J. Phys. Chem. 1996, 100, 17067-17078
17067
Excitation Wavelength Dependence of Energy Transfer and Charge Separation in Reaction Centers from Rhodobacter sphaeroides: Evidence for Adiabatic Electron Transfer 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: May 30, 1996; In Final Form: August 23, 1996X
Femtosecond transient absorption spectroscopy has been used to investigate the excitation wavelength dependence of energy transfer and initial charge separation processes in reaction centers of the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides (R-26) at room temperature. The QY transition bands of the bacteriopheophytins (H), bacteriochlorophyll monomers (B), and special pair (P) were selectively excited with pulses of 150 fs duration and 5 nm spectral bandwidth. Absorbance changes were analyzed over the entire wavelength region from 700 to 1000 nm. From this analysis we concluded the following: (1) As seen by others, energy transfer between H, B, and P is extremely fast, occurring on the 100-300 fs time scale. (2) The spectral evolution of the system is excitation wavelength dependent for picoseconds after excitation, implying that vibrational relaxation is not complete on the time scale of either energy transfer or charge separation and suggesting that the pathway of charge separation may be excitation wavelength dependent. (3) The absorbance change spectra of the initial excited states of B and H are not consistent with intensity borrowing between these bands, reopening the question of what gives rise to the complex absorbance changes normally associated with the HA- state. (4) The 10-20 ps component of the stimulated emission decay is excitation wavelength dependent and spectrally different from the dominant 2-3 ps decay of the stimulated emission. This component is unlikely to represent a static conformational heterogeneity in the reaction center charge separation rate. These conclusions lead to the proposal of the following model for energy and electron transfer in the reaction center. Energy transfer in this system is very fast because it is mediated by electron exchange interactions between cofactors (implying relatively strong electronic coupling for electron transfer) and because there is little nuclear displacement between donor and acceptor potential surfaces during energy transfer. Electron transfer is slower than energy transfer because the nuclear displacement is larger, and the rate is limited by movement along the reaction coordinate. Thus, initial electron transfer occurs in the near adiabatic limit before vibrational relaxation is complete. This model would explain many issues which have been difficult to resolve using standard electron transfer models including the difficulty in identifying the P+BA- intermediate and the insensitivity of the initial electron transfer rate to temperature and driving force.
Introduction In photosynthetic reaction centers, solar energy conversion occurs via a series of electron transfer reactions, forming a stabilized, transmembrane charge-separated state. The most extensively studied reaction centers are those from purple nonsulfur bacteria.1-7 High-resolution structures have been determined for the reaction centers from two species.8-12 The X-ray structure of the reaction center from Rhodobacter (Rb.) sphaeroides strain R-26 has shown that there are nine cofactors arranged in a C2 symmetric configuration.9 Two strongly coupled bacteriochlorophyll a molecules form a dimer (P) on the periplasmic side of the membrane with a center-to-center distance of approximately 7 Å. On each side of the dimer, there is a bacteriochlorophyll a monomer (BA and BB; the subscript A denotes the cofactor on the branch which is active in electron transfer, and B denotes the inactive side) and a bacteriopheophytin a (HA and HB). The center-to-center distances from P to B and from B to H are each about 11 Å. The quinones (QA and QB) are located near the cytoplasmic side of the complex, and an iron atom is between the two quinones. The room temperature ground state absorption spectrum of R-26 reaction centers shows three major bands in the nearinfrared region (Figure 1). The band around 760 nm originates predominantly from the QY transitions of HA and HB. The X
Abstract published in AdVance ACS Abstracts, October 1, 1996.
S0022-3654(96)01590-0 CCC: $12.00
Figure 1. Ground state absorption spectrum of isolated reaction centers from Rb. sphaeroides strain R-26 at room temperature. Vertical arrows indicate the excitation wavelengths used in the transient absorbance change measurements. Excitation was at 744, 760, 794, 800, 812, and 860 nm using 150 fs duration, 540 Hz repetition rate, 5 nm bandwidth pulses. The spectral profile of the excitation at 800 nm is shown as the dashed line.
narrow band around 802 nm is mainly due to the QY transitions of BA and BB, and the broad band at 860 nm is due to absorption by P. However, theoretical calculations considering the intermolecular interactions between reaction center chromophores, © 1996 American Chemical Society
17068 J. Phys. Chem., Vol. 100, No. 42, 1996 including charge transfer interactions, have been used to calculate the spectrum of the reaction center from Rhodopseudomonas (Rp.) Viridis13,14 and suggest that each of the nearinfrared bands in Figure 1 represents a mixture of transitions involving multiple cofactors. Photochemical hole-burning experiments have also indicated spectral overlap between the transitions associated with the different cofactors.15,16 When P is excited by light, an electron is transferred to HA, perhaps via BA, in about 3 ps. The electron is then transferred to QA on a time scale of 200 ps. There is very little detectable electron transfer involving B-side cofactors in wild type reaction centers.17 As the back-reactions are several orders of magnitude slower than the forward reactions, the yield of charge separation to P+QA- is essentially unity. (For reviews, see refs 1-3 and 7.) Both the monomer bacteriochlorophylls and the bacteriopheophytins can rapidly transfer energy to P. The kinetics of absorbance changes upon exciting the H or B band have been measured with femtosecond time resolution at both room temperature18,19 and 10 K.20 These studies suggested that much of the energy transfer between H, B, and P occurs on the 100 fs time scale. More recent femtosecond measurements of the B* decay kinetics or P* rise in reaction centers using impulsive laser excitation near 800 nm imply an energy transfer rate of roughly 100 fs between B and P.21-25 Decay kinetics measured at 1215 nm following 800 nm excitation showed a 200 fs component which was also attributed to energy transfer from B to P.26 Ultrafast energy transfer from H to B or P was implied from a study of hole burning as a function of wavelength in the bacteriopheophytin QY band at 4.2 K.16 From the insensitivity of the hole width to the burn frequency and the homogeneous broadening of the H band, the excited state lifetime of H was estimated to be less than 30 fs. Recent femtosecond measurements have implied that energy transfer from H all the way to P is considerably slower than this,23 presumably due to intermediate transfer from H to B. Based on the structure of reaction centers from Rp. Viridis and Rb. sphaeroides, time constants for energy transfer among cofactors were calculated using a Fo¨rster weak coupling model.27 The transfer rates obtained by this method were at least 1 order of magnitude slower than the experimental observations. It was suggested from this work that stronger coupling terms might need to be considered. A different point of view has been put forth by Haran et al., who have suggested that the very fast energy transfer rates could, in fact, be described by a Fo¨rster type model.25 However, Stanley et al.23 have pointed out that the calculations of Haran et al. also imply a significant temperature dependence to the energy transfer rate as spectral bands shift and narrow, which is not observed,18,21 again indicating that the Fo¨rster model is not an adequate description of energy transfer between reaction center cofactors. One question that has been raised recently which pertains to both the issues of the energy and electron transfer mechanisms in reaction centers on fast time scales is how strongly coupled the excited and charge-separated states of the reaction center are. Low-temperature kinetic measurements of R-26 reaction centers and hydrogen bond mutant reaction centers with high P/P+ midpoint potentials have suggested that the weak free energy dependence of the charge separation kinetics and the spectral evolution may be better explained by a model in which charge separation occurs near the adiabatic limit.28,29 Photochemical hole-burning data have led to a similar suggestion.30 The possibility of adiabatic electron transfer in the reaction center has also been explored theoretically and suggested to be consistent with a number of experimental observations.31 If the
Excitation Wavelength Dependence of Reaction Centers reaction does occur near the adiabatic limit, then the longstanding question of whether P+BA- serves as a real intermediate state becomes largely irrelevant,30 and one would expect the existence of a continuous nuclear potential surface for the system connecting the excited and charge-separated states, at least along the nuclear coordinates associated with certain modes. Another point recently made by measurements of vibrational coherence on the time scale of electron transfer as well as by dynamic hole-burning measurements is that the excited singlet state of P is probably not vibrationally equilibrated on the time scale of electron transfer.32-37 Though the specific modes coupled to the P to P* transition which could be measured in these studies may not themselves be coupled to electron transfer,30, 37 these studies indicate that vibrational relaxation is not rapid in the reaction center system, contrary to what one assumes in a nonadiabatic description of electron transfer.38 Additional information about the strength of interactions between cofactors and about the role of vibrational relaxation in reaction kinetics and spectral evolution can be gained by a study of time-resolved absorbance changes in reaction centers using a variety of different excitation wavelengths throughout the near-infrared region. Femtosecond kinetics of absorbance changes upon exciting reaction centers into cofactor bands other than the QY band of P have thus far only been measured at a limited number of probe wavelengths.18,19,21-23,26 Given the kinetic complexity of the overall charge separation process and the complex spectral evolution observed when exciting with narrow band excitation pulses into the QY band of P,36,37 a complete analysis of the spectral changes upon excitation into B or H as a function of time is called for. In this report, we present femtosecond transient absorption difference spectra of R-26 reaction centers over a broad probe wavelength region using narrow-band excitation at six wavelengths covering the P, B, and H near-infrared bands. Materials and Methods Reaction centers were isolated from Rhodobacter sphaeroides strain R-26 as previously described39 and suspended in 50 mM Tris-HCl (pH 8.0), 0.025% LDAO, 1 mM EDTA, and 0.5 mM terbutryne. For femtosecond transient absorbance measurements, samples were loaded in a spinning wheel with an optical path length of 2.5 mm. An optical density of roughly 1.2 at 802 nm was used. All measurements were performed at room temperature. Time-resolved absorption difference spectra were measured on a pump-probe transient absorption spectrophotometer. The layout of the system has been described previously,29,40 as has the generation and use of spectrally narrow excitation wavelengths.36,37 Excitation pulses were polarized at the magic angle with respect to the probe pulse. For each of the excitation wavelengths used, the pulse energy was 2-4 µJ per pulse over a roughly 2 mm2 area, resulting in excitation of 15-20% of the reaction centers in the sample. Transient absorption spectra were recorded at 100 time points, and data were collected over either a 140 or a 280 nm spectral window. Transient absorbance change surfaces were fit to a sum of exponential terms:
∆OD(λ,t) ) ∑Ai(λ) exp(-t/τi)
(1)
where ∆OD is the observed absorbance change at a given wavelength λ and time t. A plot of Ai(λ) vs λ gives the amplitude spectrum of the ith kinetic component with a decay lifetime of τi.29
Lin et al.
J. Phys. Chem., Vol. 100, No. 42, 1996 17069
Figure 2. Time-resolved absorbance change spectra of R-26 reaction centers with excitation at 744, 760, 794, 800, 812, and 860 nm. Time zero is defined as the point when the maximal absorbance decrease at the excitation wavelength is achieved. Spectral resolution is 2 nm. Vertical arrows in each panel indicate the wavelength of the excitation pulse.
Wavelength variation in the delay between the pump and probe pulses due to dispersion was corrected empirically as described by Peloquin et al.36,37 The amount of time delay of the signal as a function of wavelength was determined by measuring the transient rise of laser-induced birefringence in CS2.41 Results Transient absorbance changes were measured in the QY transition region from isolated reaction centers of R-26 at room temperature. Different near-infrared absorption bands of the reaction center were excited with pulses of 150 fs duration and 5 nm spectral bandwidth. Absorbance change spectra were taken either over a 9.5 ps time scale with a 95 fs interval between spectra or over a 30 ps time scale with a 300 fs interval. Figure 1 shows the ground state absorption spectrum of R-26 reaction centers at room temperature, and the arrows indicate the excitation wavelengths used (744, 760, 794, 800, 812, and 860 nm). These excitation wavelengths preferentially excite the QY absorption bands of H (744 or 760 nm excitation), B (794, 800, or 812 nm excitation), or P (860 nm excitation). A spectral profile of the excitation pulse at 800 nm is also illustrated in Figure 1. Spectral profiles of the excitation pulses at other wavelengths were very similar. Spectral Evolution as a Function of Excitation Wavelength. Figure 2 shows absorbance change spectra at various time delays after excitation. Spectra were recorded on a 9.5 ps time scale. Zero picoseconds was defined as the time when the absorbance decrease at the excitation wavelength reached
its maximum. Using 744 nm excitation, the spectrum taken at 0 ps shows a bleaching which peaks at 744 nm with a shoulder around 760 nm. At this time, a bleaching at 802 nm with a shoulder at 810 nm and a bleaching at 866 nm were also observed. Spectroscopic studies at low temperature have indicated that the bacteriopheophytin on the active and inactive side of the reaction center (HA and HB) absorb near 760 and 742 nm, respectively (for reviews, see refs 1 and 7.) Excitation at 744 nm results in excitation of both of the bacteriopheophytin QY bands though a larger fraction of HB is excited at this wavelength than at 760 nm. After 280 fs, the initial bleaching at 744 nm partly recovers, and the bleaching maximum in this region shifts to 758 nm. Because of the temporal overlap of the pump and probe beam at the excitation wavelength, a coherent artifact may contribute to the fast appearance and decay of the absorbance decrease at the excitation wavelength. Bleachings at 800 and 870 nm developed further at 280 fs. The 866 nm absorbance decrease reached its maximum and peaked around 870 nm at 950 fs. At 950 fs the 758 nm bleaching remains, but the amplitude has decreased and an overall increase in the background absorbance in this spectral region has occurred. At this time, there are also small spectral features in the 800-812 nm region, similar to those seen upon direct excitation of P (Figure 2). The spectrum taken at 8 ps shows a recovery of part of the absorbance decrease in the 870 nm region, primarily on the red side, and a shift of this band to 864 nm. By this time, the amplitudes of the bleaching at 760 and 810 nm have increased, and a positive band at 780 nm has grown in. The profile of the 25 ps spectrum is very similar to
17070 J. Phys. Chem., Vol. 100, No. 42, 1996
Excitation Wavelength Dependence of Reaction Centers
Figure 3. Comparison of the time-resolved absorbance change spectra recorded 25 ps after excitation at 760 (dashed line), 800 (dotted line), and 860 nm (solid line). Spectra are normalized at 865 nm.
those obtained with 860, 812, 800, 794, or 760 nm excitation at the same time delay and represents the primary chargeseparated state. (Results for 760, 800 and 860 nm excitation are shown in Figure 3.) The overall features of the spectral evolution using 760 nm excitation are very similar to those obtained with 744 nm excitation, except for the spectral changes in the 760 nm region itself at early times (Figure 2). Comparing 744 and 760 nm excitation, initially, 760 nm excitation produces a narrow bleaching at 760 nm with a small absorption increase at wavelengths below 750 nm. Excitation at 794 nm is on the shorter wavelength side the QY transition band of B. The 0 ps spectrum shows a narrow bleaching at the excitation wavelength and a broad weak bleaching around 870 nm (Figure 2). The spectrum of the initial bleaching near 794 nm broadens with time toward longer wavelengths, until at 280 fs, its maximum is at 798 nm. Bleaching in the 800 nm region has largely recovered by 750 fs, and bleaching of the P band at 866 nm is fully developed at this time. The 25 ps P+HA- spectrum is very similar to that obtained with other excitation wavelengths (data not shown). Excitation at 812 nm results in a 0 ps spectrum that shows a narrow bleaching at 812 nm and a broad bleaching at 864 nm (Figure 2). The amplitude of the 864 nm bleaching has already reached 50% of its maximum at this time, which is larger than is seen at 0 ps using excitation at 744, 760, or 794 nm. The bleaching of the QY band of P is fully developed at 700 fs and centered at 866 nm. The initial 812 nm bleaching has mostly recovered at this time. The spectrum observed at 25 ps is again very similar to that obtained with other excitation wavelengths (Figure 3). Excitation at 800 nm gives results intermediate between 794 and 812 nm excitation (Figure 2). In contrast to shorter wavelength excitation, excitation with spectrally narrow pulses at 860 nm results in only small absorbance changes between 740 and 830 nm at early times (Figure 2). The larger absorbance changes associated with charge separation then grow in on the several picosecond time scale. Time-resolved spectra taken at 0 and 1 ps with excitation wavelengths at 744, 760, 794, 800, 812, and 860 nm are compared directly in Figure 4 and were taken in such a way as to allow comparison of the spectral features at early times in all three of the near-infrared bands of the reaction center. Spectra are normalized at 1 ps and 865 nm. (The same normalization factors between excitation wavelengths are used at both 0 and 1 ps.) Dotted lines represent zero absorbance
Figure 4. Comparison of the time-resolved absorbance change spectra of R-26 reaction centers using different excitation wavelengths at 0 ps (solid line) and at 1 ps (dashed line) time delays. Associated dotted lines indicate zero absorbance change in each case. Spectral resolution is 2 nm per point. Spectra are normalized at 865 nm at 1 ps.
change in each case. It can be seen that, under different excitation conditions, significantly different initial excited state spectra resulted. There are several features in the 0 ps spectra which should be noticed. First, a pronounced initial bleaching around 760 nm is only seen when excitation in the bacteriopheophytin band at 744 or 760 nm is used. Instead of a bleaching band at 760 nm, a broad band absorbance increase
Lin et al. appears in this region when reaction centers are excited at other wavelengths. Second, bleaching of the P band is not fully developed at 0 ps unless the excitation is directly into the P band at 860 nm. Third, the bleaching around 800 nm appears almost instantaneously when excitation at 744 nm or at 760 nm is used. However, there is not a corresponding instantaneous bleaching observed at 760 nm when excitation into the B band at 794 or 800 nm is used. (There is a very small, but significant, absorbance dip near 760 nm when 812 nm excitation is used, probably because pumping at this wavelength excites the upper exciton band of P; see Discussion.) Excitation wavelength-dependent differences in the spectral features persist even at 1 ps after excitation (Figure 4). Using 744 or 760 nm excitation, most of the 760 nm bleaching still remains at 1 ps. In contrast, the 760 nm bleaching due to charge separation has developed to roughly 30% of its maximum amplitude at 1 ps (compared to long times, see Figure 3) when excited at other wavelengths. In addition, the 1 ps spectra using 744, 760, 794, 800, and 812 nm excitation all display some residual bleaching in the 800-810 nm region which does not fully recover; the 1 ps spectrum taken using 860 nm excitation does not show this residual bleaching. Part of this residual 800 nm bleaching appears to be excitation intensity dependent when excitation directly into the QY band of the monomer bacteriochlorophylls is used (data not shown), though the additional bleaching is substantial even at excitation intensities severalfold lower than those shown. (Similar intensity-dependent effects have been noted previously for excitation near 800 nm.22) Neither the 800 nm nor the 760 nm bleaching that persists on the 1 ps time scale when excitation into the bacteriopheophytin band is used was found to be excitation intensity dependent over the intensity range tested. Figure 5 compares the spectral differences at 0.5 and 1 ps obtained between 860 nm excitation and excitation at other wavelengths. Since the overall charge separation time starting in the state P* is about 3 ps, the reaction center is almost entirely in the P* state at 0.5 ps, and about 30% of P* has decayed 1 ps after P is directly excited. Figure 5A compares absorbance change spectra at 1 ps with 760 and 860 nm excitation. Spectra are normalized at 1 ps and 860 nm where the P band bleaching dominates. The spectra using 760 and 860 nm excitation overlap well in the P band region but are quite different at shorter wavelengths. Besides the major bleaching band around 860 nm, the spectrum obtained using 860 nm excitation shows an absorbance increase below 800 nm with small spectral features in the 765 and 800-810 nm regions, while the spectrum obtained using 760 nm excitation shows distinct bleachings at both 760 and 802 nm. Figure 5B shows the difference of the 1 ps absorbance change spectra shown in Figure 5A calculated by subtracting the 860 nm excitation spectrum from the 760 nm excitation spectrum after normalization at the P band bleaching near 860 nm. Similar difference absorbance change spectra are also shown at 0.5 ps in Figure 5B. In addition, Figure 5 shows difference absorbance change spectra obtained by subtracting spectra using 860 nm excitation from spectra using 744 nm excitation (dotted lines). The difference absorbance change spectra at the two wavelengths were normalized at 802 nm and 0.5 ps for ease of comparison. Since the absorbance change spectrum taken at 0.5 ps with 860 nm excitation is dominated by the P* contribution, the 0.5 ps difference spectra shown in Figure 5B should mainly represent differences due to early states other than P*. With 744 nm excitation (Figure 5B, dotted line), the difference spectrum at 0.5 ps shows bleaching bands centered at 750 and 802 nm. More than 65% of the amplitude at 802 nm decreased at 1 ps relative
J. Phys. Chem., Vol. 100, No. 42, 1996 17071
Figure 5. (A) Absorbance change spectra taken 1 ps after 760 nm (solid line) and 860 nm (dashed line) excitation. The two curves are normalized at 860 nm. (B) Difference between the absorbance change spectrum using 860 nm excitation and the absorbance change spectra using 744 (dotted line) and 760 nm excitation (solid line) at 0.5 and 1 ps. (C) Difference between the absorbance change spectrum using 860 nm excitation and the absorbance change spectra using 794 (solid line) and 812 nm excitation (dotted line). To generate the difference absorbance change spectra (difference-difference spectra) in (B) and (C), the absorbance change spectra at the two excitation wavelengths being compared were normalized at 860 nm before subtraction. The difference-difference spectra thus obtained for each combination of excitation wavelengths were normalized at the maximum bleaching at 0.5 ps.
to 0.5 ps while the 750 nm band only decreased 30% and shifted to 755 nm. The 0.5 and 1 ps difference absorbance change spectra between 760 and 860 nm excitation (Figure 5B, solid lines) show similar features, except that the bleaching in the bacteriopheophytin band is at about 760 nm at 0.5 ps and shifts to 758 nm at 1 ps. In Figure 5C, difference absorbance change spectra between 794 and 860 nm excitation (solid line) and between 812 and 860 nm excitation (dotted line) are compared. Spectra were calculated and normalized in the same way as those in Figure 5B. At 0.5 ps, the difference absorbance change spectrum between 794 and 860 nm excitation shows a single bleaching
17072 J. Phys. Chem., Vol. 100, No. 42, 1996
Excitation Wavelength Dependence of Reaction Centers
Figure 6. Kinetic traces of absorbance changes from R-26 reaction centers at different probe wavelengths (shown in small letters) with excitation at 744, 760, 794, 800, 812, and 860 nm (indicated in large letters). Curves are normalized at the maximum bleaching in each panel, except for the 860 nm excitation panel. In the latter case no normalization was performed. Data were recorded at 95 fs per point.
band centered at 802 nm. The 812 nm excitation minus 860 nm excitation spectrum exhibits a bleaching band at 808 nm, and the bandwidth is about 3 nm narrower than the one with 794 nm excitation. More than 50% of the bleaching has recovered at 1 ps, and the profiles of the two difference spectra become similar on the longer wavelength side. Both spectra show bleaching maxima near 800 nm at 1 ps. The difference between 794 and 812 nm excitation on the shorter wavelength side (780-800 nm) of the band becomes more pronounced at 1 ps (compared with 0.5 ps), with the 794 minus 860 nm excitation difference spectrum (solid line) at 1 ps being about 8 nm broader than the 812 minus 860 nm excitation difference spectrum (dashed line) at 1 ps. At 10 ps and later, the absorbance change spectra using different excitation wavelengths become very similar (Figure 3). However, a careful comparison of the spectra in Figure 3 still shows significant variations. Spectra were taken at 25 (
1 ps after laser excitation at 760, 800, and 860 nm with normalization at 865 nm. Significant differences can be seen in the 760 nm region where a larger bleaching is formed with 760 or 800 nm excitation than with 860 nm excitation. Small differences can also be observed near 780 and 810 nm. Kinetics of the Absorbance Change of the H, B, and P Bands. Kinetic traces of the absorbance change at selected wavelengths with 744, 760, 794, 800, 812, and 860 nm excitation are plotted in Figure 6. Traces were taken from the absorbance change surface measured at 95 fs per point over a 9.5 ps time range. (Only the first 4 ps are shown in Figure 6.) Curves were normalized at their bleaching maxima for ease of comparison (except in the case of 860 nm excitation where no normalization was performed). The kinetics at 744 nm following 744 nm excitation show a prompt absorbance decrease followed by a rapid recovery within a few hundred femtoseconds. Subsequently, a slower absorbance decrease grows in
Lin et al. with a time constant of several picoseconds. The 802 nm bleaching using 744 nm excitation reached its maximum 150 fs later than the 744 nm bleaching. The absorbance decrease at 802 nm also recovers on the few hundred femtosecond time scale and is followed by a slower absorbance decrease with a time constant of several picoseconds. The absorbance decrease at 870 nm not only occurs with a shallower slope than the 744 or 802 nm initial absorbance changes but also is initiated 100200 fs later. The kinetics using 760 nm excitation are similar to those using 744 nm excitation, except that the time lag between the initial bleaching at the excitation wavelength and that at 802 or 870 nm is less when 760 nm excitation is used (roughly 100 fs delay before bleaching at 802 nm occurs). This very short apparent energy transfer from H to B is consistent with the results from photochemical hole-burning experiments in which the excited state lifetime of H was estimated to be 30 fs.16 When excited at 794 nm, the time course at 794 nm shows a prompt bleaching followed by a several hundred femtosecond lag during which no absorbance change at this wavelength occurs. After that, the absorbance decrease recovers within several hundred femtoseconds to roughly 10% of the initial amplitude. The complexity of the kinetics at 794 nm originates in part from a fast spectral broadening of the initial bleaching at early times as can be seen in Figure 2. The appearance of the bleaching at 870 nm is delayed by about 200 fs compared with the prompt bleaching at the excitation wavelength. The kinetic trace at 810 nm using 812 nm excitation in Figure 6 shows a prompt absorbance decrease which recovers by 60% within about 300 fs of the maximum bleaching and is followed by a several picosecond absorbance decrease. The rise of bleaching at 870 nm was delayed by roughly 100 fs. The results using 800 nm excitation are intermediate between those using 794 and 812 nm excitation. Excitation at 860 nm results in only very small fast changes in the 810 nm region (Figure 6), with the dominant absorbance changes happening on the time scale of electron transfer. If the kinetics in the 760, 800, and 870 nm regions represent simply the decay of H* and B* excited states and the rise of P*, as has been suggested previously,18,19,21,26 one would expect some kinetic correspondence between the recovery of the H and B ground state absorbance changes at 760 and 800 nm with the appearance of the P* absorbance changes at 870 and 930 nm. Global analysis of the entire wavelength region was attempted using eq 1 to describe the absorbance change surface. However, it was not possible to obtain a reasonable fit of the data at all wavelengths simultaneously. Even using several exponential decay terms, there were systematic deviations between the data and the fit well outside the noise level of the measurements (not shown). Looking at the data of Figure 2, it is clear that there is substantial time-dependent band shifting and broadening at early times after excitation. In addition, there are some wavelengths such as 870 and 930 nm where there is a significant time lag before absorbance changes occurred at all. At other probe wavelengths and excitation conditions, there are rapid absorbance changes followed by a lag and then a rapid recovery (e.g., 794 nm in Figure 6). This type of kinetic behavior cannot be fit by a sum of exponential decay terms with common initiation times and decay constants at all wavelengths. Though it is not possible to analyze the data in terms of classical first-order kinetic processes, one can conclude that there is at least some extremely rapid (200 fs or less) communication between cofactors followed by a complex spectral evolution. In addition, it is generally the case that the higher the energy
J. Phys. Chem., Vol. 100, No. 42, 1996 17073
Figure 7. Kinetics of the stimulated emission decay from R-26 reaction centers at 930 nm with excitation at 760, 800, and 860 nm. Time zero is shifted for each curve so that the rising edges are aligned. The traces are normalized at time of the largest absorbance decrease.
of the exciting photon, the longer the time interval between the middle of the excitation pulse and the maximum bleaching of the QY band of P at 870 nm or the maximum stimulated emission near 930 nm. Stimulated Emission of P* and the Formation of the Charge-Separated State. If the excitation wavelength-dependent time delay before the onset of the stimulated emission is removed, the decay kinetics of the stimulated emission is not very sensitive to the excitation wavelength. Figure 7 shows kinetic traces using 760, 800, and 860 nm excitation at 930 nm. (This is not true for kinetic traces using shorter probe wavelengths; see below.) Curves are shifted to line up at their rising edge (therefore, time zero is somewhat arbitrary) and normalized at their peaks. The rise and decay profiles are similar within the time resolution and signal-to-noise ratio. Global kinetic fitting of absorbance changes between 830 and 950 nm over a 25 ps time period gave the same time constants for each excitation wavelength. (The results were a 0.5 ( 0.1 and a 3.3 ( 0.1 ps component, from a fit with two-exponential terms plus a constant and a 0.5 ( 0.1, a 2.1 ( 0.2, and a 14 ( 2 ps component from a fit with three-exponential terms plus a constant. The 0.5 ps component present in all fits was not well resolved on the 25 ps time scale and represented the rise in the stimulated emission due to energy transfer and/or vibrational relaxation.) Fitting with three-exponential decay terms was statistically superior, resulting in a >15% decrease in the chisquared error for the fit. The 2 and 14 ps lifetimes obtained from this analysis are in good agreement with previous measurements.42-45 Figure 8A shows the amplitude spectra of the picosecond exponential decay components obtained from the global fitting described above using three-exponential decay terms and a constant. For each data set, the 500 fs component exhibits a broad positive band which is centered near 880 nm using 760 nm excitation and centered around 900 nm with 800 or 860 nm excitation (not shown). Since the subpicosecond absorbance changes could not be accurately described by a sum of exponential decay components (see above), the subpicosecond events were ignored for the analysis of the long-time fits. In Figure 8A, the amplitude spectra of the 2 ps, 14 ps, and nondecaying components are plotted with normalization of the different excitation wavelength data at the peaks of the nondecaying spectra. The 2 ps spectrum shows a broad negative band around 900 nm which is due to the decay of the stimulated emission from P*. The 14 ps component shows positive
17074 J. Phys. Chem., Vol. 100, No. 42, 1996
Excitation Wavelength Dependence of Reaction Centers SCHEME 1
Figure 8. (A) Amplitude spectra of the 2 ps, 14 ps, and nondecaying components at several excitation wavelengths obtained by fitting the absorbance change surface from 830 to 950 nm over a 30 ps time range with a spectral resolution of 2 nm and kinetic data density of 0.3 ps per point. Four kinetic components were used in each fit as described in the text. The roughly 0.5 ps component is not shown. Excitation wavelengths used were 760 (dashed lines), 800 (dotted lines), and 860 nm (solid lines). The amplitude spectra at the different excitation wavelengths were normalized at the bleaching maximum of the nondecaying components. (B) Amplitude spectra of the 2 ps component for each excitation wavelength normalized at the bleaching maximum.
amplitudes at wavelengths below 880 nm and negative amplitudes above 900 nm. The profile of the amplitude spectrum associated with the 14 ps component varies substantially with excitation wavelength and is strikingly different from that of the major P* decay component of 2 ps. The amplitude spectrum of the constant term represents the ground state bleaching of P due to formation of P+. The band shape and peak position of the nondecaying spectrum are essentially the same with different excitation wavelengths. The amplitude spectrum of the 14 ps component is similar to that of a component identified previously by Nagarjan and co-workers in reaction center mutants at position M210.46 However, it is not clear whether the components described here and by Nagarajan et al. represent the same physical processes. In the M210 mutants, the component with the derivative-like amplitude spectrum was interpreted as a spectral shift of the stimulated emission and had a faster rate constant than the overall electron transfer rate constant. In Figure 8, the 14 ps component is slower than the bulk rate constant of electron transfer and thus probably does not represent a band shift. (See Discussion for further interpretation.) The peak of the amplitude spectrum of the 2 ps component is blue-shifted when shorter excitation wavelengths are used, and the overall amplitude (relative to that of the nondecaying component) is smaller (Figure 8A). An increase in the positive going portion of the 14 ps amplitude spectrum using 760 nm excitation, compared to that using 800 or 860 nm excitation, is
observed. However, the band shape of the nondecaying component is very similar under all excitation conditions. Another feature shown in Figure 8A is that the ratio of the areas of the nondecaying and 2 ps component amplitude spectra depends on the excitation wavelength, indicating a variation in the amount of P+ formed at long times vs the amount of P* formed initially. The amount of P* decayed vs the amount of P+ formed was calculated from the spectra in Figure 8A. The amount of P* decay was measured under the area of the 2 ps amplitude spectrum, and the relative amount of P+ formed was measured from the nondecaying spectrum. It has been shown previously that the yield of charge separation using excitation directly into the QY band of P is essentially 1.47 Taking the ratio of P*/P+ (the integrated 2 ps component stimulated emission spectrum vs the integrated constant bleaching spectrum) using 860 nm excitation as a reference (100%), the amount of the final charge-separated state formed directly from P* using 760 nm excitation is estimated to be 78% of the total amount of P+ formed. It is about 85% when the B band is excited. Figure 8B compares the spectral profiles of the 2 ps components obtained with different excitation wavelengths after normalization at their bleaching maxima. The bandwidth of the 2 ps spectrum with 760 nm excitation is broader on the shorter wavelength side, implying a more complicated excitation distribution at this pump wavelength. The 2 ps spectra with 800 and 860 nm excitation are essentially identical, and all the spectra are very similar on the longer wavelength side of the band. Discussion Ultrafast Spectral Evolution: Energy Transfer and Vibrational Relaxation. Past studies with limited spectral resolution have shown that excitation of the bacterial reaction center into the near-infrared bands of the monomer bacteriochlorophylls or bacteriopheophytins results in ultrafast energy transfer to the dimer followed by the normal course of charge separation reactions.18,19,21-26 Such a model is shown in Scheme 1. This scheme is consistent with the observation in this report of early time spectral evolution of short wavelength bleaching (near 760 and 800 nm) to longer wavelengths (860 nm) as shown in Figures 2 and 4 and with the insensitivity to excitation wavelength of the time course of stimulated emission decay on the picosecond time scale (Figure 7). However, there are several aspects of the spectral evolution described in this work which are not consistent with at least the simplest interpretation of Scheme 1. One would expect from Scheme 1 that the bleaching in the ground state spectral band of the donor would disappear with the same kinetics as bleaching would appear in the ground state spectral band of the acceptor. Global exponential fitting of the data during the first picosecond after excitation in the 760 or 800 nm regions was attempted, but it was not possible to obtain a fit at all wavelengths that adequately described the data even with several exponential decay terms. (There were large residual errors compared to the noise.) Looking at Figures 2 and 6, it is easy to see why
Lin et al. this would be true. Some of the early time spectral behavior of the system in Figure 2, particularly in the wavelength region near the excitation, involves shifting and broadening of spectral bands. This type of spectral evolution cannot be modeled with exponential decays. In addition, there are temporal delays between the onset of bleaching in the lower wavelength regions of the spectrum and the bleaching at 870 nm (Figure 6) or the appearance of the stimulated emission at 930 nm. This cannot be adequately modeled by simple exponential decay analysis. Past studies have measured kinetics at single probe wavelengths during one measurement. This makes it difficult to see band shifting, band broadening, or true delays in the onset of absorbance changes at different wavelengths. Note that dispersive effects of the optics in the system, which could give rise to artificial delays between absorbance changes in the long wavelength region relative to those in the short wavelength region, were removed empirically using a CS2 birefringence measurement.36 Another inconsistency between the data reported here and Scheme 1 is that in Scheme 1 one would expect the spectra to become excitation wavelength independent after a few hundred femtoseconds. By this time, essentially all of the energy should have transferred to P, and the spectral evolution of the system should not depend on the past history of the sample. This is not what is observed. Even after 1 ps, when one would expect the energy transfer to be complete, there are still major excitation wavelength-dependent differences between the difference spectra in the 760 and 800 nm regions. These differences persist throughout the first few picoseconds when most of the charge separation is occurring and to a small extent even well after charge separation. (See the 760 nm region in Figure 3.) A likely explanation for the presence of excitation wavelengthdependent absorbance changes after the initial energy transfer process is that when energy transfer occurs, it leaves the donor ground state, and possibly the acceptor excited state, vibrationally hot (or in a thermally unequilibrated distribution of conformations) after transfer is complete. Vibrational relaxation on the time scale of picoseconds and tens of picoseconds has been observed previously in porphyrin systems48,49 and could explain the additional bleaching at the wavelength of excitation in the ground state bands of H or B when these cofactors are excited directly (Figures 2 and 4). One would expect that vibrationally hot H or B molecules would absorb at a somewhat different energies than thermally equilibrated forms of H or B, leaving a hole at the excitation wavelength, and this is consistent with the observed spectral evolution. In addition, there are ground state bleaching bands that shift or broaden. Shifting and broadening of the ground state bleaching can be seen with 744, 794, and 812 nm excitation in Figure 2. As discussed previously for similar effects seen upon exciting at specific wavelengths in the QY band of P, such behavior can be explained in terms of dephasing and vibrational relaxation.36,37 Evidence for vibrational relaxation in the excited state of P can be seen in the analysis of the stimulated emission. Figure 8B shows that the amplitude spectrum of the 2 ps stimulated emission decay is successively red-shifted and broadened with lower energy excitation. (There is a 5-10 nm red shift in the peak of the 2 ps component using 860 nm excitation vs using 760 nm excitation.) This is apparently not due to photoselection of particular static ground state configurations since the spectrum of the ground state bleaching due to P+ formation is excitation wavelength independent (Figure 8A). Other studies involving coherent dynamics and transient hole burning have suggested that P* is not vibrationally relaxed on the time scale of electron transfer.32,33,35-37,42 If thermalization among local and bath
J. Phys. Chem., Vol. 100, No. 42, 1996 17075 modes in B and H is also slow, as suggested above, then it is likely that none of the states involved in initial electron transfer rapidly relax to their lowest vibrational levels during energy or electron transfer. Thus, the early spectral evolution when either H or B is excited probably consists of a combination of ultrafast energy transfer and vibrational relaxation. The idea that energy transfer between H, B, and P occurs from vibrationally unrelaxed states in this system has also been put forth by others.21 Nuclear Movement along the Reaction Coordinate May Limit the Rate of Energy and Electron Transfer. Slow vibrational relaxation and ultrafast energy transfer suggests another possible way of viewing the early spectral evolution that results in reaction center charge separation. It is unlikely that energy transfer on this time scale can be described by the weak dipole-dipole Fo¨rster interaction between the lowest energy transitions of B, H, and P.23,27 Thus, some other interaction between cofactors must be occurring. One possibility is electronic exchange. (The similarity of electron transfer and energy transfer and the involvement of electron exchange are discussed in ref 50.) This mechanism requires relatively strong electron coupling for electron transfer between cofactors. The phrase “strong coupling” needs to be defined more precisely. In this context, strong coupling will be used to mean coupling between cofactor orbitals which is strong enough to allow for rapid exchange or transfer of electrons. It is not necessarily strong enough to result in large perturbations of transition energies, though looking at the 0 ps spectra of Figure 4, 812 nm excitation (the upper exciton band of P) and 860 nm excitation (P) both result in small but significant changes in the 760 nm region (H) that could represent bandshifts or other small perturbations of the QY transition of H upon exciting P. (Instantaneous absorbance changes in the H band upon exciting P have been observed previously.51) Note that 812 nm is in the spectral region that one would expect to find the upper exciton band of P. (Exciton transfer between B and the upper exciton band of P is discussed in ref 20.) Thus, excitation at 812 and 860 nm may have similar interactions with the 760 nm band. Strong coupling between H, B, and P and slow vibrational relaxation suggest that both the energy and electron transfer process may in fact be rate limited by vibrational relaxation or nuclear motion in the system. This would explain the time delays seen in the bleaching at 870 nm or the onset of the stimulated emission when the reaction center is excited in the H or B bands. If one is limited by nuclear motion along a reaction coordinate, then the process of product formation is no longer governed by a constant instantaneous probability of transfer, resulting in an exponential decay of the donor excited state. Instead, there is a period where no transfer can occur while the system moves sufficiently far along the nuclear coordinate(s) coupled to the reaction. If energy transfer occurs via electron exchange, as suggested above, then energy and electron transfer have much in common in terms of the electronic coupling involved. However, the nuclear part of the two processes is quite different. Obviously, the energetics of energy and electron transfer will be different since electron transfer involves the separation of charge and energy transfer does not. Perhaps more importantly, if the reaction is limited by nuclear movement along the reaction coordinate, the magnitude of the nuclear displacements that the system must undergo during the process of electron transfer and subsequent relaxation events should be much larger than that necessary during energy transfer. This explains the relative rates of energy and electron transfer. Energy transfer is fast because the nuclear displacements are small. Electron transfer
17076 J. Phys. Chem., Vol. 100, No. 42, 1996 is much slower, even though both involve movement of electrons between cofactors, because the distance traveled along the nuclear coordinate is much larger. In this view, electron transfer is essentially an adiabatic process. The possibility that reaction center electron transfer is adiabatic has been suggested previously based on the weak driving force and temperature dependence of the electron transfer rate, on results from holeburning measurements, and on theoretical grounds.28-31,52 Electron Transfer along the A Branch vs the B Branch. Considering this viewpoint, it is interesting to note that the rate of energy transfer, and therefore the coupling for electron exchange, is not very different whether one excites A-side or B-side cofactors of the reaction center. By exciting on either the higher or lower energy side of the H band (at 744 and 760 nm, respectively), it is possible to change the relative distribution of excitation between HA and HB. Excitation at 760 nm favors the bacteriopheophytin on the A side and results in a narrow bleaching band at 760 nm (Figure 2). A spectrally broader absorbance decrease, with substantially more bleaching on the high-energy side, is seen following 744 nm excitation. HB is apparently on the higher energy side of the bacteriopheophytin near-infrared transition,53 and thus one would expect that 744 nm excitation would substantially favor excitation of HB (as well as generate a higher vibrationally excited initial state of HA). The kinetic traces in Figure 6 suggest that there is a slower energy transfer when 744 nm excitation is used compared to 760 nm excitation, but transfer still occurs all the way to P within a few hundred femtoseconds. This point is most easily seen in Figure 2. The 0.28 ps spectrum using 744 nm excitation involves about as much apparent energy transfer to B and P as does the 0.19 ps spectrum using 760 nm excitation. If this represents electron exchange interactions, then it implies that there is ample electronic coupling between the B-side cofactors and P for electron transfer to occur. What determines the slow rate of electron transfer along the B-branch is either the energetics or the nuclear displacement involved. Boxer and coworkers have recently arrived at a very similar conclusion by monitoring P* fluorescence with femtosecond time resolution after preferentially exciting either the A or B side cofactors.23 This conclusion is also consistent with the conclusions of Heller et al.17 and other recent work on reaction center symmetry.54-56 Charge Separation in the Adiabatic Regime. An adiabatic picture for electron transfer in the reaction center helps to explain a number of past and present observations concerning the intermediates involved in the charge separation process and their spectral properties. The amount of stimulated emission (and by inference the amount of P* formed) is not the same when shorter wavelength excitation is used compared to using 860 nm excitation. (The effect is particularly evident comparing 760 and 860 nm excitation.) This can be seen in Figure 8A, which shows the longer time scale (picosecond) global exponential decay analysis of the spectral region including P’s QY band and the stimulated emission. The amplitude and area of the 2 ps decay spectra (which represents predominantly the decay of the stimulated emission in this region) are decreased by more than 20% using 760 nm excitation compared to 860 nm excitation. (See Results section.) If one identifies the stimulated emission level with the amount of P*, this suggests that charge separation does not always occur from P* (or alternatively that the characteristics of P* depend on how the state is formed). One possibility is that this could represent charge separation directly from the excited states of H and B. Figure 5 shows the difference at 0.5 and 1 ps between excitation into H or B and excitation directly into P. In the case of H excitation (744 or 760 nm), one sees extra bleaching in both
Excitation Wavelength Dependence of Reaction Centers the H and B bands at early times during the reaction. In the case of B excitation, additional bleaching in the 800 nm B band is observed. Part of the bleaching could be explained in terms of direct formation of P+BA-, P+HA-, or B+H- from H* or B* without passing through P*. (Part is probably also due to incomplete vibrational relaxation of B and H after energy transfer, as described above.) Viewed in terms of an adiabatic reaction, one could describe the reaction by stating that the trajectory along the potential surface between H* or B* and the final charge-separated state is different from the trajectory when the reaction starts at P*. The trajectory followed when higher energy excitation is used passes through regions of the potential surface in which the relative contributions from the P* basis state (defined in terms of stimulated emission which is really just a region of the potential surface with good Franck-Condon overlap with the ground state) is smaller than is seen when 860 nm excitation is used. A multiple pathway electron transfer scheme in the purple bacterial reaction centers was suggested previously based on the electronic-vibronic coupling model. (This did not involve adiabatic electron transfer, just multiple pathways.57) Involvement of B+H- has also been considered in model calculations of electron transfer rate constants in the reaction center58,59 and to explain yield losses during B* to P* energy transfer.60 Spectral Analysis of the Complex Kinetics Associated with Early Charge Separation. One can also interpret the complex kinetic and spectral changes in the 830-950 nm region (Figure 8) in terms of adiabatic electron transfer. As has been seen previously, the decay of the stimulated emission is not single exponential.29,43-45,61 This kinetic complexity has often been attributed to a static heterogeneity in the reaction center population, such that some reaction centers decay with a 2-3 ps time constant and others with a 10-20 ps time constant. Looking, however, at the amplitude spectrum associated with the 14 ps decay component (Figure 8), the static heterogeneity explanation appears incorrect. (A similar conclusion was reached through analysis of low-temperature hole-burning measurements.62) This component has only a minor contribution in the stimulated emission region (near 920 nm) and has its major amplitude, in the positive direction, in the region of the QY band of P. What this shows is that on the 14-16 ps time scale there is a small amount of stimulated emission decay and a more significant amount of additional bleaching of the ground state band of P. It is not entirely clear what process this spectral change represents, but extending the adiabatic, nonvibrationally relaxed model described above to longer times, one might expect that with time the system would settle more and more into the lower regions of the potential energy surface. This may result in a radical pair that is more and more relaxed into a particular potential well. If moving toward the bottom of this well results in a purer and purer P+ state and less and less contribution from other basis states such as B+H- that do not involve P, then the amount of bleaching in the QY band of P will increase. At the same time, the system will be moving farther and farther from P* both in energy and in nuclear displacement, and thus there will also be some decrease in the amount of stimulated emission. This also explains why the 14 ps component’s amplitude in the 870 nm region becomes more pronounced using 760 nm excitation. As described earlier, with 760 nm excitation, charge separation appears to occur with a smaller intermediate contribution from the P* state and with a greater contribution from basis states which do not involve P. The nuclear configuration of the charge-separated state can continue to relax on longer time scales as has been discussed previously,28,29 resulting in a very nonexponential decay of the
Lin et al. emission and stimulated emission from the system. Some of these longer time scale relaxation events will involve significant activation energies and thus be temperature dependent, as has been observed.28,29,37 Slow vibrational relaxation and relatively strong coupling also offers a possible explanation for much of the temperature and driving force independence of the reaction center electron transfer rate.29,52,63-65 If energy from photon absorption (or energy transfer) is coupled into modes critical for electron transfer, then the ambient temperature will have much less effect on the overall reaction rate. Similarly, changing the energy of the crossing point between the excited state and charge-separated state potential energy surfaces by changing the reaction free energy is much less critical if electronic coupling is strong and energy remains localized in modes coupled to electron transfer for times comparable to or longer than the rate of charge separation. Anomalous Spectroscopic Properties of the State Normally Assigned to P+HA-. There are at least two aspects of the difference absorbance spectrum which appears on the several picosecond time scale which are not what one would necessarily expect from the state P+HA-. First, the absorbance decrease at 760 nm due to HA reduction appears to be smaller than would be expected from the ground state spectrum.66 This can also be seen by comparing the size of the ground state bleaching due to formation of the excited state of H immediately after 760 nm excitation to the size of the bleaching at 760 nm after charge separation (the 0 and 8 ps spectra, respectively, in Figure 2), though the comparison is complicated by the 780 nm absorbance increase upon charge separation. These two negative absorbance change spectra have about the same magnitude in Figure 2, and given the fact that H* has only a fractional population at any time due to the very rapid (less than 100 fs) rate of energy transfer from H to B, the actual bleaching that results from forming H* must be much larger than that seen after initial charge separation (when HA is presumably an anion). This suggests that in the 25 ps charge-separated state (Figure 3) there is not stoichiometric bleaching of the bacteriopheophytin ground state transition at 760 nm due to anion formation, in agreement with earlier reports.66 Second, if one generates the apparent HA- difference spectrum by photopumping53 or if one calculates the apparent HA- difference spectrum by subtracting the spectrum of P+QA- from the spectrum at 25 ps (normally assumed to represent P+HA-),7,67 one finds that the 25 ps charge separation intermediate involves not only bleaching of the 760 nm H band but an even larger bleaching of the 800 nm B band as well. Various explanations have been given for these anomalies.1 There is a small yield loss upon exciting at 760 nm,47 and this would result in a slightly smaller bleaching in the anion form of H than in the excited state. In addition, there could be compensating absorbance changes which partially cancel the ground state bleaching of the 760 nm H band due to band shifts66 or due to excited state or ion absorbances. It is not clear whether such effects could completely account for the apparently small size of the 760 nm bleaching upon charge separation. Lowtemperature measurements similar to the room temperature measurements described here are underway to investigate these possibilities. These effects probably do not account for the large bleaching near 800 nm associated with the apparent P+HA- state. Another possible explanation comes from theoretical calculations which have suggested that in the Rp. Viridis reaction center the QY band of B borrows intensity from the QY band of H,14 effecting the size of both the ground state absorbance bands of H and B. The intensity borrowing model predicts that when H
J. Phys. Chem., Vol. 100, No. 42, 1996 17077 is removed from its ground state by forming either an anion or an excited state, one should see bleaching in both the H and B bands. This is consistent with the observed bleaching in both the H and B bands when HA- is formed and with the results of 760 nm excitation in Figure 4 where essentially instantaneous bleaching of both H and B bands is observed. However, according to this model, one should be able to excite in the B band near 800 nm and see an instantaneous absorbance increase near 760 nm with the spectral shape of the H band. This is not observed in Figure 4, a fact which is not easily reconciled with intensity borrowing between H and B. The same measurements also rule out coupling between the H to H* and the B to B* transitions that is strong enough to explain the bleaching of both the H and B bands in the HA- state. If these transitions were coupled that strongly, one would expect an instantaneous decrease in both the H and B bands upon excitation of B. This reopens the question of just what interactions give rise to the spectral properties of the anion present during charge separation on the 5-50 ps time scale in bacterial reaction centers and perhaps even to the assignment of the charge-separated state on this time scale as purely P+HA-. Acknowledgment. The authors gratefully acknowledge K. Carty for preparation of reaction center samples. Also, we acknowledge Drs. W. Parson, J. Peloquin, J. Allen, and J. Williams for helpful discussions. This work was supported by Grants DMB91-585251 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. 306 from the Arizona State University Center for the Study of Early Events in Photosynthesis. 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) Martin, J.-L.; Vos, M. H. Annu. ReV. Biophys. Biomol. Struct. 1992, 21, 199. (5) Kirmaier, C.; Holten, D. In The Photosynthetic Reaction Center; Deisenhofer, J., Norris, J. R., Eds.; Academic Press: San Diego, 1993; Vol. II, p 49. (6) Zinth, W.; Kaiser, W. In The Photosynthetic Reaction Center; Deisenhofer, J., Norris, J. R., Eds.; Academic Press: San Diego, 1993; Vol. II, p 71. (7) Woodbury, N. W.; Allen, J. P. In Anoxygenic Photosynthetic Bacteria; Blankenship, R. E., Madigan, M. T., Bauer, C. E., Eds.; Kluwer Academic Publishers: Dordrecht, 1995; Vol. 2, p 527. (8) Deisenhofer, J.; Epp, O.; Miki, K.; Huber, R.; Michel, H. J. Mol. Biol. 1984, 180, 385. (9) Allen, J. P.; Feher, G.; Yeates, T. O.; Komiya, H.; Rees, D. C. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5730. (10) Chang, C.-H.; El-Kabbani, O.; Tiede, D.; Norris, J.; Schiffer, M. Biochemistry 1991, 30, 5352. (11) Chirino, A. J.; Lous, E. J.; Huber, M.; Allen, J. P.; Schenck, C. C.; Paddock, M. L.; Feher, G.; Rees, D. C. Biochemistry 1994, 33, 4584. (12) Ermler, U.; Fritzsch, G.; Buchanan, S. K.; Michel, H. Structure 1994, 2, 925. (13) Warshel, A.; Parson, W. W. J. Am. Chem. Soc. 1987, 109, 6143. (14) Parson, W. W.; Warshel, A. J. Am. Chem. Soc. 1987, 109, 6152. (15) Vermeglio, A.; Paillotin, G. Biochim Biophys. Acta 1982, 681, 32. (16) Johnson, S. G.; Tang, D.; Jankowiak, R.; Hayes, J. M.; Small, G. J. J. Phys. Chem. 1990, 94, 5849. (17) Heller, B. A.; Holten, D.; Kirmaier, C. Science 1995, 269, 940. (18) Breton, J.; Martin, J.-L.; Migus, A.; Antonetti, A.; Orszag, A. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 5121. (19) Breton, J.; Martin, J.-L.; Migus, A.; Antonetti, A.; Orszag, A. In Ultrafast Phenomena V; Fleming, G. R., Siegman, A. E., Eds.; SpringerVerlag: Berlin, 1986; p 393. (20) Breton, J.; Martin, J.-L.; Fleming, G. R.; Lambry, J.-C. Biochemistry 1988, 27, 8276.
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