changes. One-hundred spectra were taken at 67 fs intervals. The data have been corrected for dispersion as described in the text.

and prominent absorbance increase near 805 nm (Figure 1A). This initial absorbance increase is much less pronounced when 893 nm excitation is used, but grows in on the several hundred femtosecond time scale (Figure 2A). Most of the differences between 840 and 893 nm excitation disappear within the first picosecond. If this evolution toward similar difference spectra between the data taken using 840 and 893 nm excitation represents the interconversion of conformational subpopulations on the subpicosecond time scale, then measurements at low temperature, where conformational interconversion should be slower, will show the effects of conformation on the spectral evolution associated with electron transfer more clearly. Early-Time, Low-Temperature Measurements. Figures 3A, 4A, and 5A show absorption change spectra between 825 and 970 nm as a function of time between 0 ps (defined as overlap between the pump and probe pulses) and 0.75 ps for 858, 893, and 903 nm excitation, respectively. During the first few hundred femtoseconds, the spectra include a narrow bleaching centered at the excitation wavelength. In each case, the spectral bandwidth of this feature is roughly the same as that of the excitation pulse. Underneath the sharpest feature is a spectrally broader absorbance decrease. The overall width of this broader bleaching is still about 2-fold less than the 3040 nm width of the steady state bleaching of P due to P+ formation observed at this temperature.27 Local structure can be seen within the broader absorbance decrease, roughly 10 nm to either side of the excitation wavelength in each figure (about 120 cm-1). The local structure is especially pronounced in Figure 5A (903 nm excitation), where partially resolved bands are seen not only flanking the excitation wavelength but also

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Figure 2. Time-resolved absorbance change spectra of R-26 reaction centers at room temperature. Conditions are as described in Figure 1 except for the use of a 57 cm-1 wide (FWHM) 893 nm excitation.

at higher energy wavelengths (25-30 nm or about 300 cm-1 to the blue of 903 nm). Similar features were detected in the previously reported transient hole-burning experiments performed at room temperature25 but were much less well-resolved than those in Figures 3A, 4A, and 5A. The absorption change spectra during the first few hundred femtoseconds in the 740-880 nm region at 20 K (Figures 6A and 7A) show several distinct maxima and minima whose spectral positions are dependent on the choice of the excitation wavelength. This dependence is clearly seen in the 790-820 nm region where the maximum at 803 nm in Figure 6A (858 nm excitation) is shifted to 808 nm in Figure 7A (903 nm excitation) and becomes well-resolved from the 790 nm absorbance increase. The maximum which occurs at 790 nm does not appear to be dependent on the choice of the excitation wavelength. In addition to the position of the peaks, there is also a very fast time scale broad absorption increase that occurs on the few hundred picosecond time scale using 858 nm excitation, which is much less pronounced using 903 nm excitation. After about 500 fs, the absorbance decrease in the 870-890 nm region has reached its largest width (Figures 3A, 4A, and 5A). However, there are substantial differences between higher energy and lower energy excitation wavelength spectra in terms both of width and peak position. As the excitation wavelength is increased, the peak position shifts with it and the width of the absorbance decrease narrows by nearly a factor of 2. The excitation wavelength dependence of the spectra in the 800 nm region at 0.5 ps are just as dramatic. While the higher energy excitation results in a broad spectrum at this time which still resembles the excited state spectrum during the first few hundred femtoseconds and shows a positive feature between 800 and 805 nm (Figure 6A), lower energy excitation results after 500

Peloquin et al.

Figure 3. Time-resolved absorbance change spectra of R-26 reaction centers at times t < 0.75 ps (Panel A) and t > 0.75 ps (Panel B) after excitation with 64 cm-1 wide (FWHM) 858 nm excitation pulses at 20 K. These spectra represent single time slices from a surface of absorption changes. One-hundred spectra were taken at 44 fs intervals. The data have been corrected for dispersion as described in the text.

fs in a spectrum that has a strong negative feature between 800 and 805 nm (Figure 7A). Longer Time Absorption Changes at Low Temperature. Regardless of excitation wavelength, the major change in the 830-960 nm region at times longer than 0.75 ps is the recovery of the bleaching at wavelengths greater than 900 nm. This recovery is presumably due to the decay of the stimulated emission from P* during charge separation. By the time electron transfer is complete, each of the bleaching spectra has shifted about 5 nm to higher energy, but other than this small shift, the excitation wavelength dependent differences between peak position and the width of this feature present at early times remain even after electron transfer is complete. Figure 8A compares the absorption change spectra for 858, 893, and 903 nm excitation at 4.0 ps following excitation. The maxima of the bleachings are at 870, 888, and 890 nm for 858, 893, and 903 nm excitation, respectively. For 858 nm excitation, the bleaching maximum is still 870 nm after 100 ps (data not shown). Figure 8B compares the 4.0-0.75 ps differencedifference spectra for the three excitation wavelengths and should represent predominantly the spectrum of the stimulated emission. This figure shows that the wavelength of the maximum amplitude of the stimulated emission is also dependent on the choice of the excitation wavelength with maxima at 907, 915, and 917 nm for 858, 893, and 903 nm excitation, respectively, although the differences in peak position are not as large as those seen for the bleaching of the ground state spectrum (Figure 8A). In the 740-880 nm spectral region at 20 K, Figures 6B and 7B reveal that at times longer than 0.5 ps a significant bleaching occurs at 803 nm and an absorption increase occurs at 790 nm.

Excitation Wavelength Dependence of Reaction Centers

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Figure 4. Time-resolved absorbance change spectra of R-26 reaction centers at 20 K. Conditions are as described in Figure 3 except for the use of a 57 cm-1 wide (FWHM) 893 nm excitation.

Figure 5. Time-resolved absorbance change spectra of R-26 reaction centers at 20 K. Conditions are as described in Figure 3 except for the use of a 57 cm-1 wide (FWHM) 903 nm excitation.

These changes have previously been assigned to an electrochromic shift of the monomer bacteriochlorophylls upon formation of the P+HA- state (e.g., ref 5). In Figure 7B (903 nm excitation), the absorbance decrease between 800 and 825 nm is resolved into two components, although it is not clear whether these two components are two resolved absorbance decreases at 803 and 815 nm or a broad absorbance decrease with a narrow absorbance increase at 810 nm (see the Discussion). This resolved structure in the spectrum is not observed when 858 nm excitation is used (Figure 6B), although the bleaching centered at 803 nm appears to have a shoulder on the lowenergy side. With both excitation wavelengths, an absorbance decrease near 760 nm grows in during the course of the reaction, presumably from bleaching of the QY band of the A-side bacteriopheophytin upon formation of P+HA-.5,10 Kinetics of Charge Separation. The room temperature measurements of the excitation wavelength dependence of stimulated emission decay reported previously showed that, to within the signal-to-noise ratio of the data, there was little or no difference in the time course of the excited state decay between different excitation wavelengths, at least during the first 8 ps.25 Figure 9 compares the decay of the stimulated emission at 920 nm for 858, 893, and 903 nm excitation at 20 K. Time zero in these traces was set by aligning the absorbance decrease at the wavelength of excitation, after correction of the spectra for dispersion effects (see ref 25). As was true at room temperature, the early-time decay kinetics of the stimulated emission are very similar at each excitation wavelength. The signal-to-noise ratio of this data was not sufficient to resolve the longer 10-15 ps component of the stimulated emission decay, and further experiments are being performed to determine if the kinetics or relative amplitude of that component depend on excitation wavelength. The time lag before the onset of the stimulated emission decay in Figure 9 is excitation wavelength

dependent. At 920 nm, the higher energy excitation results in faster onset of the stimulated emission rise and decay than do lower energy excitation wavelengths. This is consistent with Figures 3-5 which show that the kinetic behavior of the stimulated emission during the first picosecond is quite wavelength dependent. For example, using 858 nm excitation, the 250 fs spectrum in the 890-970 nm region actually crosses the 750 fs spectrum at about 930 nm (in other words, in Figure 3A there is no net change between the stimulated emissions at 250 and 750 fs at 930 nm even though on either side of this wavelength there are either net positive or net negative changes). Similarly, the 750 fs and 1.0 ps spectra cross at about 940 nm (Figure 3B). Such features have been seen previously and interpreted in terms of coherent oscillations.28 It is clear from the work described in this report that these kinetic and spectral features are excitation wavelength dependent (see the Discussion). Excitation wavelength dependence of absorbance change kinetics is much more obvious in the 800 nm region. This is apparent from the time course of the absorbance changes at 20 K shown in Figures 6 and 7. As is evident in Figure 10, significant kinetic variation is even seen at room temperature. Figure 10 shows the room temperature kinetics at 790 nm (the amplitudes of the different kinetic curves were normalized using the 4.0 ps peak absorbance decrease in the QY band of P). This is a wavelength that has been used in the past to exemplify the complex kinetics of charge separation in the reaction center (e.g., ref 29). In general, an instantaneous absorbance increase at this wavelength is followed by a fast (roughly 1 ps) absorbance decrease and then a slow (3 ps) absorbance increase. The relative amount of the fast decrease is clearly excitation wavelength dependent, being generally more pronounced at longer wavelengths than at shorter wavelengths. The largest fast kinetic feature is seen with 878 nm excitation. Using 893

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Figure 6. Time-resolved absorbance change spectra of R-26 reaction centers at 20 K. Conditions are as described in Figure 3 except for the use of a 64 cm-1 wide (FWHM) 858 nm excitation.

Figure 7. Time-resolved absorbance change spectra of R-26 reaction centers at 20 K. Conditions are as described in Figure 3 except for the use of a 57 cm-1 wide (FWHM) 903 nm excitation.

nm excitation, the fast component is still larger than with 840 or 858 nm excitation, but the magnitude of the underlying longtime absorbance change at 790 nm decreases.

Spectral Heterogeneity of the Ground State. The persistent excitation wavelength dependence of the bleaching of the QY band of P (Figure 8A) is similar to the transient hole-burning results at room temperature,25 except that the spectral shift between the lowest and highest energy excitation is more pronounced at 20 K: 200 cm-1 at 20 K vs 80 cm-1 at room temperature. The 20 K results are comparable to the continuous, low-temperature hole-burning results of Lyle et al.22 As discussed in these previous hole-burning studies, the excitation wavelength dependence of the bleaching of P presumably arises from the photoselection of a subset of the ensemble of conformation states which make up the ground state population. Lyle et al. calculate the width of this distribution of ground state transition energies to be 150 cm-1.22 The energy difference between the central point of the bleaching at 4.0 ps using 858 nm excitation and using 903 nm excitation (see Figure 8A) is roughly 200 cm-1. While it is difficult to make a direct comparison between this value and the 150 cm-1 value obtained by Lyle et al.,22 they are clearly of the same magnitude. Early-Time Ground State Evolution. The greater excitation wavelength dependence of the bleaching spectrum of P after charge separation at 20 K vs that at room temperature is probably due to a significantly slower rate of conformational interconversion at 20 K. In the 825-965 nm region at 20 K (Figures 3-5), it appears that between 0.5 and 4.0 ps the dominant spectral change results from the decay of the stimulated emission. Only the long-wavelength side of the overall absorbance decrease in this region evolves during this time. This is in contrast to the observed spectral evolution in the QY band of P during early times at room temperature.25 In the room temperature study, it was estimated that initially after excitation there was a 160 cm-1 difference in the maximum of the bleaching using 840 vs 893 nm excitation. By 5 ps, this

Discussion The transient hole-burning experiments on R-26 reaction centers reported above and previously25 demonstrate that during charge separation the transient absorption change spectra in the 740-970 nm region are excitation wavelength dependent at both room temperature and 20 K. At 20 K, the excitation wavelength dependence of the 740-970 nm spectral changes persist well after charge separation is complete (see the 3.0-4.0 ps spectra of Figures 3-7). Measurements out to 100 ps in the QY band of P show that the strongly excitation wavelength dependent spectral changes persist on that time scale as well at 20 K. In contrast, at room temperature, the excitation wavelength dependence becomes increasingly less pronounced at times longer than 0.75 ps following excitation, leaving only small differences in the position of the absorbance decrease associated with the QY band of P at longer times (Figures 1 and 2; ref 25), presumably due to increased conformational subpopulation interconversion at the higher temperature. The wavelength-dependent spectral evolution at room temperature was explained previously by suggesting that the use of a temporally short, spectrally narrow excitation pulse results in a photoselective depletion of a subpopulation of conformations present in the ground state of P (a narrow transient hole) as well as selective population of the excited state. Subsequent spectral evolution arises from conformation relaxation and dephasing.25 A similar model can be used to explain the lowtemperature results reported here by incorporating a decreased rate of conformational interconversion at 20 K.

Excitation Wavelength Dependence of Reaction Centers

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Figure 10. Comparison of the recovery of the absorbance decrease at 790 nm for the four excitation wavelengths 840, 858, 878, and 893 nm at room temperature. These kinetic traces represent a single slice from a time vs wavelength absorbance change surface, normalized at the minimum wavelength of the QY bleaching of P at 4.0 ps.

Figure 8. (A) Comparison of the bleaching the P band 4.0 ps following excitation with 858, 893, and 903 nm pulses at 20 K. (B) Comparison of the spectrum of the stimulated emission from P* following excitation with 858, 893, and 903 nm excitation at 20 K. The spectra in panel B were generated by subtracting the 0.75 ps absorption change spectra in Figures 4, 5, or 6 for 858, 893, or 903 nm excitation, respectively, from the 4.0 ps spectra in panel A. This was done after normalizing all data sets at the minimum wavelength of the QY bleaching of P at 4.0 ps.

Figure 9. Comparison of the recovery of the absorbance decrease due to stimulated emission at 920 nm for the three excitation wavelengths 858, 893, and 903 nm at 20 K. The three curves were normalized at the minimum wavelength of the QY bleaching of P at 4.0 ps. These kinetic traces represent a single slice from a time vs wavelength absorbance change surface.

difference had decreased to 80 cm-1. The lack of a corresponding change in the ground state bleaching of the QY band of P on the picosecond time scale at 20 K is again consistent with the expected decrease in conformational interconversion at 20 vs 295 K. Although there is no large-scale conformational interconversion at 20 K, vibrational relaxation and dephasing do occur. At

very early times (0 ps in Figures 3A, 4A, and 5A), additional resolved spectral structure is observed to either side of the narrow central bleaching. This is most clearly seen when 903 nm excitation is used (Figure 5A). During the first two hundred femtoseconds following excitation, the overall width of the absorbance decrease between 825 and 970 nm increases and this additional structure disappears. Brito Cruz et al. have demonstrated that during the time that pump and probe beams are overlapped, a suppression of the inhomogeneous broadening occurs which can lead to the observation of the vibronic substructure of an absorption band.30 This explanation is consistent with the observed spectral evolution and suggests that the additional structure observed is vibronic. The recovery of the inhomogeneous broadening is due to relaxation and dephasing of the vibrational modes coupled to the P f P* transition. Lyle et al. report the observation of vibronic sidebands which build upon the zero phonon mode with a frequency separation of approximately 120 cm-1.22 Spectral features are present in Figures 3A, 4A, and 5A roughly 120 cm-1 to either side of the excitation wavelength, consistent with the notion that these represent vibronic bands. This assignment is also consistent with the observation by Vos et al. of coherent oscillation with a similar frequency.31 Structural features can also be observed farther from the excitation wavelength. The most obvious of these is the feature seen near 880 nm when 903 nm excitation is used. This corresponds to a roughly 300 cm-1 energy difference, larger than would be expected for a higher harmonic of the vibronic band. A similar feature can be seen roughly 300 cm-1 to higher energy when 858 nm excitation is used. This may represent a higher frequency vibrational mode. Ground State Absorbance of P in the 800 nm Region. The QY band of P is thought to be only one of several transitions which contain significant involvement of the special pair.32,33 If one uses a simple exciton coupling picture for the dominant interaction between the two bacteriochlorophylls of P, one predicts that a higher energy band of P should be present in the 800 nm region.34 Reddy et al. have concluded, using nonphotochemical hole-burning spectroscopy, that the upper exciton transition has sufficient oscillator strength to be visible in the absorption spectrum of Rps. Viridis and that the special pair contributes a measurable amount of oscillator strength to the BA and BB transitions.23 In their hole-burning experiments, they demonstrated that the position of a bleaching which they assigned to the upper exciton band was correlated with the burn wavelength in the lower exciton band. In Rb. sphaeroides,

14234 J. Phys. Chem., Vol. 100, No. 33, 1996 spectral congestion near 800 nm makes assignment of the bands in this region more difficult. At room temperature, it is difficult to say whether the observed early-time excitation wavelength dependence of the spectral evolution between 780 and 820 nm could be due in part to involvement of the upper exciton band of P (Figures 1 and 2). At 20 K, the excitation wavelength dependence of the spectra is more pronounced and persists for many picoseconds (Figures 6 and 7). Here, the largest difference in the spectral evolution between 858 and 903 nm excitation is in the 805 nm region. However, it is difficult to explain this difference simply in terms of a shift in the position of the upper exciton band of P with excitation wavelength. There appears to be a more complex interaction involving other transitions in this region. The possibility of substantial coupling between B* and P* is suggested by the extraordinary rate of energy transfer between them.35-38 Heterogeneity in the Excited Singlet State of P. The excitation wavelength dependence of the stimulated emission spectra in Figure 8B demonstrates that the nuclear conformations which affect the energy of the P to P* transition at low temperature also affect the energy of the stimulated P* to P transition. The peak differences between the lowest and highest energy excitation wavelengths was only about 120 cm-1 (about 10 nm, Figure 8B), roughly a factor of 2 smaller than the differences between the minima of the long-lived ground state bleaching at the same two excitation wavelengths (Figure 8A). This is consistent with the fact that at room temperature one observes much less excitation wavelength dependence of the stimulated emission spectrum than of the ground state bleaching of P.25 In fact, at room temperature there was no discernible spectral shift of the stimulated emission with excitation wavelength. The results of Figure 8B are the first direct measurement of an excitation wavelength dependence of the stimulated P* to P transition energy. The fact that the distribution of the stimulated emission energies resulting from photoselective excitation is smaller than the distribution of the ground state transition energies implies that some decoupling occurs on a very fast time scale between the modes controlling the excitation energy and those controlling excited to ground state transitions. The solvation of P* by the protein most likely leads to this decoupling of the vibrational modes and the greater spectral evolution of the excited state. As one might expect, this solvation effect apparently occurs much more quickly at room temperature than at 20 K, resulting in a long-lasting excitation wavelength dependence of the stimulated emission spectrum at low temperature. Excited state evolution has been discussed previously in the context of longer time scale changes in reaction center mutants.39,40 Dynamics of Excited State Decay and Charge Separation. One of the more surprising conclusions which is drawn from the room temperature and low-temperature transient holeburning studies is that while the spectrum of the initial and final states of the system are substantially excitation wavelength dependent (Figures 1-3 and 6-7), particularly at low temperature, there is only a very minor effect on the several picosecond time scale kinetics of stimulated emission decay (Figure 9). The bulk rate of charge separation is apparently not sensitive to the same conformational changes that strongly affect the P to P* transition energies. This was suggested from the room temperature transient hole-burning work,25 but is more evident at low temperature where the P* spectrum is clearly excitation wavelength dependent on the time scale of electron transfer. A similar conclusion was drawn from continuous hole-burning measurements.23 Since excitation with different wavelengths

Peloquin et al. should also affect the vibrational energy available before relaxation, these results also suggest that the vibrational modes coupled to the P to P* transition which apparently do not dephase and relax before electron transfer occurs41 are not the modes most strongly coupled to the electron transfer reaction itself. A similar conclusion has been reached from hole-burning data previously.42 The kinetic differences that are observed in the stimulated emission decay are almost entirely within the first 500 fs at 20 K and take the form of variable delays before the onset of the decay itself (Figure 9). These changes could be due to the kinetic complications that arise from vibrational coherence. The coherence effects are seen much more clearly using impulsive excitation.28 There, one can observe clear oscillatory movement of the stimulated emission band with time. Although the pulses used in this study were too long to be truly impulsive, some of this effect can be seen by looking at the transient absorbance change spectra as a function of time in the 900-960 nm region (see, for example, Figure 3A). One can see that during the first 0.75 ps the spectra in this region intersect, indicating that at early times the stimulated emission spectrum extended more to the red and then moved to higher energy with time. It is also possible to explain the apparent lag in the stimulated emission decay using 858 nm excitation in terms of local heating (see below). The kinetics of charge separation can also be assayed by looking in the 750-820 nm region where the monomer bacteriochlorophylls and bacteriopheophytins absorb. Throughout much of this region, the room temperature absorbance change kinetics are complex, consisting of exponential decay components with lifetimes between 0.9 and 3.5 ps (for reviews, see refs 2, 5-10). The complexity of the absorption change kinetics in this region is exemplified by the 790 nm traces shown in Figure 10. At this wavelength, one can see that the signal starts positive, drops with a time constant of roughly a picosecond, and then slowly rises with a roughly 3 ps time constant. These complex kinetics have been used as evidence for intermediate states between P* and P+HA-, such as P+BA-.29 What Figure 10 shows is that the kinetics of the absorbance changes (particularly the amount of the 1 ps component) in this region are quite excitation wavelength dependent. This observation suggests either that the dominant source of kinetic complexity in the 780-820 nm spectral region is ground and excited state evolution or that the intermediate population of P+B- or other charge-separated intermediates is excitation wavelength dependent. Excitation Wavelength and Local Temperature of P. A more careful comparison of low-temperature and room temperature spectral evolution as a function of excitation energy lends insight into the process of vibrational energy transfer from the local environment of P into the surrounding bath. Aside from the sharpening of some spectral bands at long times and the increased overall rate of charge separation, the lowtemperature spectral evolution at 858 nm is qualitatively similar at early times to that at room temperature. Both show an initial absorbance increase in the 800-810 nm region followed by the decrease in this region associated with charge separation. Both also show a broad absorbance decrease after 0.5 ps in the QY band of P. Excitation at 903 nm, on the other hand, immediately results in a spectral valley in the 800-805 nm region (though there is an instantaneous underlying broad absorbance increase) which becomes monotonically more pronounced with time. In addition, the spectrum of the P band bleaching remains narrow after 0.5 ps. Exciting at 858 nm may effectively raise the local temperature of P, resulting in a

Excitation Wavelength Dependence of Reaction Centers vibrationally hot initial donor. The transiently higher local temperature at 858 nm allows some rapid conformational interconversion to take place, resulting in a broadening and shifting of the bleaching reminiscent of the results at 295 K.25 A very interesting question is whether P remains vibrationally hot throughout the electron transfer reaction. If excess energy remained in the vibrational modes coupled to the initial electron transfer on the several picosecond time scale, it could explain the weak effect of both ambient temperature and P/P+ redox potential on the electron transfer rate.10,14,20,43-45 Conclusions. The major findings of this work are (1) it is possible to photoselect spectroscopically different subpopulations of reaction centers in both the ground and excited states by varying the excitation wavelength within the QY band of P, (2) at room temperature, these subpopulations interconvert on the picosecond and subpicosecond time scale whereas at 20 K they are largely static, (3) the photoselectable reaction center conformations all have approximately the same charge separation rate implying that the degrees of freedom which connect these subpopulations are not strongly coupled to the electron transfer reaction, (4) the complex kinetics in the 780-820 nm region are strongly excitation wavelength dependent, suggesting that either spectral evolution in the ground and excited states due to vibrational relaxation and conformation interconversion is the source of much of the kinetic complexity in this spectral region or that the population dynamics of the charge separation intermediates is dependent on the energy of excitation, and (5) local heating of P during vibrational relaxation upon excitation above the 0-0 transition energy appears to increase the distribution of conformations explored by the system on the subpicosecond time scale. It is not clear whether the system remains vibrationally hot on longer time scales. These results point out the importance of considering the effects of ground and excited state evolution and local heating in the interpretation of ultrafast spectral evolution in the reaction center. Acknowledgment. The authors thank K. Carty for preparation of reaction center samples. We also thank Drs. J. Williams and J. Allen for helpful discussions. This work was supported by Grants DMB91-58251 and MCB 9219378 from the National Science Foundation. Instrumentation was purchased with funds from NSF Grant DIR-8804992 and Department of Energy Grants DE-FG-05-88-ER75443 and DE-FG-05-87-ER75361. This is publication 296 from the Arizona State University Center for the Study of Early Events in Photosynthesis. References and Notes (1) Allen, J. P.; Feher, G.; Yeates, T. O.; Komiya, H.; Rees, D. C. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5730. (2) Feher, G.; Allen, J. P.; Okamura, M. Y.; Rees, D. C. Nature 1989, 339, 111. (3) Chang, C.-H.; El-Kabbani, O.; Tiede, D.; Norris, J.; Schiffer, M. Biochemistry 1991, 30, 5352. (4) Ermler, U.; Fritzsch, G.; Buchanan, S. K.; Michel, H. Structure 1994, 2, 925. (5) Kirmaier, C.; Holten, D. Photosynth. Res. 1987, 13, 225. (6) Parson, W. W. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, FL, 1991; p 1153. (7) Martin, J.-L.; Vos, M. H. Ann. ReV. Biophys. Biomol. Struct. 1992, 21, 199. (8) Kirmaier, C.; Holten, D. In The Photosynthetic Reaction Center; Deisenhofer, J., Norris, J. R., Eds.; Academic Press: San Diego, CA, 1993; Vol. II, p 49. (9) Zinth, W.; Kaiser, W. In The Photosynthetic Reaction Center; Deisenhofer, J., Norris, J. R., Eds.; Academic Press: San Diego, CA, 1993; Vol. II, p 71.

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