J. Phys. Chem. 1995, 99, 8910-8917
8910
The Nature and Dynamics of the Charge-Separated Intermediate in Reaction Centers in Which Bacteriochlorophyll Replaces the Photoactive Bacteriopheophytin. 2. The Rates and Yields of Charge Separation and Recombination Christine Kirmaier,? Laurent Laporte? Craig C. Schenck: and Dewey Holten*$? Department of Chemistry, Washington University, St. Louis, Missouri 63130, and Department of Biochemistry, Colorado State University, Fort Collins, Colorado 80523 Received: November 4, 1994; In Final Form: February 17, 1995@
The primary photochemistry of the (M)L214H and (M)L214W(L)E104V mutant bacterial reaction centers (RCs) from Rhodobacter sphaeroides has been investigated at room and cryogenic temperatures. In both mutants the native bacteriopheophytin electron acceptor (BPhL) is replaced with a bacteriochlorophyll (BChl) molecule denoted by p; in the double mutant a hydrogen-bonding interaction of is removed. The initial stage of charge separation, formation of an intermediate P T , is slowed somewhat in both mutants but without a detectable loss in yield. However, the yield of the subsequent stage of charge separation, P+I- P'QA-, is significantly reduced due to the combination of slower electron transfer from I- to QAand enhanced charge recombination of P+I- to the ground state. For example, in the double mutant the inherent time constant for electron transfer at 285 K is lengthened 10-fold to -2 ns and the inherent time constant for charge recombination is reduced 20-fold to -750 ps, giving a P'QA- yield of 27%. At 77 K, in both B-containing RCs electron transfer from I- to QA is slowed and the P+QA- yield reduced compared to the value at 285 K. The kinetic data and the spectroscopic results presented in the preceding article combine to require a substantial involvement of P+BChlL- in defining the character and dynamics of P+I- in the beta-type RCs. Models are considered in which P'D- and P'BChlL- are quantum-mechanically mixed or in thermal equilibrium. It is concluded that P'P- and PfBChlL- are very close in energy in the mutants and that P+BChlL- is very close in energy to the primary electron donor, P*, in both the mutant and wild-type RCs. We also propose that the energy of P+BChlL- is important not only in determining the dynamics of the initial stage of charge separation but also in dictating the rates and yields of the subsequent electron transfer and charge recombination processes.
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Introduction In the previous article we described extensive spectroscopic studies aimed at elucidating the nature of the charge-separated intermediate P+I- in several Rb. sphaeroides mutant bacterial reaction centers (RCs).' In the case of the (L)E104V mutant, where a hydrogen bond between glutamic acid L104 and the ring-V keto group of BPhL is removed, the assignment of the electron carrier as BPhL (Le., P+I- is P+BPhL-) can be easily made in analogy to wild-type RCs. In the (M)L214H mutant, where the native BPhL is replaced with a BChl molecule (denoted by p), and in the double mutant (M)L214W(L)E104V, where there is both the p substitution and change in hydrogen bonding to it, any of the models considered for P+I- requires adoption of one or more seemingly ad hoc assumptions for there to be consistency between all of the spectroscopic data. Two general classes of interpretation directly involve the BChlL monomer, namely that P+I- in the beta-type RCs either is P+BChlL- or has substantial character of both P';C- and P+BChlL-. Coupled with our previous finding in (M)L214H RCs that P+I- is 575 meV in free energy below the excited primary electron donor (P*),2 these models necessarily require that P+BChlL- is close in (free) energy to P*. To a first approximation, this important conclusion would in tum apply to wild-type RCs. Obviously the free energy of P+BChlL- in wild-type RCs is a critical issue in distinguishing between the "two-step" mechanism of initial charge separation (P* P+BChlLP+BPhL-) and the "superexchange" mechanism
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' Washington University.
* Colorado State University.
'Abstract published in Advance ACS Abstracts, May 1, 1995.
0022-365419512099-8910$09.00/0
where formation of P+BPhL- directly from P* is facilitated via quantum-mechanicalmixing of the three states. The free energy of P+BChlL- and the mechanism of initial charge separation remain highly controver~ial.~ One of the most signifcant differences between the photochemistry of (M)L214H and wild-type RCs is that in the mutant the charge recombination process P+Iground state is about a factor of 20 faster than P+BPhLground state charge recombination in wild-type RCs. This is a major factor underlying the -60% yield of P+QA- in (M)L214H RCs, compared to -100% in wild-type RCs. We invoked participation of P + B c h l ~ -via electronic mixing to account for the increased rate of the deactivation process.2 To validate any hypothesis regarding P+I- in beta-type RCs the spectroscopic studies described in the preceding paper must be united with the kinetic data for the pathways, rates, and yields of electron transfer and charge recombination. Such an analysis is undertaken here, where we compare the primary photochemistry at room and cryogenic temperatures for wild-type RCs and the (L)E104V, (M)L214H and (M)L214W(L)E104V mutants of Rb. sphaeroides. Through this analysis and in combination with the spectroscopic data we come to further insights regarding the involvement of P'P- and P + B c h l ~ in - the charge separation process in beta-type RCs with consequent implications for the involvement of P+BChlL- in the wild-type photochemistry.
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Experimental Section Preparation of the mutant strains, isolation of the RCs, and transient absorption studies were conducted as described in the preceding article.' For some measurements QA was either 0 1995 American Chemical Society
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The Charge-Separated Intermediate. 2
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Wavelength, nm Figure 1. Absorption difference spectra acquired at the indicated times following excitation of (M)L214W(L)E104V RCs at 285 K with an 150-fs excitation flash. Data from 720 to 850 nm were obtained using 867-nm excitation and data from 830 to 1000 nm with 582-nm excitation; the data were normalized in the region of overlap and combined to yield the spectra shown. Each spectrum represents the average of data acquired using -300 excitation flashes. These data and those in the figures that follow typically have a error in AA of rt0.003.
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Time, ps Figure 2. Decay of the stimulated emission measured at 965 nm elicited by excitation of (M)L214W(L)E104V RCs at 285 K with a 150-fs 867-nm excitation flash. The inset shows the first 10 ps of the data given in the main figure.
reduced with sodium dithionite or depleted (-90%) from the protein using methods described elsewhere: Static fluorescence studies employed a SPEX Fluorlog spectrofluorometer equipped with a silicon avalanche photodiode and lock-in detection.
Results The spectrum of P* observed 0.5 ps after excitation of (M)L214W(L)E104V RCs at 285 K is shown in Figure 1. The spectrum is characterized by bleaching of the ground state absorption of P near 870 nm and stimulated emission from P* at longer wavelengths, features that are also observed in the spectra ascribed to P* in wild-type, (L)E104V, (M)L214H, and several other mutant^.',^.^-'^ The stimulated emission decay profiles were fit to the cross correlation of two pulses plus an exponential plus a constant; typical data and fit are shown in Figure 2. The resulting time constants at 285 K (867-nm excitation) are 3.5, 5.3,5.8, and 5.8 ps for wild-type, (L)E104V, (M)L214H, and (M)L214W(L)E104V RCs, respectively (Table l ) . I 3 The P* lifetime in wild-type RCs is the same as reported previously.6-'o The P* lifetime in (L)E104V RCs is slightly longer, as was found previously for the Rb. capsulatus (L)E104L mutant.I4 At 77 K the P* lifetimes in the two P-containing
J. Phys. Chem., Vol. 99, No. 21, 1995 8911 mutants are shorter than at 285 K (see Table l), similar to what has been previously reported for wild-type R C S . ~ , ' , ~ , ' ~ During the course of P* decay there is no appreciable return to the ground state in wild-type RCs or any of the three mutants. This fact is evident from the constant amplitude of bleaching of the 870-nm ground state band of P (compare the 0.5- and 45-ps spectra of (M)L214W(L)E104V RCs in Figure 1, and see inset to Figure 3). This finding also holds true at low temperature for all four RCs. We interpret these observations to mean that PfI- forms with a quantum yield of essentially 100% at room and low temperature in all four RCs. The subsequent decay of P+I- (P+BPhL-) in wild-type and (L)E104V RCs also does not lead to significant ground state recovery but leads exclusively to formation of P+QA-. This result mirrors that found previously for wild-type RCs and for a number of mutants including Rb. capsulatus (L)E104L R C S . ~ - " , ' ~In contrast, there is a substantial branching of the decay of P+I- in both beta-type RCs, as we reported previously for the (M)L214H mutant.2 At 285 K the decay of PfI- in (M)L214W(L)E104V RCs is accompanied by a 73% decrease in the bleaching in the 870-nm ground state absorption of P (compare the 45-ps and 2.7-11s spectra in Figure 1). This means that P+I- decays 73% via charge recombination to the ground state and 27% via electron transfer to yield PfQA-. An average PfI- lifetime of 550 ps is found for decay of P's bleaching between 840 and 870 nm (Figure 3) or for decay of the broad anion band between 660 and 710 nm (see Figure 4 of the previous paper). We previously found a similar branching of the photochemistry at P+I- for (M)L214H RCs, but with a shorter P+I- lifetime (350 ps) and a higher quantum yield (60%) of P'QA- formatiom2 The P+I- lifetime lengthens and the P+QA- yield decreases in both beta-type RCs at low temperature (Table 1). For example, the P+I- lifetime is 950 ps and the P+QA- yield is 18% for (M)L214W(L)E104V RCs at 77 K. Overall, the results on the two beta-type RCs demonstrate a dramatic change in photochemistry compared to wild-type and (L)E104V RCs, where P+I- (P+BPhL-) lifetimes of 100-200 ps and a P+QAyield of -100% are observed at both room and cryogenic temperatures (Table 1). Measurements of the formation and decay of P+I- were also made in the BChl QYregion (740-820 nm); as in Figure 3, data out to -3 ns were obtained. Using the standard simple model P* P+I- -,P'QA- as the zeroth-order description of the charge separation sequence, the data in the QYregion were analyzed using the dual-exponential function AA = a be-'/'l ce-"Q (plus pulse convolution). Here, zl reflects the P* lifetime and z2 reflects the P+I- lifetime. The values of both zl and z2 obtained from this zeroth-order analysis vary significantly with detection wavelength across the QYregion, as we noted previously for (M)L214H RCs2 and described in detail for Rb. sphaeroides R-26 RCs9 A portion of the data at 285 K is shown for (M)L214H and (M)L214W(L)E104V mutants in Figure 4 parts A and B, respectively. Over the range 770-820 nm, zl varies from -3.5 to -6.5 ps and t 2 from -200 to -550 ps for (M)L214H RCs. A similar trend, but with slightly longer values of zl and z2, is found for the double mutant. Over this same wavelength interval zl varies from -1 to -3.5 ps and z2 from -100 to -350 ps for R-26 R C S . ~We also find wavelength-dependent time constants for the Rb. sphaeroides (L)E104V mutant (data not shown) and for Rb. capsulatus wild-type and (L)E104L RCs.I6 Rough estimates for the free energy gap between P* and P+Iin the mutants were obtained at room temperature from the increase in the (delayed) steady-state fluorescence (880- 1000 nm) resulting from thermal repopulation of P* from P+I-. The
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8912 J. Phys. Chem., Vol. 99, No. 21, 1995
Kirmaier et al.
TABLE 1: Rates and Yields of Electron Transfer in Reaction Center Mutants and Wild-Type"
(M)L214H (M)L2 14H (M)L2 14H (M)L2 14H (Q reduced) (M)L2 14H (Q reduced) (M)L214H (Q-depleted) (M)L2 14W(L)E 104V (M)L2 14W(L)E 104V (M)L2 14W(L)E104V (Q-reduced) (M)L2 14W(L)E 104V (Q-depleted) (L)E104V (L)E104V wild-type' wild-type' wild-type' wild-typek (Q-blocked)
5.8 3.3 2.6 nm nm nm 5.8 4.2 7.7 nm 5.3 nm 3.5 2.4 1.4 4.3
285 77 5 295 77 295 285 77 295 295 285 77 285 77 5 295
350 605 800 820 950 1060 550 950 720 680 240 180 200 100 100 -15 ns
60 50 25 na na na 27 18 na na 100 100 100 100 100 na
0.58 1.2 3.2 na na na 2.0 5.3 na na 0.24 0.18 0.20 0.10 0.10 na
na na na 5 9 5 na na 7 6 na' na' na na na 15
0.88 1.2 1.1 0.86 1.o 1.1 0.75 1.2 0.77 0.68 na' na' na na nm 20
a Lifetimes have a relative error of 1 1 0 % (e.g, 5.8 i 0.6 ps), and yields have an absolute error of f58 (e.g., 60% i 5%). nm = not measured; na = not applicable. Reaction centers were studied with either QA in its normal (unreduced) state or with QA removed or reduced with sodium dithionite. The P* lifetimes are those determined from decay of stimulated emission from P*. In all cases the yield of P+I- (or P+BPhr-) was found to be -100%. The lifetime of the radical pair state was determined from the decay of bleaching of P, except in the case of (L)E104V RCs where the time constant for electron transfer from BPhL- to Q was determined from the decay of the BPhL anion absorption and/or BPhL QX bleaching. e The P'QA- yield was determined from the ratio of amplitude of the long-time bleaching of P (in state P'QA-) and the initial amplitude P+QA-, using the data in columns 4 of P bleaching (in states P* and P+I-). fcalculated time constant for electron transfer, P+BPhL- (or P+I-) ground state, using, for samples with functional and 5: r[Q = &bJ(4//100). E Calculated time constant for charge recombination, P+BPhL- (or P+I-) QA, the data in columns 4 and 5 : t l ~= s,~J((lOO-~)/lOO).For Q-reduced/Q-depleted samples, TIG is calculated from the data in columns 4 and 8 with the assumption that the asymptote of the decay (column 8) represents formation the triplet state PR. in the Q-reduced/Q-depleted samples will be slightly lower (equal to the tabs given in column 4) if the asymptote represents a fraction of unreducedundepleted RCs. Observed asymptote following charge recombination in Q-reduced RCs [lo0 x AA(O)/AA(IO ns)]. This asymptote reflects either formation of PR and/or P+QA- in a fraction of RCs with Q unreduced/undepleted (see also footnote g ) . ' As in wild-type RCs, charge recombination to the ground state in (L)E104V RCs is not observed and presumably requires on the order of 10-20 ns. 1 Typical values for wild-type RCs, as taken from refs 3, 5-9. Typical values for Q-reduced and/or Q-depleted wild-type RCs as reported in refs 7, 17, and 18. See also ref 23 for a discussion of the decay pathways of P+BPhL- in wild-type RCs leading to an -20 ns inherent lifetime for charge recombination to the ground state.
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Figure 3. The time evolution of bleaching at 855 nm in the ground
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state absorption band of P for (M)L214W(L)E104V RCs at 285 K following excitation with a 150-fs 582-nm excitation flash. The inset shows the first 15 ps of the data, demonstrating that there is no decay of P bleaching (Le., no loss of quantum yield) during the P* lifetime.
ratio of the static fluorescence amplitudes relative to wild-type (all RCs with Qn in its normal unreduced state) are found to be 1.2 f 0.1 for (L)E104V RCs, 7 f 3 for (M)L214H RCs, and 16 f 2 for (M)L214H/(L)E104V RCs. These ratios alone do not reflect the relative free energy gaps between P* and P+Isince the lifetimes of the two states vary among the RCs (Table 1). The simplest way to take these factors into account is to use a two-state model. In this way, we estimate free energy gaps of 220,75, and 50 meV (f30%) for (L)E104V, (M)L214H, and (M)L214W(L)E104V RCs, respectively, assuming a free energy gap of 250 meV" for wild-type RCs. If AG -150 meV is used instead for wild-type RCs,I8 the estimate for (L)E104V RCs is reduced by about half and those for the two beta-type
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Figure 4. Wavelength dependence of the fit time constants for the P* P+I- process (open circles) and for the P+I- P+Q+.- process (filled circles) for (M)L214H RCs (A) and (M)L214W(L)E104V RCs (B) at 285 K using 150-fs 870-nm excitation flashes.
mutants by about 25%;the values are reduced further if an even lower value for the native system is employed.Ioa The free energy gap we obtain for (M)L214H RCs is the same as the value of 70 f 30 meV obtained from our previous time-resolved delayed fluorescence studies2 The important point from this
The Charge-Separated Intermediate. 2 analysis, which ignores numerous potential complexities, does not lie in numerical values themselves, but in the general trend to smaller free energy gaps in the beta-type mutants. In particular, the modest increase in the free energy of P+I- in the double mutant compared to (M)L214H RCs is consistent with the difference observed between wild-type and (L)E104V RCs and the predicted effect of removing a hydrogen bond to ring V.I9 Thus, a higher free energy for P+p- in (M)L214W (L)ElO4V RCs compared to (M)L214H RCs is corroborated by the fluorescence studies and can be incorporated into analysis of the kinetic data.
J. Phys. Chem., Vol. 99, No. 21, 1995 8913 A Beta Scheme 1
B Beta Scheme 2
C General Scheme
Ground State
Discussion The kinetics of the absorption changes associated with the charge separation events are complex, as readily seen in the QY region of the spectrum. This is true on both the picosecond time scale for the formation of P+I- and on the hundreds of picoseconds time scale for its decay (Figure 4). As we noted previously for wild-type RCs? a wavelength dependence of the apparent time constants necessarily means that the photochemistry is more complex than described by the simple kinetic scheme P* P+IP+Qa- in a homogeneous system; the true kinetic form of the raw data for each step clearly is not, in fact, single exponential. Possible origins of the wavelength dependence of the kinetics include additional states in a linear charge separation sequence (such as the involvement of P+BChlL- in the initial stage of charge separation) or static or dynamic inhomogeneities involving populations of multiple conformationdenergy "substates" of the system. Such possibilities have been considered previously for wild-type RCs9 and were enforced by other recent work including the finding that the P* emission kinetics are not single exponential.6,'0b*''b$20 Complex relaxation kinetics also may arise from correlated fluctuations of the energy gaps between the states that persist for long times after excitation.22 Rather than applying more complex fitting schemes at the outset, which necessarily have built-in model-dependent biases also, we begin analysis of the photochemistry and the nature of P+I- in the four RCs under investigation using the "average" values for decay of P* and decay of P+I- listed in Table 1. These values come from measurements of the decay of stimulated emission (for the P* lifetime) and the decay of the anion-absorption band and/or P bleaching (P'I- lifetime). Note that the values in Table 1 fall within the respective ranges of the T I and t2 values shown in Figure 4; they should reflect the composite differences in the photochemistry of the two mutants. Since, in all four RCs, the yield of P+I- is -1, the P* lifetimes in Table 1 basically reflect the average inherent time constants for P'I- formation. Similarly, the observed P+I- lifetime and P+Qa- yield in Table 1 together with the scheme in Figure 5C allow calculation of the average inherent time constants for the electron transfer ( T ~ Q )and charge recombination (ZIG) decay pathways of P+I-. Here, Z ~ Gincludes the direct decay of Pepand/or P+BChlL- to the ground state and any thermal routes involving these states andor P*. The calculated values of TIG and T ~ Qare given in Table 1. Deactivation times (ZIG) on the order of 1 ns are obtained for beta-type RCs. This value was corroborated in measurements of the P+I- lifetime in (M)L214H and (M)L214W(L)E104V RCs in which electron transfer to QA was blocked by its chemical reduction or removal (Figure 6 and Table 1). As we have discussed before,2 one of the most significant differences from wild-type RCs is that the -1-ns inherent time constant for deactivation of P+I- to the ground state in the beta-type mutants is significantly shorter than the value of -20 ns for P'BPhL- .23
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Figure 5. Schemes for the energy ordering of states in wild-type and beta-type RCs. P'BChl- is placed below P* throughout for consistency and for reasons discussed in this and the preceding article. Panels A and B represent two scenarios for beta-type RCs, differing in the relative ordering of P+BChlL- and P'P-. The boxes enclosing these states indicate that P'I- could be either the lower energy of the two states or a composite of the two states involving quantum-mechanical mixing or thermal equilibration. Individual kinetic routes involving P+BChlLand P'P- are not shown, but the overall electron transfer to QA and deactivation to the ground state are illustrated. Panel C gives a general framework for discussing the results on both wild-type and mutant RCs, with the modeled time constants for electron transfer and charge , The deactivarecombination of P+I- denoted Z ~ Qand t , ~respectively. tion of P+I- encompasses both the direct return to the ground state and thermal repopulation involving P*. r r
tl
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Time, ps
Figure 6. Measurement of the P+I- lifetime in (M)L214H RCs in which QA is reduced with sodium dithionite. Open (295 K) and closed (77 K) circles show the decay of bleaching of the ground state absorption band of P at 855 nm (295 K) or 885 nm (77 K); the data were acquired using 30-ps pump/probe flashes with excitation at 532 nm. The solid and dashed lines are fits to a pulse convolution plus a single exponential plus a constant. The resulting lifetimes are given in
Table 1. An important consideration for the nature and dynamics of P+I- in beta-type RCs is the energy ordering of states P'pand P+BChlL- (Figure 5). Along these lines, we considered in the preceding article whether the spectroscopic data for (M)L214H and (M)L214W(L)E104V RCs are most consistent with P+I- being P+BChlL-, P+p-, or a mixture of the two. We now consider these possibilities with respect to the kinetic data presented here and assess the implications of the combined results. In our original study of (M)L214H RCs we suggested that P+I- largely had the character of P+p-, but with some mixingin of P+BChlL- in order to explain the dramatically enhanced rate of charge recombination (TIG).* However, much new spectral and kinetic data is now at hand that requires assumptions to be made if P+I- in the mutants indeed largely has the character of P+p-. As discussed in the previous paper, the
8914 J. Phys. Chem., Vol. 99, No. 21, 1995
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-AG (cm-') Figure 7. The dependence of rate on driving force for electron transfer from I- to QA at 285 K in R-26 wild-type RCs (circles) containing the native UQlo (1) or reconstituted with other quinones (2-9) and three mutants containing the native UQlo (squares). The mutants are (L)E104V (v), (M)L214H @), and (M)L214W(L)E104V (vp). The quinones reconstituted into wild-type RCs are 9,lO-anthraquinone (2), 1-methoxyanthraquinone (3), 1-methylanthraquinone (4), 2-ethylanthraquinone (5), 2-methylanthraquinone (6), 2,3-dimethylanthraquinone (7), 2,7-dimethylanthraquinone(8), and 2-aminoanthraquinone (9). These data are in good agreement with those derived from measurements of the P+QA- yield in Q-substituted R C S . Also ~ ~ shown are three simulations of the data for the Q-substituted wild-type RCs using an electronic coupling of 3.2 cm-' : (solid curve) semiclassical theory25a with 1 = 650 cm-l, (dotted curve) quantum mechanical theoryZSbwith one quantum mode (150 cm-I and S = 35) and a solvent mode (10 cm-I, S = 2); and (dashed curve) quantum-mechanical theory25cwith two quantum modes (1600 cm-I and S = 1; 120 cm-' and S = 37). The two-mode simulation has the same vibrational parameters as those used in the previous study of the rate of electron transfer from BPhLto QA and of P'BPhL- charge recombination in Q-substituted wildtype R C S . ~ ~
transient anion-region dichroism data likely would require that ,8 be physically rotated -20" from the position of BPhL.' Additionally, it is not straightforward to see how rotation of ,!? alone could simultaneously slow electron transfer from P* to p and enhance charge recombination of P'P- to the ground state since the rates depend similarly on orbital overlap between P and P. Furthermore, the magnitude of the falloff in the rate of electron transfer to QAis more dramatic than expected, implying that more has changed among the RCs than a simple change in the free energy of P+I-. The latter point is made in Figure 7, where the inverse of TIQ in Table 1 is plotted as a function of the estimated driving force for wild-type (point l), (L)E104V (point v), (M)L214H (point P), and (M)L214W(L)E104V (point vp) RCs along with data for a series of Rb. sphaeroides R26 RCs in which the native ubiquinone was replaced with quinones of varying redox potential (points 2-9).4a324 The deviation of the data for the beta-type RCs, especially the double mutant, from the theoretical prediction^^^ based on curves appropriate for BPhL-containing R C S ~can ~ be , ~explained ~ if there is a decrease in the electronic coupling and/or reorganization energy for electron transfer from I- to QA. Analysis of these data in conjunction with results on the Q-substituted beta-type RCs suggests that both of these effects may operate and that a likely origin is a substantial involvement of P+BChlL- in P+I- in the mutants.26 In order to explore the electronic coupling arguments, consider the extreme case if PfI- were pure P+BChlL- in the beta-type RCs. The unpaired electron on B c h l ~ -will be farther from QA compared to the distance between /3 (or BPhL) and QA. Since P+BChlL- would lie below P'P- in this assignment, electron transfer from B c h l ~ -to QAshould be assisted by P'P-
Kirmaier et al. via a superexchange mechanism, with the extent of the mixing dependent on factors such as the energy gap between P+BChlLand P'p-. The increased free energy of P'P- further above that of P+BChlL- in (M)L214W(L)E104V vs (M)L214H RCs would diminish the superexchange mixing and slow the rate of electron transfer from I- to QA in the double mutant, as is observed. Similarly, the enhanced deactivation of PfI- to the ground state in the mutants compared to wild-type RCs (ZIG -1 ns vs -20 ns) would arise because of increased electronic coupling due to the shorter distance between the unpaired electron and the hole in state P+BChlL- compared to PfBPhL-. An inherent time constant for charge recombination of P+Bchl~of 1 ns or less is reasonable considering the difference in edgeto-edge distances between P and BChlL vs BPhL (r 3.5 vs 7.5 and an exponential distance dependence of the rate of electron transfer [ k = exp(br), with b-' 1 A],28 assuming comparable Franck-Condon factors in the two cases. However, the idea that PfI- basically has the character of P+BChlL- with little or no involvement of P'P- has several shortcomings with respect to the spectral data (as is true of the other models as well).' This interpretation also does not provide a ready explanation for the reduction in the rate and yield of the P+I- P+Q*- electron transfer process found in both betatype RCs at low temperature. One would have to propose, for example, that the superexchange role of P+P- is temperature dependent due to a temperature-dependent energy (or enthalpy) gap between P+BChlL- and P'P-. There have been several recent discussions of a temperature-dependent free energy (and perhaps enthalpy) gap between PfI- and P* in various RCs, 10%1 829
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Many of the favorable aspects of a role for P+BChlL- are also incorporated in a model in which P+I- is a quantummechanical mixture of P+P- and P+BChlL-. Comparable contributions of the two states to P+I- can explain the 46" polarization angle observed for the anion transition in the betatype RCs.' A key factor required by this model is that P'Pand P+BChlL- must be essentially degenerate in order to obtain a substantial mixing (substantial contributions of both states to P+I-) since the electronic coupling is expected to be small. To a first approximation the electronic coupling can be estimated to be only -50 cm-'. This value comes from calculations of the electronic coupling between P+BPhL- and P+BChlL- based on orbital overlap of BPhL and BChlL in the wild-type crystal structure,30and we note that the overlap involving the anions may be larger than for the neutrals due to the more diffuse nature of the orbitals. The possibility of significant mixing between various states (even between P* and P+BPhL-) also has been raised from studies on mutants in which P has an increased oxidation p ~ t e n t i a l . ' ~ . ~ ~ If the wave function of I- is spread over both 4, and BChlL due to strong quantum-mechanical mixing between P+BChlLand P'P-, then initial electron transfer proceeds over a different effective distance in the beta-type mutants than in wild-type RCs. Thus, raising the energy of P+BPhL-IP+P- could affect the relative importance of one-step and two-step processes, thereby altering the mechanism of P+I- formation. As noted above, some investigators have proposed that in wild-type RCs the mechanism proceeds by both A modified wave function and electronic density distribution of I- in beta-type vs wild-type RCs can also explain the altered rates of the decay pathways of P+I- in these systems (Table 1). The shift of some electron density of I- onto BChlL from P-IBPhL- would bring the electron cloud in the reduced acceptor closer to P+ in the mutants compared to in wild-type RCs, increasing the effective electronic interaction for charge recombination. Such a shift in electron density onto BChlL in the
The Charge-Separated Intermediate. 2
ground state Figure 8. Scheme used for analysis of the kinetic data for the betatype RCs assuming that P+I- involves a thermal equilibrium between P'BChlL- and P'P-. P* is placed at 1.4 eV and P+QA-at 0.6 eV; the energy gaps for P'P- and P+BChlL- are discussed in the text. The following parameters were found to reproduce the experimental results in Table 1 for (M)L214H RCs at 285 K: llkl = 35 ps, llk-1 = 79 ps, Ilk2 = 7 ps, Ilk-2 = 99 ps, Ilk3 = 200 ps, Ilk4 = 20 ns, llks = 2 ps, llk-5 = 12 ps, llk6 = 200 ps, llk7 = 700 ps, Ilks = 1 ns. This set of parameters is not unique in providing a reasonable reproduction of the data. Variations in the parameters used to reproduce the data for (M)L214H RCs at other temperatures and for the (M)L214W(L)E104V mutant are discussed in the text.
reduced acceptor would simultaneously move the electron cloud farther from QA,decreasing the effective interaction for electron transfer from I- to QA. Although the quantum-mechanicalmixing model qualitatively explains many of the key observations, difficulties arise in the quantitative aspects and in reconciling the interpretations with the finding in the previous paper of the same anion-region dichroism angle for both beta-type RCs at both 285 and 77 K. It would seem that a specific set of circumstances needs to exist in order to explain the spectral data under this model; namely, P'p- must lie slightly below P+BChlL- in (M)L214H RCs and slightly above it in (M)L214W(L)E104V RCs so that the small relative change in their contributions to P+I- would give rise to the same polarization angle within experimental error. If P+I- is an -5Ol50 mixture of P+p- and P'BChlL-, then to a first approximation the electronic coupling with P'QA- should be reduced by a factor of 2 compared to wild-type RCs, corresponding to a factor of 4 decrease in rate. The rate of electron transfer from I- to QAin the double mutant is reduced by a factor of 10 compared to wild-type RCs at 285 K and by a factor of 50 at 77 K. Thus, in order to explain the differences in the rate of electron transfer to QA among the RCs, a substantial change in the Franck-Condon factor must occur as well, and it is not clear that such a change is reasonable to postulate. Additionally, the temperature dependence of the time constant for electron transfer from I- to QA is difficult to understand simply on the basis of the extent of mixing between P'p- and P+BChlL-. One could consider a temperaturedependent energy (or enthalpy) gap between P+BChlL- and P'p-, thus giving rise to a temperature-dependent nature of P'I-. Again, it is not easy to reconcile this interpretation with the temperature independence of the anion-region dichroism angle in the previous paper.' Another way to explore the temperature dependence of the rate and yield of P+IP'QA- electron transfer is to place PfBChlL- and P'P- in close thermal equilibrium. Figure 8 shows a scheme in which P+BChlL- lies below P+p-. This energy ordering follows from the ideas discussed above-that P'BChlL- should have a slower rate of electron transfer to QA and a faster rate of charge recombination to the ground state. A model having P+BChlL- lower in energy can account for a decrease in the rate and yield of electron transfer from I- to
-
J. Phys. Chem., Vol. 99, No. 21, 1995 8915 QA at low temperature for both beta-type RCs. This energy ordering also accounts for the longer P+I- lifetime in (M)L214W(L)E104V compared to (M)L214H RCs given that P'pis farther above P+BChlL- in free energy in the double mutant. Simulations of the thermal equilibration model in Figure 8 were carried out. The legend to Figure 8 lists a set of rate constants that reproduce the data for (M)L214H RCs at 285 K. It should be noted at the outset that there are uncertainties in many of the rate constants and given the number of parameters involved the simulations are not unique. The rate constants kl, k2, k-1, and k-2 were chosen to reproduce the observed P* lifetime in accord with the choice of the (P* - P+BChlL-) and (P* - PcP-) free energy gaps discussed below. The decay of P* to the ground state was fixed at Ilk3 = 200 ps on the basis of work on RCs in which electron transfer is b l ~ c k e d . ~The ' rate of charge recombination of P'p- to the ground state was set equal to llk4 = 20 ns for P+BPhL- in wild-type R C S . The ~~ rate of charge recombination of P+BChlL- was set at llkg 1 ns, in accord with the electronic coupling arguments outlined earlier, previous work on (M)L214H R C S , ~and other recent s t ~ d i e s . ~Electron ~ , ~ ~ transfer from BChlL- to QA should be slower than from p- to QA,although the difference may not be as great as simply reflected in the difference in distances involved since the P'BChlLP'QA- process should be assisted by P'p- via superexchange. Values of llk6 -200 ps (the wild-type value) and llk7 of -700 ps were used as part of the set of parameters that reproduce the data for (M)L214H RCs at 285 IC, but again, these choices are not unique. Turning to the free energy gaps used, P'BChlL- was placed 65 meV below P* for (M)L214H RCs in accord with our fluorescence data, previous estimates used in a thermal equilibrium model for (M)L214H R C S , analysis ~~ of recent kinetic data on (M)210 and RCs in which BPhL is replaced by p h e ~ p h y t i n . ' ~(Placing , ~ ~ P+BChlL- close in free energy to P* is in accord with some calculation^^^ and is inconsistent with others.36) Simulations in which P'p- is varied from 20 to 45 meV above P+BChlL- reproduce the data for (M)L214H RCs with appropriate adjustments in the rate constants. However, this free energy gap must be confronted with the spectroscopic data in the preceding article.' In particular, we have found that the anion-region absorption has an angle of 46 f 3" with respect to the 870-nm band of P independent of temperature for both beta-type mutants. The expected angle (for BChlL-) is -30" (see previous paper). Thus, for the equilibrium model in Figure 8 to be applicable, it is required that (i) the transition direction of BCML must be greater than expected by about 15" and (ii) P+P- must be sufficiently far above P'BChlL- in (M)L214H RCs at 285 K that the equilibrium population of P'p- is not spectroscopically significant. This would allow for no detectable change in the anion angle either at 77 K or in the double mutant. With P'p- 45 meV above P+BChL- (and 20 meV below P*) the equilibrium population of P'p- is 17% that of P+BChlLwith the rate parameters (more specifically their ratios) used in Figure 8 for (M)L214H RCs at 285 K. Thus at low temperature or in going to the double mutant at either 285 or 77 K there would be a reduction of this 17% contribution of P'p-. Such a change is probably borderline in terms of an experimentally detectable change in the anion angle. In this regard a 45-meV (P'P- - P+BChlL-) free energy gap works. Placing P+pcloser to P+BChlL- is less consistent with the dichroism data, although as noted, at the minimum a perturbation of the direction of the BChlL anion transition is required in the first place, independent of the choice of this free energy gap. The values of the free energy gaps and rate constants detailed here give an excellent reproduction of the kinetic data for (M)L214H RCs at 285 K. However, variations of 50% or more
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8916 J. Phys. Chem., Vol. 99, No. 21, 1995
Kirmaier et al.
the decay of P* and P+I- in wild-type or mutant RCs to energy in the values of some rate constants along with changes in the substates of P+BPhL-.11bs21The increased free energy of P'PP*, P+BChlL-, and P'P- free energy gaps also are consistent in the beta-type RCs could give rise to overlapping distributions with the observed kinetics and yield of P+QA- formation. An of energy substates of P'P- and P+BChlL-. Thus, depending interesting finding, however, is that in going from the single to on the precise mean positions and widths of the P'P- and the double mutant simply raising the free energy of P'P- by P + B c h l ~ distributions, individual conformers (RCs) may differ 20 meV does not reproduce the overall time or yield for P'QAin the spacing and perhaps ordering of these two basis states formation unless the inherent rate of electron transfer from and thus in the character of P+I- and the detailed mechanisms BChlL- to QA (k7) is also reduced. The latter is physically of its formation and decay. Such a possibility, as well as reasonable (assuming the ordering of the states as shown in relaxations of the charge-separated intermediates following their Figure 8) since the superexchange contribution of P+P- to may contribute to the difficulties in explainelectron transfer from BChlL- to QAis expected to decrease as ing some of the spectral and kinetic data in terms of the thermal the energy gap between P+P- and P+BChlL- is increased, as it equilibrium and quantum-mechanical-mixing models for P+Iis in the double mutant. Furthermore, because of this superin the beta-type RCs. exchange contribution of P'P-, the yield of P+QA- should not go to zero even when the temperature is reduced sufficiently All of these factors lead to inherent uncertainities with regard that the thermal population of P+p- is negligible, and the lowto modeling the combined spectral and kinetic data for temperature asymptotic value of the yield should be larger for (M)L214H and (M)L214W(L)E104V RCs using techniques (M)L214H RCs than for the double mutant RCs. The latter is such as global analysis. Components derived from singularconsistent with the trends in the data seen in Table 1. value decomposition must also, in the end, be subjected to a Nonetheless, for both the single and double beta-type mutant model. The number of parameters involved raises concerns RCs the observed lifetimes and yields at low temperature are about the uniqueness of any such modeling, as we have noted not reproduced using the 285-K parameters unless, for example, above in simulating the kinetic data on the basis of an the rate of the superexchange-assisted process (k7) is progresequilibrium model (and ignoring distributed kinetics and sively reduced. Again, one would need to consider a temperrelaxation effects). However, we believe that our results and ature-dependent energy gap between P+BChlL- and P+P- in discussion indicate that detailed and physically realistic modeling order to account for such an effect. This possibility warrants of the data for the beta-type mutants must incorporate a serious consideration in view of indications of a temperaturesubstantial contribution of P+BChlL- in state P+I- and a precise dependent free energy (and perhaps enthalpy) gap between P+Inature of P+I- that may (i) differ for (M)L214H and (M)L214W and P* for wild-type RCs and several mutant R C S . ~ ~ ~ .(L)E104V ~ ~ , ~ ~RCs, (ii) change as a function of temperature, and However, a better understanding of the potential effects of (iii) differ among the members of a distribution arising from temperature on the energy gaps, on the RC structure, and on static andor dynamic inhomogeneities. the population of conformationallenergy substates is needed We have noted here and in the previous article that no one before the predictions of the thermal equilibrium model of Figure of the assignments for P+I- in the beta-type RCs can uniformly 8 can be evaluated at a quantitative level. At present we can explain all the spectral and kinetic data. In addition to models say that this model is qualitatively consistent with the effects in which P+I- involves both P+BChlL- and P+P-, a number of of temperature on the rates and yields of charge separation and additional factors such as temperature-dependent (free) energy with the changes in photochemistry observed between (M)L214H gaps and statiddynamic inhomogeneities have been mentioned. and (M)L214W(L)E104V RCs. As we have seen, however, Nonetheless, in both the mutant and wild-type RCs many of even within the thermal equilibrium model some degree of the difficulties in assessing the fundamental details of the quantum-mechanical mixing involving P+BChlL- and P'Pprimary photochemistry would seem to be a consequence of seems to be required to explain certain rate and yield data. the RC design, most notably stemming from a close energy Additionally, one still must rationalize the anion dichroism angle position of P*, P+BChlL-, and P+BPhL- (P+P-). In short, the reported in the previous paper. main conclusions from this work are that P + B c h l ~ -must lie Some of the difficulties with either the thermal equilibrium close in free energy to P* and that this state plays a pivotal or quantum-mechanical mixing models, particularly the effects role in beta-type RCs both in the initial stage of charge of temperature, also can be rationalized if one folds in the separation and in the subsequent electron transferlcharge complexities associated with presence of static and/or dynamic recombination reactions that dictate the overall yield of P+QA-. inhomogeneities involving conformational/energy substates of Furthermore, our analyses suggest that even within the framethe RC. Such substates may differ in structurdenergetic factors work of a thermal equilibrium model some degree of quantumthat in tum would affect the electronic couplings andor Franckmechanical mixing between P+BChlL- and P+P- and perhaps Condon factors for electron transfer and charge recombination. other states as well must occur to explain the observed rates The substates may exist prior to excitation or develop as a and yields of electron transfer. We believe that these key consequence of excitation or electron transfer, and they may conclusions transcend the uncertainties in arriving at a precise interconvert with rates much different than the rates of electron assignment of P+I- in the beta-type RCs. The conclusion that transfer. Inhomogeneities and relaxation effects have been P+BChlL- lies below P* does not prove that P+BChlL- serves considered in order to interpret a variety of observations on the as a chemical intermediate in a two-step mechanism for P+BPhL- formation in wild-type RCs. The uncertainties RC photochemistry using a variety of techniques probing various time scales.6.8-10,11b,17b-18,20,21-37In view of all of these studies discussed in the literature for arriving at such an assignment from the available spectral and kinetic data remain, and some and the vast literature on the protein substates of heme proteins,38 of these issues have been noted here with respect to the betawe believe that it is physically unrealistic to consider the RC type RCs. to be a homogeneous system characterized by a simple set of rate constants for a linear electron transfer sequence. Our findings give design criteria for the choice of chromophores to serve as intermediate relays in a charge separation Of particular relevance are our workg attributing wavelengthsequence, both in proteins and model systems. In particular, dependent kinetic data in wild-type RCs (analogous to that seen the choice of chromophores and their environment must be based in Figure 4) to structural and/or energetic factors associated with not only on how the (free) energy gaps involving the chargeP* and P+BPhL- and recent work associating complexities in
The Charge-Separated Intermediate. 2 separated intermediates affect the Franck-Condon factors for electron transfer and charge recombination but also on how the energy gaps affect the electronic interactions between the states. For example, our results suggest that utilizing BPh in place of BChlL as a bridging cofactor between P and BPhL, as in using a BChl in place of the BPhL transient acceptor, would be disadvantageous since electronic mixing and/or thermal equilibration between the close-lying charge-separated states would enhance deactivation to the ground state and diminish the yield of charge separation to give PfQ*-.? Similarly, it seems clear that protein modifications that would raise the free energy of PfBPhL- in order (presumably) to reduce the Franck-Condon factor for charge recombination via the inverted-region effect may give just the opposite result, namely increased deactivation due to a smaller energy separation and increased involvement of the higher energy P+BChlL- state.2 Enhanced deactivation rates due to smaller free energy gaps between states also have been found in heterodimer RCs,29,39other mutants in which the oxidation potential of P is increased,I0 pheophytin-substituted R C S , ' ~and , ~ ~model systems.40 These results are of general importance in considerations of how energy gaps need to be balanced in order to control the relative rates of charge separation and recombination in multistep solar-energy conversion systems.
Acknowledgment. This work was supported the National Science Foundation (Grant MCB9405248 to D.H. and C.K.), by the National Institutes of Health (Grants GM38214 and GM48254 and Research Career Development Award GM00536 to C.C.S.), and by the Colorado Agricultural Experiment Station (Grant 632 to C.C.S.). References and Notes (1) Kirmaier, C.; Laporte, L.; Schenck, C. C.; Holten, D. J. Phys. Chem. 1995, 99, 8903 (previous paper in this issue).
(2) Kirmaier, C.; Gaul, D.; DeBey, R.; Holten, D.; Schenck, C. C. Science 1991, 251 922. (3) For overviews of the primary photochemistry see The Photosynthetic Reaction Center; Deisenhofer, J., Noms, J. R., Eds.; Academic Press: New York, 1993; Vol. 2. (4) (a) Gunner, M. R.; Robertson, D. E.; Dutton, P. L. J. Phys. Chem. 1986, 90, 3783. (b) Liu, B.-L.; Yang, L.-H.; Hoff, A. J. Photosynth. Res. 1991, 28, 51. ( 5 ) Breton, J.; Martin, J.-L.; Fleming, G. R.; Lambry, J.-C. Biochemistry 1988, 27, 8276. (6) H a " , P.; Gray, K. A.; Oesterhelt, D.; Feick, R.; Scheer, H.; Zinth, W. Biochim. Biophys. Acta 1993, 1142, 99. (7) Woodbury, N. W.; Becker, M.; Middendorf, D.; Parson, W. W. Biochemistry 1985, 24, 75 16. (8) Nagarajan, V.; Parson, W. W.; Davis, D.; Schenck, C. C. Biochemistry 1993, 32, 123. (9) (a) Kirmaier, C.; Holten, D. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3552. (b) See chapter 2 in ref 3. (10) (a) Peloquin, J. M.; Williams, J. C.; Lin, X.; Alden, R. G.; Taguchi, A. K. W.; Allen, J. P.; Woodbury, N. W. Biochemistry 1994,33, 8089. (b) Woodbury, N. W.; Peloquin, J. M.; Alden, R. G.; Lin, X.; Lin, S.; Taguchi, A. K. W.; Williams, J. C.; Allen, J. P. Biochemistry 1994, 33, 8101. (11) (a) Chan, C. K.; DiMagno, T. J.; Chen., L. X. Q.;Noms, J. R.; Fleming, G. R. Proc. Nail. Acad. Sci. U.S.A. 1991, 88, 11202. (b) Jia, Y.; DiMagno, T. M.; Chan, C. K.; Wang, Z.; Du, M.; Hanson, D. K.; Schiffer, M.; Noms, J. R.; Fleming, G. R.; Popov, M. S. J. Phys. Chem. 1993, 97, 13180. (12) Shkurapotov, A. Y.; Shuvalov, V. A. FEBS Lett. 1993, 322, 168. (13) Somewhat longer time constants are obtained for the beta-type mutants when 582-nm excitation flashes are employed. For example, the time constant for the (M)L214H mutant is 8.1 ps using visible excitation. This difference likely reflects energy transfer from one or more of the BChls to P. (14) Bylina, E. J.; Kirmaier, C.; Holten, D.; Youvan, D. C. Narure 1988, 336, 182. (15) Lautenvasser, C.; Finkele, U.; Scheer, H.; Zinth, W. Chem. Phys. Lett. 1991, 183, 471. (16) Kirmaier, C.; McDowell, L. M.; Holten, D. unpublished results. (17) (a) Ogrodnik, A.; Volk, M.; Letterer, R.; Feick, R.; Michel-Beyerle, M. E. Biochim. Biophys. Acta 1988, 936, 361. (b) Goldstein, R. A.; Takiff L.; Boxer S. G. Biochim. Biophys. Acta 1988, 934, 253. ~
J. Phys. Chem., Vol. 99, No. 21, 1995 8917 (18) Woodbury, N. W. T.; Parson, W. W. Biochim. Biophys. Acta 1984, 767, 345.
(19) (a) Hanson, L. K.; Thompson, M.; Fajer, J. In Progress in Photosynthesis Research; Biggens, J., Ed.; Martinus Nijhoff Boston; 1987; Vol. 3, p 31 1. (b) Michel-Beyerle, M. E.; Plato, M.; Deishenhofer, J.; Michel, H.; Bixon, M.; Jortner, J. Biochim. Biophys. Acta 1988, 932, 52. (20) (a) Martin, J. L.; Lambry, J. C.; Ashokkumar, M.; Michel-Beyerle, M. E.; Feick, R.; Breton, J. In Ultrafast Phenomenon III ; Harris, C. B., Ippen, E. P., Mourou, G. A,, Zewail, A. H., Eds.; Springer: Berlin; 1990, p 514. (b) Becker, M.; Nagarajan, V.; Middendorf, D.; Parson, W. W.; Martin, J. E.; Blankenship, R. E. Biochim. Biophys. Acta 1991, 1057, 299. (c) Mueller, M. G.; Griebenow, K.; Holzwarth, A. R. Chem. Phys. Lerr. 1992, 199, 465. (21) Ogrodnik, A.; Keupp, W.; Volk, M.; Aumeier, G.; Michel-Beyerle, M. E. J. Phys. Chem. 1994, 98, 3432. (22) Gehlen, J. N.; Marchi, M.; Chandler, D. Science 1994, 263, 499. (23) (a) State P+BPhL- in wild-type RCs decays by charge recombination to the ground state and by formation of the triplet state of P, combining to give a lifetime of 10-15 ns for the state. The inherent time constant of the ~ Parson, ~ ~ - ~ pathway for deactivation to the ground state is -20 n ~ . (b) W. W.; Clayton, R. K.; Cogdell, R. J. Biochim. Biophys. Acta 1975, 387, 265. (c) Budil, D. E.; Kolaczkowski, V.; Noms, J. R. In Progress in Photosynthesis Research; Biggens, J., Ed.; Nijhoff Dordrecht, 1987; Vol. 2, p 245. (d) Chidsey, C. E. D.; Kirmaier, C.; Holten, D.; Boxer, S. G. Biochim. Biophys. Acta 1984, 766, 424. (24) Kirmaier, C.; Gunner, M. R.; Holten, D.; Dutton, P. L. Unpublished results. (25) (a) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985,81,265. (b) Jortner, J. J. Chem. Phys. 1976, 64, 4860. (c) Sarai, A. Chem. Phys. Lett. 1979, 63, 360. (26) Laporte, L.; Kirmaier, C.; Schenck, C. C.; Holten, D. Chem. Phys., in press. (27) (a) Deisenhofer, J.; Michel, H. Annu. Rev. Biophys. Biophys. Chem. 1991, 20, 247. (b) Yeates, T. 0.; Komiya, H.; Chirino, A.; Rees, D. C.; Allen, J. P.; Feher, G. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 7993. (c) El-Kabbani, 0.; Chang, C. H.; Tiede, D.; Noms, J. R.; Schiffer, M. Biochemistry 1991, 30, 5361. (28) (a) Closs, G. L.; Miller, J. R. Science 1988, 240, 440. (b) Moser, C. C.; Kenske, J. M.; Warncke, K.; Farid, R. S.; Dutton, P. L. Nature 1992, 355, 796. (29) Laporte, L.; McDowell, L. M.; Kirmaier, C.; Schenck, C. C.; Holten, D. Chem. Phys. 1993, 176, 615. (30) (a) Warshel, A.; Creighton, S.; Parson, W. W. J. Phys. Chem. 1988, 92, 2696. (b) Scherer, P. 0. J.; Fischer, S. F. J. Phys. Chem. 1989, 93, 1633. (c) Plato, M.; Mobius, K.; Michel-Beyerle, M. E.; Bixon, M.; Jortner, J. J. Am. Chem. SOC.1988, 110, 7279. (31) Bixon, M.; Jortner, J.; Michel-Beyerle, M. E. Biochim. Biophys. Acta 1991, 1056, 301. (32) (a) Schenck, C. C.; Parson, W. W.; Holten, D.; Windsor, M. W. Biochim. Biophys. Acta, 1981, 635, 383. (b) Breton, J.; Martin, J.-L.; Lambry, J.-C.; Robles, S. J.; Youvan, D. C. in Reaction Centers of Photosynthetic Bacteria; Michel-Beyerle, M. E., Ed.; Springer-Verlag: New York, 1990; Springer Series in Biophysics, Vol. 6, p 293. (33) Bixon, M.; Jortner, J.; Michel-Beyerle, M. E. in The Photosynthetic Bacterial Reaction Center 11;Breton, J., Vermeglio, A., Eds.; Plenum: New York 1992; p 291. (34) Schmidt, S.; Ark, T.; Hamm, P.; Huber, H.; Nagele, T.; Wachtveitl, J.; Meyer, H.; Scheer, H.; Zinth, W. Chem. Phys. Lett. 1994, 223, 116. (35) (a) Parson, W. W.; Chu, Z. T.; Warshel, A. Biochim. Biophys. Acta 1990, 1017, 251. (b) Warshel, A.; Chu, Z. T.; Parson, W. W. J. Photobiochem. Photobiol. A: Chem. 1994, 82, 123. (361 (a) Thomoson. M. A.; Zemer, M. J. Am Chem. SOC. 1991, 113, 8210. (b) Marchi, M.;Gehlen, J. N.; Chandler, D.; Newton, M. J. Am. Chem. SOC.1993, 115, 4178. (37) (a) Kleinfeld, D.; Okamura, M. Y.; Feher, G. Biochem. Biophys. Acta 1984, 765, 406. (b) Dijkman, J. A.; Den Blanken, H. J.; Hoff, A. J. lsr. J. Chem. 1988, 28, 141. (c) Kirmaier, C.; Holten, D.; Parson, W. W. Biochim. Biophys. Acta. 1985,810,33. (d) Tiede, D. M.; Kellog, E.; Breton, J. Biochim. Biophys. Acta 1987, 892, 294. ( e ) Franzen, S.; Goldstein, R. F.; Boxer, S. G. J. Phys. Chem. 1993, 97, 3040. (f)Gao, J.-L.; Shopes, R. J.; Wraight, C. A. Biochim. Biohys. Acta 1991, 1056, 259. (8) Baciou, L.; Rivas, E.; Sebban, P. Biochemistry 1990,29,2966.(h) Small, G. J.; Hayes, J. M.; Silbey, R. J. J. Phys. Chem. 1992, 96, 7499. (38) Frauenfelder, H.; Steinback, P. J.; Young, R. D. Chem. Scr. 1989, 29A, 145-150. (39) McDowell, L. M.; Kirmaier, C.; Holten, D. J. Phys. Chem. 1991 95, 3379. (40) (a) Warman, J. M.; Smit, K. J.; Jonker, S. A,; Verhoeven, J. W.; Oevering, H.; Kroon, J.; Paddon-Row, M. N.; Oliver, A. M. Chem. Phys. 1993, 170, 359. (b) Wasielewski, M. R.; Johnson, D. G.; Niemczyk, M. P.; Gaines, G. L., 111. O'Neil, M. P.; Svec, W. A. J. Am. Chem. SOC. 1990, 112, 6482. JP943009U
