J. Phys. Chem. 1995,99, 1349-1356
1349
Excitation Wavelength Dependence of Bacterial Reaction Center Photochemistry. 1. Ground State and Excited State Evolution? 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 85287-1 604 Received: August 16, 1994; In Final Form: November 3, 1994@ The effect of excitation wavelength on the ground state absorption, excited state stimulated emission, and the electron transfer process in reaction centers from the R-26 carotenoidless strain of the bacterium Rhodobacter sphaeroides was studied using time-resolved hole-burning spectroscopy. The P* state was prepared using 838, 858, 878, and 892 nm excitation pulses which had a temporal width of approximately 150 fs and a spectral width of about 60 cm-'. At early time, the bleaching of the Qy band of P is centered near the excitation wavelength and significantly narrowed relative to its width at long time. Within 1 ps, this bleaching broadens to nearly the entire width of the ground state band. However, even after 5 ps the wavelength of maximum absorbance decrease in this spectral region remains excitation wavelength dependent, indicating P* transition energies in the ground state population that there exists a roughly 80 cm-' distribution of P on the time scale of charge separation. The majority of the stimulated emission from P* moves to wavelengths greater than 890 nm, within the first 200 fs following excitation. Neither the spectrum of the stimulated emission, whose maximum is at 905 nm, nor its roughly 3.5 ps decay kinetics is significantly dependent on the excitation wavelength after the first 500 fs following excitation. This excitation wavelength insensitivity implies that the overall electron transfer rate from P* to P'HA- is largely independent of the manner in which P* is prepared at room temperature. If conformational subpopulations do exist in the excited state with different rates of electron transfer, they appear to be distinct from the conformational subpopulations in P* transition energies. the ground state which give rise to the distribution of P
-
-
-
Introduction When light is absorbed by the bacterial photosynthetic reaction center, excitation energy is transferred to the lowest excited singlet state (P*) of a dimer of bacteriochlorophylls (P) which serves as the primary electron donor in the reactions that follow (for reviews see refs 1-7). At room temperature, this state decays to the charge-separated state P+HA- (possibly via the state P+BA-) with an overall time constant of about 3.5 ps. HA is a bacteriopheophytin located on one of two symmetryrelated branches of reaction center cofactors, and BA is a bacteriochlorophyll situated roughly between P and Studies of both the spontaneous emission decay from P* and the absorbance changes associated with charge separation have shown that the kinetics of charge separation is complex, requiring multiple exponential decay components for an accurate One possible model of the complex early time kinetics associated with electron transfer proposes that there is a static distribution of protein conformations which have different rates of electron t r a n ~ f e r . ' ~The ~ ~ existence ~~'~ of a distribution of protein conformations around the initial electron donor has been implied by steady state nonphotochemical and photochemical hole burning experiments at 4.2 K on the QY band of P in Rhodopseudomonas viridis and Rhodobacter sphaeroides.20-22 These experiments have been interpreted in terms of a 140 cm-' inhomogeneous contribution to the line width of this band arising from different 0-0 energies of the P t 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 no. 224 from the Arizona State University Center for the Study of Early Events in Photosynthesis. The Center is funded by DOE Grant DE-FG02-88-ER13969 as a part of the USDA/DOE/NSF Plant Science Centers Program. * Corresponding author. @Abstractpublished in Advance ACS Abstracts, January 1, 1995.
0022-3~~54/95/20991349$09.OO/O
P* transition in different conformations. This distribution is, however, not broad enough to explain the complexity of the electron transfer kinetics.20 These experiments also indicated that the rate of electron transfer was independent of the specific excitation wavelength within the QY band of P at cryogenic temperature. As yet there has been no room temperature estimate of the degree of static inhomogeneity of the Qy transition of P on the time scale of electron transfer nor of the effect of different excitation wavelengths on the rate of electron transfer or spectral evolution of the excited state. Another issue relevant to the early time kinetics in reaction centers is the nature of the vibrational modes coupled to the P to P* transition. Hole burning measurements have implied that the QY band of P is broadened in part by a progression of vibrational bands including displaced low-frequency modes.22-26 These measurements have been interpreted in terms of lowfrequency phonons centered at approximately 30 and 120 cm-' which are strongly coupled to the P to P* transition.20,22Lowfrequency modes have also been observed in resonance Raman experiments including a prominent mode near 120 ~ m - ' . *The ~ dynamic effects of the strongly coupled 30 and 120 cm-' modes have recently been observed in transient absorbance change measurements and fluorescence decay m e a ~ u r e m e n t s . ~Vos ~~~~-~~ et al.30-32used 40 fs pulses to impulsively prepare the P* state and observed oscillations in the stimulated emission signal from P* as a function of time. They proposed a model in which, following excitation, the system moves coherently along the vibrational coordinates strongly coupled to the P to P* transition. Fourier transform analysis of the modulations reveals a number of vibrational modes with the dominant modes having frequencies near 30 and 120 cm-'. (The exact values depend on the sample and conditions.) The coherent motion of the wave packet does not dephase until more than a picosecond after excitation. The oscillatory nature of the stimulated emission spectrum at early times suggests that, in functional reaction
0 1995 American Chemical Society
1350 J. Phys. Chem., Vol. 99, No. 4, 1995 centers, the P* state requires at least a picosecond to attain thermal equilibrium, a time comparable to the rate of electron transfer at low temperature.' This implies that a significant portion of the electron transfer process at low temperature occurs from a nonthermalized P* state.30 At room temperature the situation is less clear, since the 1-2 ps duration of the spectral oscillation^^^^^^ represents a lower limit to the dephasing and thermalization time of the excited state, and the bulk of the electron transfer takes places on the several picosecond time scale. If dephasing and vibrational relaxation of modes coupled to P* formation and charge separation take place on a time scale similar to electron transfer, then one would expect that the spectral evolution of the system during the electron transfer reaction would be dependent on the energy, spectral bandwidth, and duration of the excitation pulse. In this report, we present transient absorption measurements of the room temperature photochemistry which is initiated by 150 fs, narrow spectral bandwidth (3-5 nm full width at halfmaximum) excitation pulses at several wavelengths covering the QY band of P. Detailed absorption change spectra were monitored throughout the same spectral region, including the region of excitation. In this way, it was possible to monitor the spectral evolution of both the ground and excited states of the reaction center as a function of time and as a function of excitation wavelength on the subpicosecond and picosecond time scales. This has allowed both an evaluation of the excitation wavelength dependence of the excited state evolution and an estimate of the static heterogeneity in the ground state on the time scale of electron transfer at physiological temperatures.
Materials and Methods All experiments were performed at room temperature on reaction centers isolated from photoheterotrophically grown Rhodobacter sphaeroides strain R-26 bacteria.34 The reaction centers were suspended in a buffer of 50 mM Tris-HC1 (pH K O ) , 0.025% LDAO, 1 mM EDTA, and 0.5 mM terbutryn. The sample cell was a spinning wheel with a path length of about 3 The absorbance at 800 nm of the sample in the cell was about 1.2- 1.4. A pump-probe transient absorption a p ~ a r a t u s ' was ~ , ~ utilized ~ to measure the spectral changes in the stimulated emission. The short laser pulses were generated by a dye laser synchronously pumped by a frequency-doubled, mode-locked Nd:YAG laser (Spectra Physics). Both the Nd:YAG laser pulse train and the dye laser pulse train were compressed by fiber pulse compressors (Spectra Physics). The final pulse output was a 590 nm, 80 fs, 0.5 nJ pulse. This pulse was amplified in a three-stage pulse amplifier pumped by a frequency-doubled (532 nm) 540 Hz repetition rate, 10 mJ regeneratively amplified (Continuum) Nd:YAG pulse seeded by the mode-locked ND:YAG laser. The output of the dye amplifier was roughly a 200 pJ, 590 nm pulse with a pulse duration of approximately 150 fs, which was split into two equal parts. Each half of the pulse was used to generate a continuum. One of these continuum pulses was split again and used as the sample and reference probe beams. The other half was sent through an interference filter (either 838, 858, 878, or 892 nm) with a spectral bandwidth of 3-5 nm and reamplified in a prism amplifier stage (Santa Ana Lasers) pumped with 2 mJ of 532 nm light from the 540 Hz regenerative Nd:YAG amplifier (Continuum). The dye used for reamplification was LDS867 (Exciton). The resulting approximately 10 pJ pulses were passed through a polarizer and a half-wave plate to define the polarization and then focused to a 1-2 mm diameter spot on the sample. The final excitation pulses were roughly 5 pJ. For all experiments reported, the relative polarization of the pulse and the probe beams was at the magic
Peloquin et al.
838 nm excitation 0.00
-0.03 -0.06 Q,
-0.09
M
' 9
a2
-0.12
-0.16
0
9
0.00
g
-0.09
a &
a Q
-0.08 -0.08
-0.12 -0.16
826
850
876
800
926
960
Wavelength (nm) u
0.00
M fl - 0 . 0 3
cd
6
-0.06
0)
-0.08
e
-0.12
0
m
a 4
-0.16 ~
-1
0
1
2
3
4
6
6
Time (pa) Figure 1. Time-resolved absorbance change spectra of R-26 reaction centers at times t 0.5 ps (panel A) and times t > 0.5 ps (panel B) after excitation with 55 cm-' wide (full width at half-maximum) 838 nm excitation pulses. These spectra represent single time slices from a surface of absorption changes. A total of 100 spectra were taken at 67 fs intervals. The data have been corrected for dispersion as desnibed in the text. These spectra and the corresponding spectra in Figures 2-4 have all been normalized so that the absorbance change at 850 nm and 5 ps is the same in each figure. The curve in panel A with diamonds is the spectrum of the excitation pulse. Panel C shows kinetic traces of the absorption change at selected probe wavelengths which have been normalized so that each trace has the same maximum bleaching.
angle. After collection all spectra were corrected for any temporal dispersion of the probe pulses, as described in the Results section. The spectrum of the excitation pulses was determined by sending a portion of the excitation pulse along the path of one of the probe beams.
Results The early time transient absorbance change over the spectral range between 825 and 965 nm was studied as a function of excitation wavelength in R-26 reaction centers from Rhodobacter sphaeroides. Difference spectra were taken at 67 fs intervals over a 6 ps time scale. Figures 1-4 summarize the results of these studies using narrow bandwidth (3-5 nm full width at half-maximum), 150 fs excitation pulses at 838 nm
Excitation Wavelength Dependence of Reaction Centers
J. Phys. Chem., Vol. 99, No. 4, 1995 1351
868 n m excitation 0.00
-
-0.03
-
878 n m excitation I
0.00 -0.03
-0.06
-0.06
-0.09 .
P)
P)
M
's 9
' 9
-0.12 -0.15 :
Ll
Y
0
e a .e
-0.09
BD
-0.12
-0.15
0,
I
___--___--.
0.00 -0.03
-
-0.06
-0.09 -
0.00 -0.03
a .e
-0.06
-0.12
i
1,
-0.15
9
e
-0.09
.
-0.12
0
825
860
876
925
900
950
-0.15 825
850
Wavelength (nm) 0 hD
875
900
925
950
Wavelength (nm)
0.00
aa
-0.03
f!
-0.06
Q
0
9
-O.O9
f0
-0.12
f
-0.15
m
-
1
0
1
2
3
4
6
8
Time (pa) Figure 2. Time-resolved absorbance change spectra and kinetics of R-26 reaction centers. Conditions as described in Figure 1 except using 67 cm-' wide (full width at half-maximum), 858 nm excitation.
(Figure 1), 858 nm (Figure 2), 878 nm (Figure 3), and 892 nm (Figure 4). Each of these wavelengths results almost exclusively in direct excitation of the initial electron donor. The curve with diamonds in each of Figures 1-4 is the spectrum of the excitation pulse. For ease of comparison the absorption change spectra in Figures 1-4 are normalized such that the absorption at 850 nm 5 ps following excitation is the same for each of the four excitation energies. Narrow Absorption Decrease at the Excitation Wavelength. In each case, a narrow absorption decrease is observed at early times (less than 0.5 ps) when the pump and probe pulses are temporally overlapped (Figures 1-4). This feature is centered at the wavelength of excitation and has a spectral width roughly equivalent to that of the excitation pulse. Careful observation of the spectral features surrounding this narrow absorption decrease reveals local structure in the spectrum. Sidebands are evident 5-10 nm to either side of the central absorption decrease, particularly at very early times. Comparison of the absorption change spectra with the spectrum of the excitation pulse shows that there is a strong excitation wavelength dependence of the absorption change spectra even at wavelengths well beyond the spectral band of the excitation
2 3 4 6 e Time (pa) Figure 3. Time-resolved absorbance change spectra and kinetics of R-26 reaction centers. Conditions as described in Figure 1 except using 64 cm-' wide (full width at half-maximum), 878 nm excitation. pulses. In all cases, the most narrow feature of the absorption change spectrum has essentially disappeared within 0.5 ps after excitation. Spectral Evolution. During the initial 0.5 ps, the absorption change spectrum (see Figures lA, 2A, 3A, and 4A) evolves from a narrow absorbance decrease centered near the excitation wavelength to a much broader bleaching centered near 865 nm. (The exact peak position depends on the excitation wavelength; see below.) For example, using 838 nm excitation (Figure lA), one can see a dramatic evolution in the shape of the overall absorption change spectrum toward longer wavelengths between spectra taken near zero time and the spectrum at 0.5 ps. Even ignoring the narrow spectral feature within the region of excitation, the peak of the broader spectral band moves from 848 nm near zero time to 864 nm at 0.5 ps. The lag in the absorption decrease at wavelengths distant from the excitation wavelength is shown most clearly in Figure lC, which compares the kinetics of the absorption changes at a number of wavelengths. The absorption decrease observed at 838 nm (the peak of the excitation pulse spectrum) and at 850 nm (outside the spectral region of the excitation pulse) occur substantially (100150 fs) before the absorption decreases at longer or shorter wavelengths.
-1
0
1
1352 J. Phys. Chem., Vol. 99, No. 4, 1995 892
Peloquin et al.
nm excitation
..............
._.. ........
0.00 1
I
0.00
-0.03
-0.03
-0.06
-0.06 Q
-0.09
M
!-0.12
-0.09
M
9
-0.12
f
-0.16
f
e, -0.16 0
a
0
9
0.00
tg -0.03
a rl
a -0.06
-0.06
-0.12
-0.12
-0.16
B 826
-0.16
860
626
876
900
926
0.00
960
880 n m
-0.03
.......... 9 2 0 n m
d
.-..
....
....
-0.06
I
I -1
0
1
2
3
4
6
876
900
926
960
Figure 5. Comparison of the absorption change spectrum 1 ps (panel A) and 5 ps (panel B) after excitation at the four excitation wavelengths. The absorption change spectrum of R-26 reaction centers due to steadystate illumination (S.S.) is shown for comparison. The time-resolved spectra are normalized as in Figure 1.
830 n m
M
860
Wavelength (nm)
Wavelength (nm)
9
O.OO -0.03
-0.09
-0.09
Q
............
i!
0,
0
Time (pa) Figure 4. Time-resolved absorbance change spectra and kinetics of R-26 reaction centers. Conditions as described in Figure 1 except using 57 cm-' wide (full width at half-maximum), 892 nm excitation.
Using 892 nm excitation, one sees similar phenomena (Figure 4). At zero time, there is a narrow bleaching at 892 nm. By 0.5 ps, the narrow feature has essentially disappeared, but the maximum of the bleaching is at 874 nm, which is 160 cm-' to lower energy than the 0.5 ps spectrum using 838 nm excitation (Figure 1A). Figure 4C shows the kinetics of the absorption changes at a number of wavelengths following 892 nm excitation. Again, it can be seen that the bleaching near the excitation wavelength precedes the bleaching at other wavelengths. The lag between the bleaching at 892 and 830 nm is approximately 300 fs, and the lag between 892 and 920 nm is about 100 fs (Figure 4C). Figures 2A and 3A show the early time evolution of the absorption change spectrum following 858 and 878 nm excitation, respectively. In both cases, the initial narrow bleaching centered at the excitation wavelength broadens into a spectrum by 0.5 ps which is similar to that using other excitation wavelengths, though spectrally shifted. The maximum of the bleaching at 0.5 ps is 864 and 870 nm with 858 and 878 nm excitation, respectively. Figures 2C and 3C show the kinetics of the absorption changes at a number of wavelengths following 858 and 878 nm excitation, respectively. Again one observes
a lag between the onset of the absorption decrease near the excitation wavelength and that at wavelengths farther away. Excitation Wavelength Dependence of Long-Lived Absorbance Changes. Figure 5 compares the absorption change spectra 1 and 5 ps after excitation for each of the four excitation wavelengths. As was observed at 0.5 ps (Figures lA, 2A, 3A, and 4A), the wavelength of the maximum absorbance decrease shifts to lower energy with increasing wavelength of excitation. However, at both 1 and 5 ps the excitation wavelength dependence of the spectral changes is less pronounced than at 0.5 ps. The position of the maximum absorbance decrease at 1 ps is 864,864,868, and 870 nm and at 5 ps is 858,862,864, and 864 nm for 838, 858, 878, and 892 nm excitation, respectively. Comparing the 5 ps absorption change spectra using different excitation wavelengths, one can also see that the width of the four absorbance change spectra in Figure 5B is slightly different. The absorbance decrease due to 838 nm excitation is slightly wider than that using longer excitation wavelengths. The overall shift to shorter wavelengths of the bleaching between 1 and 5 ps results from the loss of stimulated emission from P*. The magnitude of the apparent blue shift due to P* decay is similar for each of the different excitation wavelengths, suggesting that the majority of the spectral evolution is complete within 1 ps. Figure 6 compares the kinetics of the recovery of the bleaching at 920 nm for each of the four excitation energies. It can be seen that the rate of recovery is nearly identical in the four curves, though the magnitude relative to the 5 ps bleaching at 850 nm changes significantly with excitation wavelength. Thus, while changing the excitation wavelength significantly alters the spectral evolution of the system during the first picosecond after excitation, it does not appear to have dramatic effects on the kinetics of excited singlet state decay on the 1-5 ps time scale. Dispersion Corrections. Obviously, one artifact that can give rise to both spectral and kinetic distortion of transient
J. Phys. Chem., Vol. 99, No. 4, 1995 1353
Excitation Wavelength Dependence of Reaction Centers
I
I
- 1 ' 0
1
2
3
4
6
8
Time (pa) Figure 6. Comparison of the recovery of the absorbance decrease due to stimulated emission at 920 nm for the four excitation wavelengths. These kinetic traces represent a single slice from a time vs wavelength absorbance change surface. As in Figure 1, the absorbance change surfaces were normalized at 5 ps and 850 nm.
absorption data on subpicosecond time scales is wavelength dispersion. In most condensed media such as glass and water, longer wavelength photons travel faster, giving rise to a temporal lag in the appearance of features on the red end of the transient absorption spectrum relative to the blue end. Without correction either optically or experimentally, the dispersion in the 800900 nm spectral region would be about 2.7 fshm in the experimental system used for these measurements. Two approaches were taken to correct for dispersion. The data shown in Figures 1-4 were taken using optics not corrected for dispersion and then were corrected after data collection using a dispersion correction curve derived from looking at an instantaneous, spectrally very broad, roughly 100 fs duration transmittance change signal obtained by exciting benzonitrile with 590 nm pulses. Similar measurements have been made by monitoring the birefringence of CS2 with essentially the same results. For comparison, some of the data was retaken after correcting for the dispersion optically using a prism pair. This also yielded essentially the same spectral features. A comparison of these correction techniques has indicated that the corrected spectra of Figures 1-4 should have less than f 0 . 2 fshm dispersion. In any case, one would not expect to find a change in the direction of the dispersion effect with excitation wavelength. It is clear from comparing Figures 1 and 4 that at these two excitation wavelengths the spectra evolve in opposition directions.
Discussion The 0.0 ps spectrum in each of Figures 1-4 shows a narrow bleaching at the excitation wavelength accompanied by a broader absorption decrease in the surrounding spectral region. The source of the spectrally narrow bleaching observed during the time that the probe and pump pulses are overlapped is difficult to assign unambiguously. One obvious possibility is that this is due to a coherent artifact caused by interaction between the pump and probe pulses. One would expect such an artifact to be more pronounced when off-resonance excitation wavelengths are used.36 This does not appear to be the case (Figures 1-4). Also, the kinetic manifestation is not what one normally observes for such an artifact. This is particularly evident when 892 nm excitation is used. A pronounced narrow spectral feature is observed at this excitation wavelength (Figure 4A), but the rapidly forming and decaying spike expected during the pump/probe overlap in the kinetic trace (Figure 4B) is essentially absent. The narrow spectral features could be due to nonsta-
tionary ground state absorbance and excited state stimulated emission as described for the broader spectral features below. However, given the difficulty in assigning the source of this feature, the analysis below will concentrate on the broader spectral features observed. One important benefit of using spectrally narrow excitation pulses is that only the spectral region within about 5 nm of the central excitation wavelength would be significantly affected by any coherent artifact. (The spectra of the excitation pulses are shown in Figures lA, 2A, 3A, and 4A.) Thus, the analysis of spectral features outside of this range is essentially independent of any coherent artifact which may exist. At all excitation wavelengths, the difference spectra at times less than 1 ps have minima nearer to the excitation wavelength than do the spectra at 5 ps. In addition, the width of the bleaching is narrower at early times than in the 5 ps spectra. The absorbance decrease observed during the lifetime of P* is a combination of ground state bleaching, excited state absorbance, and stimulated emission. By 5 ps, primarily the ground state bleaching remains. However, the spectral broadening with time is just the opposite of what would result from loss of the stimulated emission with time, and thus it is very unlikely that the time-dependent spectral shifting and broadening is due to decay of the excited state during electron transfer. The spectral broadening is essentially complete by 1 ps, and at this time the spectral width of the absorbance decrease following excitation at any wavelength measured is roughly what has been obtained previously using spectrally broad excitation pulses.'~~This evolution of the width of the absorption loss at early times suggests that the preparation of P* with short, spectrally narrow pulses leads to the creation of nonstationary ground and excited states and that at least hundreds of femtoseconds is required for complete dephasing to occur. This is consistent with previous results using temporally shorter, spectrally broader excitation. 17,28-33 The width of an absorption or emission band arises from a combination of three source^.^^.^^ There is broadening due to the vibronic sublevels of the ground and excited states. Vibronic sidebands in the transient absorbance spectra occur at higher energies relative to the 0-0 transition energy and are offset by multiples of the frequency of the excited state vibrational modes strongly coupled to the transition. Likewise, emission sidebands will occur offset to lower energy from the 0-0 transition by multiples of the ground state vibrational frequencies. Raman spectroscopy, coherence sensitive absorption and fluorescence spectroscopy, and steady-state hole-burning experiments have shown that vibrational modes strongly coupled to the P P* transition occur at frequencies of approximately 30 and 120 cm-' in both the ground and excited states.20,22,27,31-33 Lifetime broadening, due to the uncertainty relationship between time and energy, will largely determine the width of the 0-0 transition and each of the vibronic sidebands for any particular reaction center in the population. At 4.2 K, steady state hole-burning spectroscopy has shown that for the QYband of P the lifetime broadening should be on the order of tens of wavenumbers. Such a width is consistent with a P* lifetime of about 1 ps at this temperature.20s21 Lastly, because the equilibrium position of the excited state is displaced relative to the ground the actual population distribution of reaction centers along vibrational coordinates of the ground and excited state contributes to the width of the absorption transition. The displacement creates a situation where the energy of the vertical transition between the ground and excited state surfaces is different for different points along the vibrational coordinate^.^^ The vibrational motion can be
-
1354 J. Phys. Chem., Vol. 99, No. 4, 1995
Peloquin et al.
provisionally partitioned into dimer modes (pseudolocalized Given the 150 fs duration of the excitation pulses used, over modes associated with P) and bath modes.20~21~37~38 The dimer 80% of the excited state should be formed by 0.13 ps (0 ps is modes are typically solute (i.e., P) centered, underdamped, and defined as the middle of the excitation pulse), but it is clear strongly coupled to the electronic transition and are the origin from Figure 4A that substantial evolution of the bleaching to of the vibronic sidebands. On the other hand, the bath modes wavelengths shorter than 878 nm occurs between 0.13 and 0.5 are solvent (Le., protein) centered and overdamped and colps. A similar evolution can be seen between 0.13 and 0.5 ps lectively give rise to a set of interconverting solvent configurawhen 838 nm excitation is used, but in the opposite direction tions which are commonly described in terms of a distribution (Figure 1A). This spectral evolution most likely arises from in the zero-point energy of the P* state and thus create a the interconversion of reaction center conformations via motion distribution of 0-0 transition energies. The broadening due to of the ground state along the bath modes of the protein. the dimer modes is usually termed the homogeneous broadening Comparison of Figures 1C and 4C shows that there is a much while that due to the bath modes is termed the inhomogeneous larger lag between the appearance of the bleaching at 892 and b r ~ a d e n i n g . ~Another ~ , ~ ~ important difference between the two 830 nm following 892 nm excitation, about 300 fs, than between types of vibrational motion is that the interconversion of reaction 838 and 830 nm following 838 nm excitation, about 150 fs. center conformations via the bath vibrational motion should be Because 892 nm excitation excites conformations with lower temperature dependent. Consequently, what is a dynamic transition energies than 838 nm excitation, it is expected that it perturbation of the zero-point energy at room temperature would take longer for the higher-energy side of the P band to becomes a static distribution of zero-point energies at cryogenic bleach following 892 nm excitation than following 838 nm temperatures. Nonphotochemical hole-burning experiments at excitation. Examination of the lag between the bleaching at low temperatures have shown that this distribution is about 140 the excitation wavelength and that at 830 nm in Figures 1-4 cm-l in the reaction centers from Rhodopseudomoms viridis.20 shows that the lag increases with increasing wavelength of In a time-resolved experiment where the pulses are shorter excitation. However, with all four excitation wavelengths, by than the periods of some of the coupled vibrations in the system 1 ps after excitation, the higher-energy side of the absorption and the excited state is displaced relative to the ground state, a decrease is very similar to that of the 5 ps difference spectra spectrally narrow pulse will select only a small subset of the (Figure 5 ) . It is thus likely that by 1 ps the majority of the reaction center conformations present in the ground ~ t a t e . ~ ~ ,evolution ~~ of the ground state which occurs within the time frame An important consequence of this is that there will be a of these measurements is complete. nonequilibrium depletion of the reaction centers distributed Comparing spectra taken using different excitation wavealong the ground state coordinates and selective excitation of a lengths shows that there is an excitation wavelength-dependent conformational subpopulation of reaction centers into a specific spectral variation at both 1 and 5 ps following excitation (Figure region of the excited state potential surface. The instantaneous 5 ) . Although the spectra taken with different excitation bleaching of the absorption band will be centered at the wavelengths have nearly the same line shapes, the position of excitation frequency, and its spectral width will be determined the overall ground state bleaching appears to be shifted to longer by that of the excitation pulse. Thus, the spectral width of the wavelengths when longer excitation wavelengths are used, as initial absorption decrease can be substantially narrower than 5 ps spectra of Figure 5B. The 80 cm-' can be seen in the that obtained by steady-state or broad-band illumination. difference in position of the maximum absorbance decrease Likewise, the emission spectrum of P* will be centered at the between the 1 ps spectra following 838 nm excitation and that excitation wavelength initially, and its width will also be dictated following 892 nm excitation (Figure 5A) is still present in the by the spectral width of the excitation pulse. Only after the 5 ps spectra using these two wavelengths (Figure 5B). This ground and excited states begin to evolve along their respective suggests that there is a component of the ground state conforvibrational coordinates will the homogeneous and inhomogenmational interconversion which is slow on the few picoseconds eous widths begin to be recovered. time scale. The rate at which the ground state approaches an Analysis of the manner in which the spectra in Figures 1-4 equilibrium conformational distribution appears to be waveevolve toward their equilibrium positions yields information on length dependent. The 5 ps spectra for 892 and 878 nm the frequency of the dimer modes of the electronic transition, excitation and to a lesser extent 858 nm excitation are similar their dephasing time, and the longitudinal relaxation time of to the steady state difference spectrum, but the 5 ps spectrum the bath vibrations in the ground and excited state populations. for 838 nm excitation differs significantly from the steady state The remainder of the discussion will be devoted to using the spectrum (Figure 5B). spectral changes in Figures 1-4 to investigate vibrational One important conclusion from this work is that the inhorelaxation and dephasing of the ground and excited states. mogeneous broadening of the P band which has been seen in Ground State Evolution. The absorption decrease at all low-temperature hole-burning studiesZoand apparently persists wavelengths shorter than 892 nm in the -0.13 and 0.0 ps spectra up to roughly 200 K39is at least partially present at room in Figure 4A (892 nm excitation) is consistent with the results temperature and persists throughout the electron transfer reacof hole-burning measurements which found that the prominent tion. This shows that part of the conformational interconverdimer mode undergoes dephasing within about 200 fs.20 sion which underlies the inhomogeneous broadening of the Photochemical steady state hole-burning experiments have dimer Q y band occurs on time scales longer than picoseconds shown that, following excitation into the low-energy side of even at 295 K. Note that caution must be used in any the P band, the maximum of the bleaching is at the 0-1 quantitative comparison of the 80 cm-' distribution of peak transition and not the 0-0 transition.20s21The small shoulder transition energies in Figure 5 with the 140 cm-' inhomogenwhich occurs 8-10 nm to shorter wavelength from the excitation eous broadening quoted by Small and co-workers,20since the wavelength in Figure 4A may be the manifestation of this 0-1 80 cm-' value obtained here simply reflects the spread of peak transition. This feature occurs roughly 100 cm-' to higher bleaching energies at a particular time derived from a small energy than the 892 nm excitation and is thus consistent with number of selected excitation wavelengths. Qualitatively, the the 120 cm-' frequency of the dimer mode predicted for Rb. two results are of similar magnitude. sphaeroides.
Excitation Wavelength Dependence of Reaction Centers
J. Phys. Chem., Vol. 99, No. 4, 1995 1355
does not appear to move more than 5 nm. Thus, any evolution of the excited state which is occurring on the 500 fs to 5 ps time scale has only minor effects on the stimulated emission spectrum. In addition, the peak wavelength of the stimulated emission spectrum does not depend significantly on the excitation wavelength at 1 ps (Figure 7) as did the ground state spectra on the same time scale (Figure 5). Even on time scales as long as nanoseconds, the spectrum of the emission from P* (measured by time resolved detection of spontaneous is not dramatically different from that shown in Figure 7. All of this suggests that past the first roughly 500 fs there is little change in the average energy of the emission from P. Vos et al.32concluded that dephasing of the prominent I' , , I I. I dimer mode is not complete until after 1 ps at room temperature, (126 860 075 e00 OP6 060 8P6 660 I T 6 BOO 9Bb 960 Wavelength (nm) Wavelength (nm) implying that vibrational relaxation of the excited state should take at least this long. Apparently, any vibrational relaxation Figure 7. Comparison of the spectrum of the stimulated emission at or excited state evolution which does occur past the first 500 fs three times following excitation calculated by subtracting the 5 .O ps absorption change spectrum from the 0.5, 1.0, and 3.0 ps absorption results in only very small changes in the average energy of change spectra for 838, 858, 878, and 892 nm excitation: (-) 5.0allowed transitions between the excited state and the ground 0.5 PS, (- -) 5.0-1.0 PS, 5.0-3.0 PS. state. It can be seen in Figure 6 that the decay kinetics of the Excited State Spectral Evolution. Figure 1C (838 nm stimulated emission at 920 nm are not significantly dependent excitation) shows that the time delay between the initial on the excitation wavelength. This independence is consistent bleaching at 838 nm and the appearance of the stimulated with the results of photochemical hole-burning experiments at emission at 920 nm is about 200 fs. Previously, Vos et al.30 K which showed that the width of the zero-phonon hole, 4.2 showed at 10 K that following 878 nm excitation it took about and consequently the lifetime of P*, was independent of the 150 fs for the full Stokes shift of the stimulated emission to excitation wavelength.20s21As the lifetime of P* is determined appear. The rapid appearance of the stimulated emission at 920 mostly by the electron transfer reaction P* P+HA-, it can be nm in Figures 1-4 following formation of P* suggests that the concluded that the rate of the overall electron transfer reaction formation of the Stokes shift is as fast at room temperature as between P* and P+HA- at room temperature is not significantly it is at 10 K. This is in agreement with very recent room temperature work by Vos et al.32 Vos et al.30332have interpreted affected by the way in which P* is prepared. In addition, while this in terms of a coherent movement of the wave packet along the excitation wavelength dependence of the spectra in Figure the displaced potential surface of the excited state. In each of 5 suggests that there is a distribution of P P* transition Figures 1-4, following 0.25 ps after excitation, an increase and energies on the order of 80 cm-', which is essentially static on then rapid decrease of the simulated emission are seen at the time scale of electron transfer, it does not appear that this wavelengths around 940 nm, consistent with the observation distribution is a major factor in determining the complex decay of coherent movement of the stimulated emission first to kinetics of P*. If the complex decay of the stimulated emission wavelengths longer than its final peak wavelength and then back did arise from a distribution of zero-point energies of P*, to shorter wavelength^.^^,^^ This spectral movement indicates then the red and blue sides of the observed stimulated emission that at room temperature the excited state is still evolving in a spectrum would be expected to have different distributions of coherent manner even after the initial fast displacement process. decay rates. This is not observed in R-26 reaction centers, where This has been interpreted in terms of incomplete vibrational the stimulated emission spectrum is nearly independent of time relaxation of the excited state on the time scale of electron past the first 500 fs (e.g. Figure 7), though a wavelength transfer.32 dependence of the P* decay kinetics has been reported in a If one assumes, as suggested above from the excitation mutant with a slow rate of electron tran~fer.~' wavelength dependence of the QY bleaching of P, that the The zero-point energy of P* is not the only factor which ground state spectrum does not evolve rapidly between 1 and 5 determines the rate of electron transfer. The electronic couplings ps, then the 5 ps spectrum will be a good representation of the between P* and P+HA-, the reorganization energy, and perhaps contribution of the Qu bleaching to the absorption change most importantly the zero-point energy of the P+HA- state spectrum at times between 1 and 5 ps. Subtraction of the 5 ps contribute to the rate of electron transfer. Reddy et suggest spectrum from absorption change spectra which precede it will that the insensitivity of the P* lifetime to the excitation yield the approximate spectrum of the stimulated emission. (The wavelength at 4.2 K may arise because of a distribution of contribution between 850 and 1000 nm from excited state electronic couplings. Although the spectrally narrow excitation absorbance is expected to be small.) Figure 7 shows that the pulses prepare only a narrow distribution of P* energies, the maximum of the stimulated emission spectrum calculated in this excitation pulse may not affect the initial distribution of other way is about 905 nm at times longer than 0.5 ps following each parameters which are important for electron transfer, such as of the excitation wavelengths, confirming the notion that the Stokes shift of the stimulated emission forms r a p i d l ~ . ~ ~ . the ~ ~ coupling between P* and the charge-separated state(s). Another important consideration is the rate at which the protein Between 0.5 and 1 ps, changes on the higher energy side of the solvates P+HA-. The rate at which [P+HA-]/[P*] approaches spectra can be seen for 878 and 892 nm excitation, probably its equilibrium value Keq is what ultimately determines the rate representing the evolution of the ground state on this time scale. of decay of P*. Fluorescence measurements suggest that the Careful examination of the spectra in Figure 7 shows that, with increasing time, the width of the stimulated emission spectra solvation of P+&- occurs over many time scales.'* The nuclear becomes slightly narrower, but the position of the maximum motions that are strongly coupled to electron transfer may well 898
,
868 nm
nm
,
,
,
,
(.*a)
-
-
Peloquin et al.
1356 J. Phys. Chem., Vol. 99, No. 4, 1995
-
be orthogonal to those coupled to the P P* transition, making their initial distribution essentially excitation wavelength independent. One of the most interesting questions which has arisen from the observation of coherence in excited state spectral changes is, does the coherent evolution of the excited state play a role in electron t r a n ~ f e r ? ~ OThe - ~ ~fact that there is no large change in the decay rate of the excited state when the excitation wavelength is changed means that the rate does not significantly depend on where one initiates the system along the vibrational coordinates which are strongly coupled to the P to P* transition. However, it does not speak to the issue of whether the subsequent coherent displacement along those coordinates is a necessary step in the electron transfer process or simply an unrelated spectral change associated with a mode which is not involved in electron transfer. In summary, the evolution of the ground state occurs in two phases. First, a rapid dephasing occurs on about the 200 fs time scale along the vibrational modes strongly coupled to the P P* transition. This evolution does not lead to the recovery of the full spectral width of the ground state absorption. Instead, the full width of the ground state absorption decrease is not attained until the initially depleted distribution of reaction center conformations has evolved into the equilibrium distribution through the relaxation of the bath vibrations of the protein. Most of this relaxation occurs within 1 ps. However, even 5 ps after excitation, there remains a roughly 80 cm-' difference between the maximum of the bleaching following 838 and 892 nm excitation, suggesting that a significant ground state conformational distribution persists past the first few picoseconds. The spectral evolution of the stimulated emission is essentially complete in 500 fs, and on the time scale of initial electron transfer (-3.5 ps) there is little influence of the manner in which P* is prepared on the maximum wavelength of the stimulated emission from P* or on the overall kinetics of the charge separation reaction. At room temperature, if conformational subpopulations do exist in the excited state with different rates of electron transfer, then they appear to be distinct from the conformational subpopulations in the ground state which give rise to the distribution in the P P* transition energies. Though, the evolution of the ground state will have no effect on the electron transfer reaction, the complexity of the ground state absorbance change during the first picosecond is striking and could account for some of the complex kinetic features observed at early times in transient absorption measurements.
-
-
References and Notes (1) Kirmaier, C.; Holten, D. Photosynth. Res. 1987, 13, 225. (2) Feher, G.; Allen, J. P.; Okamura, M. Y.; Rees, D. C. Nature 1989, 339, 111. (3) Parson, W. W. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, FL, 1991; p 1153. (4) Kirmaier, C.; Holten, D. In The Photosynthetic Reaction Center; Deisenhofer, J., Noms J. R., Eds.; Academic Press: San Diego, 1993; Vol. 11, p 49. (5) Martin, J.-L.; Vos, M. H. Annu. Rev. Biophys. Sfmct. 1992, 21, 199. (6) Zinth, W.; Kaiser, W. In The Photosynthetic Reaction Center; Deisenhofer, J., Noms, J. R., Eds.; Academic Press: San Diego, 1993; Vol. 11, p 71.
(7) Woodbury, N. W.; Allen, J. P. In Anoxygenic Bacteria; Blankenship, R. E., Madigan, M. T., Bauer, C. E., Eds.; Kluwer Academic Publishing: Dordrecht, in press. ( 8 ) Deisenhofer, J.; Epp, 0.;Miki, K.; Huber, R.; Michel, H. J . Mol. Bioi. 1984, 180, 385. (9) Allen, J. P.: Feher, G.; Yeates, T. 0.;Rees, D. C.; Deisenhofer, J.; Michel, H.; Huber, R. Proc. Natl. Acad. Sci. U S A . 1987, 84, 5730. (10) Chang, C.-H.; El-Kabbani, 0.;Tiede, D.; Noms, J.; Schiffer, M. Biochemistry 1991, 30, 5352. (1 1) Holzapfel, W.; Finkele, U.; Kaiser, W.; Oesterhelt, D.; Scheer, H.; Stilz, H. U.; Zinth, W. Chem. Phys. Lett. 1989, 161, 1. (12) Chan, C.-K.; DiMagno, T. J.; Chen, L. X.-Q.; Noms, J. R.; Fleming, G. R. Proc. Natl. Acad. Sci. U S A . 1991, 88, 11202. (13) Du, M.; Rosenthal, S. J.; Xie, X.; DiMagno, T. J.; Schmidt, M.; Hanson, D. K.; Schiffer, M.; Noms, J. R.; Fleming, G. R. Proc. Natl. Acad. Sci. USA. 1992, 89, 8517. (14) Muller, M. G.; Griebenow, K.; Holtzwarth, A. R. Chem. Phys. Left. 1992, 199, 465. (15) H a " , P.; Gray, K. A.; Oesterhelt, D.; Feick, R.; Scheer, H.; Zinth, W. Biochim. Biophys. Acta 1993, 1142, 99. (16) Kirmaier, C.; Holten, D. Proc. Natl. Acad. Sci. USA. 1990, 87, 3552. (17) Vos, M. H.; Lambry, J.-C.; Robles, S. J.; Youvan, D. C.; Breton, J.; Martin, J.-L. Proc. Natl. Acad. Sci. USA. 1991, 88, 8885. (18) Peloquin, J. M.; Williams, J. C.; Lin, X.; Alden, R. G.; Taguchi, A. K. W.; Allen, J. P.; Woodbury, N. W. Biochemistry 1994, 33, 8089. (19) Woodbury, N. W.; Peloquin, J. M.; Alden, R. G.; Lin, X.; Lin, S.; Taguchi, A. K. W.; Williams, J. C.; Allen, J. P. Biochemistry 1994, 33, 8101. (20) Reddy, N. R. S.; Kolaczkowski, S. V.; Small, G. J. J . Phys. Chem. 1993, 97, 6934. (21) Lyle, P. A.; Kolaczkowski, S. V.; Small, G. J. J . Phys. Chem. 1993, 97, 6924. (22) Reddy, N. R. S.; Lyle, P. A.; Small, G. J. Photosyn. Res. 1992,31, 167. (23) Meech, S. R.; Hoff, A. F.; Wiersma, D. A. Chem. Phys. Left. 1986, 121, 287. (24) Boxer, S.G.; Lockhart, D. J.; Middendorf, T. R. Chem. Phys. Lett. 1986, 123, 476. (25) Middendorf, T. R.; Mazzola, L. T.; Gaul, D. F.; Schenck, C. C.; Boxer, S. G. J . Phys. Chem. 1991, 95, 10142. (26) Hayes, J. M.; Small, G. J. J . Phys. Chem. 1986, 90, 4928. (27) Cherepy, N. J.; Shreve, A. P.; Moore, L. J.; Franzen, S.; Boxer, S. G.; Mathies, R. A. J. Phys. Chem. 1994, 98, 6023. (28) Vos, M. H.; Lambry, J.-C.; Robles, S. J.; Youvan, D. C.; Breton, J.; Martin, J.-L. Proc. Natl. Acad. Sci. USA. 1992, 89, 613. (29) Vos, M. H.; Rappaport, F.; Lambry, J.-C.; Breton, J.; Martin, J.-L. Nature 1993, 363, 320. (30) Vos, M. H.; Jones, M. R.; Hunter, C. N.; Breton, J.; Lambry, J.C.; Martin, J.-L. Biochemistry 1994, 33, 6750. (31) Vos, M. H.; Jones, M. R.; M c G l y ~P.; , Hunter, C. N.; Breton, J.: Martin, J.-L. Biochim. Biophys. Acta 1994, 1186, 117. (32) Vos, M. H.; Jones, M. R.; Hunter, C. N.; Breton, J.; Martin, J.-L. Proc. Natl. Acad. Sci. USA.,in press. (33) Stanley, R. J.; Boxer, S. G. Personal communication. (34) Feher, G.; Okamura, M. Y.The Photosynthetic Bacteria; Clayton, R. K., Sistrom, W. R., Eds.; Plenum F'ress: New York, 1978; p 349. (35) Taguchi, A. K. W.; Stocker, J. W.; Alden, R. G.; Causgrove, T. P.; Peloquin, J. M.; Boxer, S. G.; Woodbury, N. W. Biochemistry 1992, 31, 10345. (36) Cong, P.; Yan, Y. J.; Deuel, H. P.; Simon, J. D. J . Chem. Phys. 1994, 100, 7855. (37) Loring, R. F.; Yan, Y. J.; Mukamel, S. J . Chem. Phys. 1987, 87, 5838. (38) Scherer, N. F.; Jonas, D. M.; Fleming, G. F. J . Chem. Phys. 1993, 99, 153. (39) Hayes, I. M.; Lyle, P. A.; Small, G. J. J . Phys. Chem. 1994, 98, 7337. (40) Woodbury, N. W.; Parson, W. W. Biochim. Biophys. Acta 1984, 67, 345. (41) Nagarajan, V.; Parson, W. W.; Davis, D.; Schenk, C. C. Biochemistry 1993, 32, 12324. JP942198D
