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Excitation Wavelength Dependence of Primary Charge Separation in Reaction Centers from Rhodobacter sphaeroides Haiyu Wang, Su Lin, and Neal W. Woodbury* Biodesign Institute at Arizona State UniVersity and Department of Chemistry and Biochemistry, Arizona State UniVersity, Tempe, Arizona 85287-5201 ReceiVed: July 3, 2008; ReVised Manuscript ReceiVed: September 8, 2008
The excitation wavelength dependence of the initial electron transfer rate in both wild type and mutant reaction centers from Rhodobacter sphaeroides has been studied between 840 and 920 nm as a function of temperature (10-295 K). The dynamics of primary charge separation show no resolvable excitation wavelength dependence at room temperature over this spectral range. A small variation in rate with excitation wavelength is observed at cryogenic temperatures. The low temperature results cannot be explained in terms either of a nonequilibrium model that assumes that the primary charge separation starts from a vibrationally hot state or a model that assumes a static inhomogeneous distribution of electron transfer driving forces. Instead these results are consistent with the concept that primary charge separation kinetics are controlled by the dynamics of protein conformational diffusion. Introduction The initial energy conversion event in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides involves electron transfer from the excited singlet state of a bacteriochlorophyll dimer (P*) via a monomer bacteriochlorophyll (BA) to a nearby bacteriopheophytin (HA). This reaction shows an unusual temperature and driving force dependence in which the electron transfer rate increases slightly in wild type reaction centers with decreasing temperature1,2 while simultaneously responding only weakly to changes in driving force.3 In addition, the kinetics of electron transfer in essentially all of these mutants and at all temperatures is kinetically heterogeneous.4-6 Recently, this laboratory has used tryptophan absorbance in RCs as a natural probe of ultrafast protein conformation diffusion coupled with a reaction diffusion formalism to quantitatively describe the detailed kinetics of electron transfer as a function of temperature in wild type reaction centers and a series of mutants with altered driving force values for the primary charge separation.7 It was possible to fit this data varying only the driving force between the different mutants and the reorganization energy as a function of temperature. In these fits, the driving force was essentially constant with temperature for any particular mutant and other model parameters (vibrational frequency and coupling) were held constant in all samples at all temperatures. According to this model, the initial charge separation in photosynthesis is limited by protein dynamics, rather than by a static electron transfer rate. While the reaction diffusion model is very successful at describing the complex electron transfer kinetics of the bacterial photosynthetic reaction center, it is not the only possible model for describing kinetically complex electron transfer. One thing that differentiates these models from one another is the source of the heterogeneity in the kinetic decay (static conformational differences, equilibrium between multiple forms, dynamic progression of conformational forms, and so forth). Depending on the type of heterogeneity, detailed reaction center kinetics * To whom correspondence should be addressed. E-mail: Nwoodbury@ asu.edu. Tel: 480-965-3294. Fax: 480-727-0396.
should be more or less sensitive to the wavelength of excitation, particularly at low temperature. Here, a detailed exploration of the temperature dependence as a function of temperature is performed for wild type and several mutants. Static Heterogeneity. One key aspect of the reaction diffusion model is that protein dynamics occurs on the time scale of electron transfer. One could instead consider a model in which reaction centers have many conformational substates that are static on the time scale of electron transfer. Such a model results in a distribution in the population of energy levels and in a static inhomogeneous distribution of rates.4,6,8 At cryogenic temperatures, the homogeneous line width associated with the lowest energy transitions of the initial electron donor narrows, and it generally becomes possible to selectively excite different substates with a spectrally narrow laser pulse. Therefore, this model predicts a substantial excitation wavelength dependence of the electron transfer rate. Nonequilibrium Excited State. It is also possible that charge separation occurs from one or more unrelaxed vibrational states of the excited electron donor. This would result in a timedependent charge separation rate which would give rise to the observed kinetic heterogeneity of P* decay in the reaction center. This concept is supported by measurements using 30 fs excitation pulses which have suggested that the vibrational relaxation may not be complete during electron transfer.9-14 Further, in mutants that greatly decrease the overall free energy change for electron transfer and thus would be expected to have an increased activation energy using conventional electron transfer models (e.g., Marcus Theory15), rapid electron transfer at cryogenic temperature is still observed. Again one possible explanation of this behavior is that electron transfer takes place from vibrationally hot states. A number of nonequilibrium theories have been developed to address charge separation initiated from vibrationally hot states.16,17 The most general theory, in this respect, arises from the so-called density matrix method.18,19 This considers multivibrational modes involved in the charge separation process, and the mode parameters are obtained by molecular dynamics simulations. This approach has been reasonably successful at explaining the unusual temperature
10.1021/jp8058799 CCC: $40.75 2008 American Chemical Society Published on Web 10/22/2008
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dependence of the charge separation rate.18,19 Recent calculations using this approach indicate that the charge separation rates should be strongly dependent on the wavelength of excitation in the dimer band, at least at cryogenic temperatures. To further clarify these electron transfer mechanisms in RCs, the wavelength and temperature dependence of the initial electron transfer rate has been explored in detail, particularly on the red edge of the P band at low temperatures. Materials and Methods The preparation of His-tagged Rhodobacter sphaeroides wild type reaction centers expressed from a pRK-based plasmid was performed as described previously.20 The construction and expression of the mutants L131LfH(L131LH), L131LfH/M160LfH (L131LH+M160LH), L153HfS(L153HS), M203GfL(M203GL), and M210YfF(M210YF) were also described previously.3,21-23 For room temperature measurements, all reaction centers were suspended in 15 mM Tris-HCl (pH 8.0), 0.025% N,N-dimethyldodecylamine-N-oxide (LDAO), 1 mM EDTA, and 0.1 mM orthophenanthroline. Room temperature samples were loaded in a spinning wheel with an optical path length of 1.2 mm. A final optical density in the 1.2 mm path of roughly 1.0 at 802 nm was used. For low temperature experiments, the reaction center quinones were removed as described previously24 and the samples were suspended in 15 mM Tris-HCl (pH 8.0), 0.025% LDAO, 1 mM EDTA and then mixed with 67% (v/v) glycerol. The samples were placed between two glass plates with a rubber spacer of 1.2 mm and then attached to the coldfinger of a closed circulated helium displex (APD) capable of holding temperatures between 300 and 10 K. The femtosecond transient absorption spectroscopy was performed as follows. A titanium sapphire oscillator (Tsunami, Spectra-Physics) was used to generate 100 fs, 800 nm laser pulses at a repetition rate of 82 MHz. These pulses were used to seed an optical regenerative amplifier system (Spitfire, Spectra-Physics), resulting in pulses of approximately 0.8 mJ at a repetition rate of one KHz. Part of the pulse energy (∼10%) was then used to generate a white light continuum by focusing the beam into a sapphire plate, and this was used for the probe beams. The remainder of the amplified 800 nm pulse was used to pump an optical parametric amplifier (OPA-800, SpectraPhysics) generating excitation pulses at wavelengths between 840 and 920 nm (second harmonic of idler beam). To get spectrally narrow pulses, 5 or 8 nm band-pass interference filters were used in the excitation beam; the final full width at halfmaiximum (fwhm) of excitation pulse spectrum was less than 8 nm. Transient absorption changes were measured at 930 and 950 nm (stimulated emission from P*) using a monochromator (SP150, Action Res. Corp.) and a diode detector (Model 2032, New Focus Inc.). The relative polarization of the excitation and probe beams was set to the magic angle. The excitation intensities were kept below 500 nJ per pulse for all measurements reported here (except 920 nm excitation, where 2 µJ per pulse was used because of the very low extinction coefficient of the sample at this wavelength), and the excitation spot size was 0.5 mm in diameter. Results Wild Type. Figure 1a shows the time course of the transient absorbance signal at 930 nm from wild type Rhodobacter sphaeroides reaction centers using different excitation wavelengths at room temperature. The kinetics are essentially invariant with excitation wavelength and fit well to two
Figure 1. Time dependent changes in absorbance probed at 930 nm (stimulated emission of P*) at room temperature (top) and 10 K (bottom). The excitation wavelengths are also shown. The curves are offset for clarity.
TABLE 1: The Kinetic Fitting Parameters Obtained upon the Analysis of 930 nm Transients (Stimulated Emission of P*) from Wild Type Reaction Centers Using Different Excitation Wavelengths at Room Temperaturea excitation (nm)
τ1 (ps)
A1 (%)
τ2 (ps)
A2 (%)
840 860 880 905 920
2.27 ( 0.06 2.32 ( 0.03 2.29 ( 0.03 2.17 ( 0.06 2.1 ( 0.1
66 70 70 67 67
6.5 ( 0.3 6.5 ( 0.2 6.3 ( 0.2 6.9 ( 0.4 6.6 ( 0.7
34 30 30 33 33
a
The errors of the amplitudes A1 and A2 are less than 5%.
exponentials with time constants (and relative amplitudes) of 2.3 ps (67%) and 6.5 ps (33%) (Figure 1a and Table 1). Previous measurements of P* kinetics using either transient absorption or fluorescence up-conversion have also shown a similar nonexponential decay.2,4,5 However, the reported time constants and amplitudes differ significantly from one report to another. This is a natural consequence of differences in the signal-tonoise ratio of the measurement as well as the time scale over which measurements are taken (particularly in terms of the time constant for the long component and its relative amplitude). Measurements of the antenna decay kinetics in LH1/RC preparations also show a biexponential phase that is reaction center dependent.25 This suggests that the kinetic complexity is not simply an artifact of reaction center isolation, but is present even in intact photosynthetic membranes.26 The independence of the kinetics with excitation wavelength indicates that at room temperature the entire spectral band is approximately homogeneous with respect to the kinetic decay. Figure 1b shows the low temperature (10 K) kinetics probed at 930 nm using different excitation wavelengths. As seen at room temperature, the decay profile of each transient can be fitted well to two exponentials (Table 2). The shorter of the two time constants is more than 2-fold faster at low temperature than at room temperature, consistent with previous temperature dependent studies.1,2 The slower time constant has a value of approximately 9 ps which is more or less constant within the noise (this component is hard to measure because it is a small fraction of the overall decay). In contrast to the results at room temperature, a small but significant excitation wavelength dependence of the relative amplitudes of the two components
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Figure 2. Temperature dependence of the fast charge separation time constants and amplitudes of the slow component at 905 (top) and 860 nm excitation (bottom).
is observed as the excitation wavelength approaches the red edge of the spectrum. The amplitude of the slower component is about 8-9% at the blue side of the P-band and becomes undetectable at the far red side; the trace obtained using 920 nm excitation is well fitted by single exponential. The complete temperature dependence of the P* (stimulated emission) decay was measured in detail using two excitation wavelengths, 860 and 905 nm (Figure 2). A striking aspect of this dependence is that as the temperature decreases, the decrease in the faster time constant closely tracks the decrease of the amplitude of the slower component. The temperature dependence study described above was repeated measuring the stimulated emission at 950 nm instead of 930 nm and nearly the same time constants and amplitudes were observed as a function of excitation wavelength and temperature. This is also true for the measurements in the mutants we used (see below). Previous spontaneous emission decay measurements of P* by fluorescence up-conversion methods have also given time constants that were nearly independent of the detection wavelength.4 Mutants. In addition to wild type, temperature dependent measurements were also performed on five reaction center mutants. The electron transfer rate of mutant L153HS clearly accelerates at lower temperature.7 The electron transfer rates of the mutants L131LH, M210YF, M203GL, and L131LH+ M160LH slow down substantially at low temperature and the kinetics become highly nonexponential with components on all time scales from several picoseconds to several hundred picoseconds. The overall electron transfer kinetics (the value obtained by dividing the total area of the decay curve by the initial amplitude) between the different mutants measured varies by more than 1 order of magnitude at room temperature and by 2 orders of magnitude at cryogenic temperature. The excitation wavelength dependence was measured at room temperature (Figure 1) and 10 K for each mutant (Figure 3). At room temperature, the rates are excitation wavelength independent for all mutants (data not shown), as was the case in wild type. At 10 K, as in wild type, the overall electron transfer rates become faster when the excitation wavelength increases for all mutants except L131LH. The effect is more pronounced in the mutants with slower charge separation rates. In the mutant
Figure 3. Excitation wavelength dependent changes in the kinetics of absorbance changes probed at 930 nm (stimulated emission of P*) for various mutants at 10 K.
L131LH, the electron transfer shows no excitation wavelength dependence at either temperature. Discussion The unusual temperature dependence and nonexponetial charge separation kinetics of reaction centers have been extensively studied by various theoretical models. These can be largely divided into two categories: models using nonequilibrium states,16-19,27 and models involving inhomogeneous line shapes.6,28 Inhomogeneous Model. The inhomogeneous model considers that reaction centers have many conformational substates that are static on the time scale of electron transfer. The distribution in the population of energy levels created by the conformational substates can be represented by a Gaussian function. For each substate in this model, the charge separation process is described by Marcus theory. The evolution of P*(t) as a function of time can be written as6,28
P*(t) ) )
∞ d(∆G)g(∆G)exp[-k(∆G)t]k(∆G) ∫ -∞
[
(∆G + λ)2 2π V2 exp p 4πλk T 4λkBT √ B
]
(1)
where V is the electronic coupling matrix element, λ is the reorganization energy, ∆G is the free energy gap between the reactants and the products, and g(∆G) is a Gaussian distribution of ∆G. In general, this model gives a heterogeneous decay function for P*. This model was proposed based on several experimental observations including wavelength dependent dynamics and the heterogeneity suggested by the spectral line width of P.4,8 In this model, any particular excitation wavelength encompasses a fraction of the ∆G distribution in eq 1. Given the degree of kinetic heterogeneity observed at all temperatures, particularly for some of the mutants, one would expect that if eq 1 holds, a strong dependence of electron transfer kinetics on excitation
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TABLE 2: The Kinetic Fitting Parameters Obtained upon the Analysis of 930 nm Transients (Stimulated Emission of P*) from Wild Type Reaction Centers Using Different Excitation Wavelengths at 10 Ka excitation (nm)
τ1 (ps)
A1 (%)
τ2 (ps)
A2 (%)
840 860 880 894 905 920
1.08 ( 0.01 1.03 ( 0.01 1.04 ( 0.01 1.05 ( 0.01 1.04 ( 0.01 1.01 ( 0.04
92 91 92 95 96 100
9(1 8.7 ( 0.7 8.7 ( 0.6 9(1 9(1
8 9 8 5 4
a
The errors of the amplitudes A1 and A2 are less than 5%.
wavelength would result, particularly at low temperature. Instead, what is observed is that electron transfer has essentially no excitation wavelength dependence at room temperature and only a weak dependence at low temperature. In addition, particularly toward the blue end of the spectrum, even excitation with spectrally narrow light generally results in multiple different exponential decay components (Tables 1 and 2 and Figure 3). This is most evident in the mutants with slower overall electron transfer rates (Figure 3). Thus, it is unlikely that a simple model involving static distributions of substates is the primary explanation for the nonexponential kinetics associated with initial electron transfer. The fact that the mutant L131LH shows no excitation wavelength dependence in its rate constant at all, further supports this conclusion; it would be very difficult to imagine how the L131LH mutation would eliminate the excitation wavelength dependence but not reduce the kinetic heterogeneity in the context of a static heterogeneity model. This model also does not explain the correlation between the temperature dependence of the time constant for the fast component of electron transfer and the amplitude of the slow component (Figure 2). Nonequilibrium Excited-State Model. Another way to explain the kinetic heterogeneity of the P* decay is through a nonequilibrium state model, in which the charge separation occurs from an unrelaxed vibrational state of the excited electron donor. In general, this would result in a time-dependent charge separation rate and thus a nonexponential decay of P*. First, consider a simple treatment using an elementary timedependent perturbation theory, which gives a clear physical picture of the nonequilibrium state model. Assuming that the bath consists of harmonic oscillators and that the modes are linearly coupled to the reactant and the product states, the timedependent charge separation rate can be expressed as16
k(t) )
[
2π [∆G + -Q(t)]2 exp 2 < U2>
]
(2)
Where U represents the magnitude of the bath fluctuation energy (at the classical or high temperature limit, U∼2kBTλ), and Q(t) is the time-dependent reorganization energy (relaxation of the nonequilibrium state). This differs from the Marcus expression is that the reorganization energy is time-dependent. According to this model, the slower decay rate of P* observed should approximately represent the charge separation rate after reaching the final vibrationally equilibrated state. Since the charge separation starts from a nonequilibrium state, the initially prepared state (nuclear coordinate) will strongly influence the kinetics of charge separation. Thus, one would expect to observe a clear excitation wavelength dependence in the kinetics of P* decay, especially
at cryogenic temperatures. There is essentially no wavelength dependence in any sample tested at room temperature, even at the very red edge of the P band. Though some excitation wavelength dependent behavior is seen in the low temperature kinetics of Table 2 and Figure 3 (in terms of the amplitude of the long component), the dependence is generally weak for all samples tested. In addition, the trend in the dependence is opposite of what one would predict. In particular, the kinetics using 920 nm excitation for wild type contains only the fast component of electron transfer, no slow component (see Figure 1b). One would expect that excitation at the red end of the P band would result in an initial state that is closer to being vibrationally relaxed and thus favor the slower (8-9 ps) electron transfer rate, opposite of what was observed. A similar trend is seen in the mutants (Figure 3); as the excitation wavelength is moved to the red, the faster components become more prominent. A more general treatment of nonequilibrium theory using density matrix methods considers the effects of multiple vibrational modes in the charge separation process. In this case, the mode characteristics are determined through molecular dynamics simulations.18,19 This approach has provided a plausible explanation of the unusual temperature dependence of the charge separation rate.18,19 However, as with the simple nonequilibrium model described above, this model results in heterogeneous P* decay kinetics and the prediction that the slowest rate will occur on the red edge of the spectrum, contrary to observation. This model, like the static heterogeneity model, also offers no explanation for Figure 2. Reaction Diffusion Model. In the reaction diffusion model, the heterogeneous electron transfer kinetics are due to the fact that the electron transfer rate is limited by the exploration of protein conformational space. Effectively, conformational diffusion occurs until a configuration with a low activation energy for electron transfer is achieved (the driving force and the free energy cancel in the Marcus formalism). One would not expect a strong excitation wavelength dependence to the electron transfer kinetics from this model. The extent to which any wavelength dependence would be observed should depend on the coupling between excitation wavelength and either the driving force or the reorganization energy. Thus, the fact that there is no wavelength dependence at room temperature and only a weak excitation wavelength dependence at low temperature is consistent with this model. Figure 2 now also makes sense. As described in more detail elsewhere,7 the reaction diffusion model accurately predicts the overall temperature dependence of electron transfer. Wild type reaction centers start out very close in conformational space to the point where the activation energy is zero; thus, at lower temperatures it becomes more difficult to diffuse away from this region. This results in an apparent decrease in the value of the fast time constant and a decrease in the relative amplitude of the slow component as the temperature is decreased. Conversely, as the temperature is increased there is greater diffusion away from this region resulting in the longer components of the electron transfer kinetics. In addition to being generally consistent with the weak excitation wavelength dependence (Figure 1) and the detailed kinetics as a function of temperature (Figure 2), this model also makes a testable prediction. In the context of the reaction diffusion model, the observation of a weak but significant excitation wavelength dependence at low temperature would mean that excitation wavelength is coupled to either the driving force or the reorganization energy. This effectively moves the initial position in conformational space relative to the point
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Figure 4. Comparison of the electron transfer kinetics of mutants M210YF and M203GL using different excitation wavelengths at 10 K. The same electron transfer kinetics can be obtained from different mutants by varying excitation wavelength.
where the activation energy is near zero. As a result, the time course for electron transfer changes. If this is true, there is not much difference between changing the wavelength and changing between two mutants with different driving forces (both change the sum ∆G + λ and thus the distance in conformational space that must be traversed before the activation energy for electron transfer is near zero). This predicts that it should be possible to shift the excitation wavelength used for one of the mutants until the value of ∆G + λ is the same for both mutants. If this model is correct (if the initial value of ∆G + λ is all that matters), then the electron transfer kinetics for the two mutants with appropriately shifted excitation wavelengths should overlap essentially perfectly. As shown in Figure 4, this is exactly what is observed. The kinetic traces of M210YF and M203GL can be quantitatively overlapped by shifting the excitation wavelength of M203GL to the red relative to that of M210YF (this is shown for two sets of excitation wavelengths). Another point on the excitation wavelength dependence in the context of the reaction diffusion model is that it appears likely that excitation wavelength is coupled more strongly to the reorganization energy at low temperature than to driving force. Had the excitation wavelength been coupled to driving force, one would have expected that the initial driving force would be larger at shorter wavelengths (because shorter wavelengths would favor excitation of reaction center conformations in which P* is at higher energy). As shown previously in wild type reaction centers, increasing the driving force speeds up the overall reaction,1,2 presumably because ∆G + λ is smaller under these conditions. Starting with a higher driving force conformation thus means the path to the region of near zero activation energy is shorter. If this were true, faster overall electron transfer should be observed using higher energy excitation, opposite of what is observed. However, if excitation wavelength was coupled more strongly to reorganization energy (to the equilibrium position on the reaction coordinate of the potential wells), it would be quite possible for a lower excitation energy to result in overall faster electron transfer, which is what is observed. Finally, it is interesting that the mutant L131LH shows no excitation wavelength at all for any temperature measured. This may provide additional clues into the mechanistic nature of the coupling between excitation wavelength and the energetic parameters associated with charge separation. Stark spectroscopy analyses have shown that the excited-state of P has substantial charge transfer character,29 arising predominantly through mixing of the local excited-state with an intradimer charge transfer state. If changing the excitation wavelength effectively changed the degree of mixing, this could certainly change the
Wang et al. equilibrium position of the potential well associated with P* and thus the effective reorganization energy associated with electron transfer. The mutant L131LH is known to substantially decrease both the asymmetry of the electron hole distribution in P+ 31 and presumably the charge transfer character of the excited state. Thus, it is understandable that changing the excitation wavelength might have less effect in the L131LH mutant than in mutants that have a greater charge transfer character. A more comprehensive study of the relationship between the charge transfer character of the excited-state and the electron transfer properties in the context of the reaction diffusion model is underway. Conclusions The nonexponential kinetics of the primary charge separation in reaction centers show only a weak excitation wavelength dependence at low temperature and essentially no excitation wavelength dependence at room temperature. This is not easily reconciled either with a model for nonexponential kinetics involving static distributions of reaction center free energies or a model in which electron transfer and vibrational relaxation are occurring on similar time scales. The weak excitation wavelength dependence is much more consistent with a model recently proposed in which the primary charge separation is controlled by protein relaxation. In this view, the complex charge separation process in RCs reflects diffusion through protein conformational space. This results in a complex electron transfer decay profile which would not necessarily be coupled in a direct way to the excitation wavelength dependence. Acknowledgment. We thank Dr. Evaldas Katilius and Christa Laser for their help with the reaction center preparation and Professor Steven Boxer for the gift of M210YF strain. This research was supported by NSF Grant MCB0642260. The transient spectrometer used was funded by NSF Grant BIR9512970. References and Notes (1) Fleming, G. R.; Martin, J. L.; Breton, J. Nature 1988, 333, 190. (2) Huber, H.; Meyer, M.; Scheer, H.; Zinth, W.; Wachtveitl, J. Photosynth. Res. 1998, 55, 153. (3) Wang, H. Y.; Lin, S.; Allen, J. P.; Williams, J. C.; Blankert, S.; Laser, C.; Woodbury, N. W. Science 2007, 316, 747. (4) Du, M.; Rosenthal, S. J.; Xie, X.; DiMagno, T. J.; Schmidt, M.; Hanson, D. K.; Schiffer, M.; Norris, J. R.; Fleming, G. R. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 8517. (5) Hamm, P.; Gray, K. A.; Oesterhelt, D.; Feick, R.; Scheer, H.; Zinth, W. Biochim. Biophys. Acta 1993, 1142, 99. (6) Jia, Y.; DiMagno, T. J.; Chan, C.-K.; Wang, Z.; Du, M.; Hanson, D. K.; Schiffer, M.; Norris, J. R.; Fleming, G. R.; Popov, M. S. J. Phys. Chem. 1993, 97, 13180. (7) Wang, H. Y.; Lin, S.; Katilius, E.; Allen, J. P.; Williams, J. C.; Laser, C.; Woodbury, N. W. J. Phys. Chem. B, in press. (8) Kirmaier, C.; Holten, D. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3552. (9) Shuvalov, V. A.; Yakovlev, A. G. FEBS Lett. 2003, 540, 26. (10) Vos, M. H.; Jones, M. R.; Breton, J.; Lambry, J. C.; Martin, J. L. Biochemistry 1996, 35, 2687. (11) Vos, M. H.; Lambry, J.-C.; Robles, S. J.; Youvan, D. C.; Breton, J.; Martin, J.-L. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 8885. (12) Vos, M. H.; Rischel, C.; Jones, M. R.; Martin, J. L. Biochemistry 2000, 39, 8353. (13) Yakovlev, A. G.; Shkuropatov, A. Y.; Shuvalov, V. A. Biochemistry 2002, 41, 2667. (14) Yakovlev, A. G.; Shkuropatov, A. Y.; Shuvalov, V. A. Biochemistry 2002, 41, 14019. (15) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265. (16) Cho, M. H.; Silbey, R. J. J. Chem. Phys. 1995, 103, 595. (17) Ando, K.; Sumi, H. J. Phys. Chem. B 1998, 102, 10991. (18) Parson, W. W.; Warshel, A. Chem. Phys. 2004, 296, 201. (19) Parson, W. W.; Warshel, A. J. Phys. Chem. B 2004, 108, 10474.
Excitation Wavelength Dependence (20) Goldsmith, J. O.; Boxer, S. G. Biochim. Biophys. Acta 1996, 1276, 171. (21) Lin, X.; Murchison, H. A.; Nagarajan, V.; Parson, W. W.; Allen, J. P.; Williams, J. C. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10265. (22) Katilius, E.; Babendure, J. L.; Lin, S.; Woodbury, N. W. Photosynth. Res. 2004, 81, 165. (23) Treynor, T. P.; Yoshina-Ishii, C.; Boxer, S. G. J. Phys. Chem. B 2004, 108, 13523. (24) Okamura, M. Y.; Isaacson, R. A.; Feher, G. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 3491. (25) Laible, P. D.; Greenfield, S. R.; Wasielewski, M. R.; Hanson, D. K.; Pearlstein, R. M. Biochemistry 1997, 36, 8677.
J. Phys. Chem. B, Vol. 112, No. 45, 2008 14301 (26) Beekman, L. M. P.; Van Stokkum, I. H. M.; Monshouwer, R.; Rijnders, A. J.; McGlynn, P.; Visschers, R. W.; Jones, M. R.; van Grondelle, R. J. Phys. Chem. 1996, 100, 7256. (27) Aagaard, A.; Brzezinski, P. FEBS Letters 2001, 494, 157. (28) Small, G. J.; Hayes, J. M.; Silbey, R. J. J. Phys. Chem. 1992, 96, 7499. (29) Lockhart, D. J.; Boxer, S. G. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 107. (30) Rautter, J.; Lendzian, F.; Schulz, C.; Fetsch, A.; Kuhn, M.; Lin, X.; Williams, J. C.; Allen, J. P.; Lubitz, W. Biochemistry 1995, 34, 8130.
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