Primary Events in Photosynthetic Reaction Centers with Multiple

J. Phys. Chem. B 2001, 105, 5575-5584

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Primary Events in Photosynthetic Reaction Centers with Multiple Mutations near the Photoactive Electron Carriers James A. Roberts,† Dewey Holten,* and Christine Kirmaier* Department of Chemistry, Washington UniVersity, St. Louis, Missouri 63130 ReceiVed: January 23, 2001; In Final Form: April 4, 2001

We have investigated the primary charge separation reactions in two Rhodobacter capsulatus bacterial reaction center (RC) mutants that have multiple mutations near the L-side bacteriochlorophyll (BChlL) and bacteriopheophytin (BPhL) cofactors. Both RCs contain the “beta” mutation L(M212)H that results in incorporation of a BChl molecule, denoted β, in place of the native BPhL. The mutants additionally contain one or two newly introduced aspartic acid residues. In the triple mutant F(L97)V/F(L121)D/L(M212)H, an Asp replaces Phe at L121 (near BPhL), while in the quadruple mutant F(L97)V/F(L121)D/G(M201)D/ L(M212)H aspartic acid residues are introduced at L121 and at M201 (near BChlL). These mutants also incorporate a Val residue at L97 (near BPhL) that is photochemically silent but appears to increase RC protein stability. Femtosecond transient absorption studies reveal a longer P* lifetime in both the triple mutant (10 ( 2 ps) and quadruple mutant (19 ( 3 ps) compared to wild-type (4.3 ( 0.3 ps). In the quadruple mutant, P* decay occurs via a combination of electron transfer to the L side to give an intermediate P+I- (involving P+β- and P+BChlL-) in 67% yield, decay to the ground state in 15% yield, and electron transfer to the M side to form P+BPhM- in 18% yield. Key observations on the quadruple mutant include significant bleaching in the 530-nm QX absorption band of BPhM, and distinctive biexponential decay kinetics in the 600-700-nm anion region with time constants of 200 ( 30 ps (associated with decay of P+I-) and 1-4 ns (associated with charge recombination of P+BPhM-). These findings are evidence for parallel primary charge transfer to the L-side and to the normally inactive M-side. Similar results are obtained for the triple mutant, but the yields of electron transfer to the M side (12%) and P* decay to the ground state (5%) are lower and the yield of electron transfer to the L side correspondingly higher (83%). One of the most interesting differences between the two mutants is that even though the initial yield of electron transfer from P* to the L side is higher in the triple mutant, the yield of the final L-side state P+QA- is smaller than in the quadruple mutant (38% versus 48%). This distinction derives from differences in the nature of P+I- and the rates of its decay pathways in the two mutants. These and other results are discussed in terms of the effects that the Asp residues appear to have on the free energies of the charge-separated states, namely raising P+β- (Asp L121) and P+BChlL(Asp M201). The results obtained here complement those on related mutants in providing a consistent picture of the free energies of the states and the primary events in this family of RCs and in the native system. One conclusion is that P+BChlL- in wild-type RCs probably lies 80-100 meV below P*, which is a slightly larger free energy gap than previously estimated.

Introduction The bacterial reaction center (RC) is a membrane-bound pigment-protein complex that converts absorbed light energy to chemical potential energy.1 This photoconversion occurs via sequential electron transfer through a series of cofactors, ultimately separating charge across the photosynthetic membrane. The crystal structures of RCs from Rhodopseudomonas Viridis and Rhodobacter sphaeroides show that the isolated complex has a macroscopic C2 symmetric arrangement of the L and M polypeptides and the associated bacteriochlorophyll (BChl), bacteriopheophytin (BPh), and quinone (Q) cofactors (Figure 1).2 Upon excitation, the special pair (P, a dimer of BChls) is raised to its singlet excited state (P*). An electron is transferred from P* in ∼4 ps to the L-side BPh molecule (BPhL) followed by electron transfer to the neighboring quinone (QA) in ∼200 ps (Figure 2A). In wild-type (wt) RCs, this overall * Authors to whom correspondence should be addressed. † Current Address: Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Building 5, Room 135, Bethesda, MD 20892-0520.

Figure 1. Arrangement of the RC cofactors given by the X-ray structures from Rb. sphaeroides and Rps. Viridis (see ref 2).

10.1021/jp010280k CCC: $20.00 © 2001 American Chemical Society Published on Web 05/17/2001

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Figure 2. Schematic state summary diagrams for the various mutants. The abbreviations B ) BChl and H ) BPh have been used for simplicity. The values next to the arrows are the inverse of the rate constant for a given process, calculated from the lifetime of the state and the yield of the process (see text).

charge separation process (P* f P+BPhL- f P+QA-) occurs with a yield of ∼1. Many recent investigations have focused on the role of BChlL in initial L-side charge separation. Central to this question are the free energies of P*, P+BChlL-, and P+BPhL-. It is generally thought that in wt RCs, P+BChlL- lies slightly (0.05-0.08 eV) below P* in free energy,3-10 that P+BPhL- is below P* by ∼0.28 eV11,12a (or less if unrelaxed13), and that P+QA- is below P* by ∼0.8 eV, which itself lies ∼1.4 eV above the ground state.13 Calculations have suggested that P+BChlM- and P+BPhM- lie at higher free energies than their L-side counterparts.10,14 Experimental information on the free energies of the M-side states is indirect and comes from a handful of studies where electron transfer to the M side is observed.7,15,16 Insights into many fundamental properties of the RC and the rates and yields of the primary charge separation reactions have come from studies on RCs that have amino acid mutations near the chromophores. Mutants have generally targeted metal ligation or hydrogen bonding involving an amino acid and a

chromophore, as well as amino acid size, polarity, polarizability, and charge sall with the intent of perturbing the free energies and nature of the charge-separated states and/or P*.3,5-8,16-20 Selection of the mutations used in the two RCs reported here derive from previous studies of RCs that incorporate individually the same changes: F(L97)V,6a F(L121)D,6b L(M212)H,7 G(M201)D,6b,7and G(M201)D/L(M212)H7 in Rb. capsulatus and the analogous L(M214)H5 and G(M203)D18b in Rb. sphaeroides. Thus, we have constructed in Rb. capsulatus the F(L97)V/ F(L121)D/L(M212)H triple mutant and the F(L97)V/F(L121)D/ G(M201)D/L(M212)H quadruple mutant. These will be referred to as the VDH and VDDH mutants, respectively. Both the VDH and VDDH mutants have as a key foundation the so-called “beta” mutation that has been studied extensively in both Rb. capsulatus and Rb. sphaeroides.5,6 This mutation replaces the native Leu at M212 (M214 in Rb. sphaeroides) with His over one face of BPhL. As a result, BPhL is replaced with a BChl, denoted β. The photochemistry of the L(M212)H mutant is depicted in Figure 2B. The altered electron-transfer

Photosynthetic Reaction Centers with Multiple Mutations properties of the mutant derive primarily from the fact that P+βis at higher free energy than P+BPhL- in wt and thus much closer to P+BChlL-. The resulting quantum/thermal admixture of P+BChlL- and P+β- produces the intermediate designated P+I- that forms from P* in ∼ 9 ps in L(M212)H RCs. The primary effect of the “beta” mutation is revealed in the subsequent decay of P+I-. Both a longer time constant for electron transfer to QA (∼280 ps versus ∼180 ps for wt) and a dramatically shorter time constant for charge recombination to the ground state (∼1 ns versus ∼20 ns for wt) are found. The combined effects give a significantly reduced overall yield of P+QA- (∼70% versus ∼100% for wt). Similar branched photochemistry at P+I- leading to reduced P+QA- yields (with differences in detail) is also found for the analogous L(M214)H Rb. sphaeroides mutant,5 for other Rb. capsulatus mutants that contain β as a result of incorporation of His at a position other than M212,6a and chemically modified RCs in which another pigment is substituted for BPhL.4 The F(L121)D component of the VDH and VDDH mutants also has been studied previously.6b The F(L121)D RC contains an Asp residue near ring V of BPhL, and has photochemistry similar to that in the L(M212)H RC. However, the F(L121)D mutant retains the native BPhL. As is shown in Figure 2C, we have proposed that the effect of the Asp residue introduced at L121 is to raise the free energy of P+BPhL-, which could occur by several mechanisms depending on the protonation state of the Asp. Resonance Raman experiments are most consistent with Asp L121 being ionized.21 Based on modeling of the kinetic data and yields of the charge-separated states at room and low temperature, we have proposed free energy orderings of the two states comprising P+I- in the various mutants exhibiting “beta-type” photochemistry (i.e., branching at P+I-).5b,6b We believe that P+BPhL- is slightly below P+BChlL- in free energy in the Rb. capsulatus F(L121)D mutant (Figure 2C), that P+β- is essentially degenerate with P+BChlL- in the Rb. capsulatus L(M212)H mutant (Figure 2B), and that the reverse ordering (P+β- above P+BChlL- but still below P*) occurs in the Rb. sphaeroides L(M214)H mutant. The ordering also may vary in different RCs in which another pigment has been chemically substituted for BPhL.4 To obtain further information along these lines, and more generally to alter the free energy of P+β- (hopefully raising it above P+BChlL- or perhaps even above P*) and further explore the consequences on the primary events, we have combined the L(M212)H and F(L121)D mutations to make the VDH triple mutant. (The V mutation at L97 is photochemically silent but appears to have a role in the stability of the RC protein.) Similarly, we have added F(L121)D to the previously studied G(M201)D/L(M212)H mutant. G(M201)D introduces an Asp residue near ring V of BChlL. In the G(M201)D/L(M212)H mutant the free energy of P+BChlL- appears to be significantly increased (likely ∼100 meV or more) raising this state well above P+β- and probably above P*.7a,22 This change slows electron transfer to the L side, allowing competing electron transfer to BPhM in ∼15% yield and deactivation to the ground state in ∼15% yield (Figure 2D). Subsequent decay of the L-side intermediate (presumably largely P+β-) occurs mostly (g90%) via electron transfer to QA with little parallel charge recombination to the ground state. Our goal in adding the F(L121)D mutation to G(M201)D/L(M212)H is again to explore the changes in the electron-transfer pathways that derive. At the extreme, if both P+BChlL- and P+β- are raised sufficiently above P* one might expect little if any charge separation to the L-side of the RC and a higher yield of electron transfer to the

J. Phys. Chem. B, Vol. 105, No. 23, 2001 5577 M side. The results reported here on the VDH and VDDH mutants thus give further insights into how the free energies of the L-side charge-separated states influence the L-side chargeseparation and charge-recombination processes as well as the yield of electron transfer to the normally inactive M branch. Experimental Methods Construction of the F(L97)V, F(L121)D, G(M201)D, and L(M212)H mutants has been reported previously.6,7 All of these mutants were assembled in the pU2924 plasmid originally devised by Youvan and Bylina.23 Our initial intent here was to study the F(L121)D/L(M212)H and the F(L121)D/G(M201)D/ L(M212)H mutants. To make the former of these, we took our previously constructed pU2924 vector containing the M-gene L(M212)H mutation7a and spliced in the KpnI-BamHI L-gene fragment having the Asp mutation at L121.6b The F(L121)D/ G(M201)D/L(M212)H mutant was assembled similarly starting with our previously constructed pU2924 plasmid having both the G(M201)D and L(M212)H mutations.7a It later proved difficult to study RCs from these two mutants; the yields of protein were low and/or it degraded fairly rapidly. Hence we re-made the target mutants but this time splicing in an L-gene fragment that contained F(L97)V/F(L121)D.6a In previous work we noted that changing Phe L97 to a Val seems to impart greater stability to the protein for some mutants that have additional changes near BPhL.6a (The Phe at L97 is near ring IV of BPhL.) This is the case, for example, for the F(L121)H mutant which by itself gives little or no yield of RCs from standard isolation procedures but when combined with F(L97)V gives good yields of protein. We have speculated that this may be due to a volume effect although this point is not clear. In any case, the phenomenon proved true here again and we were able to isolate RCs in good yields from both the VDH and VDDH mutants, although the latter was still somewhat unstable. RCs were isolated and purified using established procedures from cultures grown semi-aerobically in the dark for 72 h.6,7 Absorption spectra were recorded on a Perkin-Elmer 330 spectrometer. The purity of RCs was determined by the ratio of absorbances at 280 and 800 nm, with a general finding of A280/A800 ∼ 1.7. The yields of isolated RCs for the VDH and VDDH were ∼50% and ∼30%, respectively, of the yield of RCs from the same volume of wt cells. All measurements were done on freshly prepared RCs in 10 mM potassium phosphate buffer pH 7.6 containing 0.05% LDAO and 1 mM EDTA. Our standard isolation procedures yield RCs that are >90% depleted of QB. The results obtained here are consistent with this fact although we did not independently verify it in millisecond time scale measurements. The primary electron-transfer reactions were investigated on flowed samples at ∼285 K via transient absorption spectroscopy using an Ar+-pumped regeneratively amplified Ti:sapphire OPA system operated at 10 Hz. The samples were excited with ∼130-fs excitation pulses at 850 or 760 nm and probed with ∼130 fs “white-light” flashes. The excitation pulses were defocused and/or attenuated such that ∼30% of the RCs in the excited region were pumped on a single flash. Further details of the transient absorption apparatus, data acquisition, and data analysis methods have been described elsewhere.7 Results The VDH Mutant. The results of femtosecond transient absorption measurements on the VDH mutant are presented using the model given in Figure 2E. Formation of P* is indicated by bleaching of the 850 nm absorption band of P, with stimulated emission from P* appearing as an additional “bleaching”

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Figure 3. Transient absorption difference spectra and kinetic data in the region of P bleaching and P* stimulated emission acquired using 130-fs excitation flashes at 760 nm. The lines through the data in the main portion of panels B and D are fits to the P* stimulated emission decay at 910-920 (B) or 900-910 nm (D) nm using a single-exponential decay component (plus the instrument rise and a constant), giving the time constants given in Table 1. The solid lines in the insets to panels B and D are fits to the P-bleaching data at 830-840 nm to a three-exponential function (plus the instrument rise and a constant). The time constants are discussed in the text.

TABLE 1: Transient-State Lifetimes for Mutant RCs sample

P* (ps)

P+I- (ps)

wt a F(L97)Vb F(L121)Da L(M212)Ha G(M201)Da G(M201)D/L(M212)Hc VDHd VDDHe

4.3 ( 0.3 4.5 ( 0.3 5.8 ( 0.6 8.5 ( 0.8 7.6 ( 0.5 15 ( 2 10 ( 2 19 ( 1

175 ( 15f 185 ( 20f 155 ( 20g 210 ( 30h 160 ( 15f 150 ( 30i 190 ( 30h 200 ( 30h

a From ref 6b. b From ref 6a. c From ref 7b. d F(L97)V/G(M201)D/ L(M212)H mutant. e F(L97)V/F(121)D/G(M201)D/L(M212)H mutant. f Here, P+I- is essentially P+BPh -. g Here, P+I- is an admixture of L P+BPhL- and P+BChlL-. h Here, P+I- is an admixture of P+β- and +BChl -. i Here, P+I- is largely P+β-. P L

(actually a probe gain) on the long-wavelength (870-950 nm) side of the P-bleach signal (0.5-ps spectrum in Figure 3A). A fit of the stimulated emission decay between 890 and 920 nm to a single-exponential plus a constant yields a P* lifetime of 10 ( 2 ps (Table 1). The results for the 910-920-nm region are shown in the main panel of Figure 3B. In wt RCs, a constant magnitude of P bleaching is seen in the transient spectra acquired during the P* lifetime and at subsequent delay times to many nanoseconds after excitation. This observation is consistent with the formation of the chargeseparated state P+QA- with essentially unity yield starting from P*. However, for the VDH mutant, the amplitude of P bleaching in the transient spectrum at 3.3 ns (Figure 3A) is reduced by about 60% compared to its initial magnitude. The extent of the P-bleaching decay, which directly reflects return of RCs to the ground state, is best monitored at 830-850 nm (on the blue side of the P absorption band), where the contribution of P* stimulated emission to the transient difference spectrum is minimal or absent. The P-bleaching decay in this region is quite

complex and requires fitting with three exponentials plus a constant in order to obtain satisfactory fits and time constants that are consistent with those obtained in other regions of the spectrum. Thus, RCs return to the ground state in the VDH mutant via three channels. Specifically, in the following presentation of results in other spectral regions it will be shown that return to the ground state takes place during the decay of (1) P* (10 ps lifetime), (2) the L-side intermediate P+I- (200 ps lifetime), and (3) the M-side state P+BPhM(1-4 ns lifetime). Transient difference spectra measured following P* decay have a small but distinct bleaching near 530 nm (in the QX region of BPhM), as shown in the spectrum taken at 35 ps (Figure 4A). We have reported a similar bleaching associated with the formation of P+BPhM- in the G(M201)D/L(M212)H and S(L178)K/G(M201)D/L(M212)H) mutants.7 On the basis of this finding and the other results presented below, we assign the 530-nm Qx bleaching in the VDH mutant to the formation of P+BPhM-. To determine the yield of P+BPhM-, we compared the integrated magnitude of bleaching at 530 nm in the VDH mutant with that found in the G(M201)D/L(M212)H and S(L178)K/G(M201)D/L(M212)H) mutants, where the P+BPhMyields have been measured both with respect to each other and to the ∼100% P+BPhL- yield in wt RCs.7 The spectra utilized in this comparison were acquired at delay times equivalent to three to four P* lifetimes as appropriate to each RC. The spectra at these times provide a good measure of the maximal concentration of P+BPhM- in each sample. Because slightly different experimental conditions were used for the VDH mutant and the other two RCs, the data sets were first normalized to the same initial concentration of P* as measured by the earlytime bleaching in the 600-nm (or 850-nm) band of P. Subsequent comparison of the integrated bleachings at 530 nm in the spectra

Photosynthetic Reaction Centers with Multiple Mutations

Figure 4. Transient absorption difference spectra in the QX and anion regions acquired using 120-fs excitation flashes at 850 nm.

following P* decay gives a P+BPhM- yield of 12 ( 3% in the VDH mutant. The time-evolution of the spectra in the anion region (Figures 4A and 5A) also demonstrate the formation of the M-side state P+BPhM- in parallel with the formation of the L-side intermediate P+I- (involving P+BChlL- and P+β-). The spectra observed immediately following excitation contain bleaching at 600 nm and positive absorption to longer wavelengths typical of P* (1-ps spectrum). Spectra shortly following P* decay (35-ps spectrum) have a broad transient absorption with two maxima centered near 640 and 690 nm. Following the procedure we have used previously to analyze the decay kinetics in this region,6,7 points before and during the excitation flash and during the P* lifetime were removed from the data sets (reducing the number of parameters in the fits), and the time-evolution of the absorbance changes were analyzed on data acquired from ∼35 ps to several nanoseconds. As is seen in the main part of Figure 5A and the plot of the residuals (inset), the time evolution of ∆A between 640 and 650 nm is not well described by a singleexponential plus a constant (dashed line) but requires a fit to the sum of two exponentials plus a constant (solid line). The same is true of the data throughout the entire region between 620 and 720 nm, with average fitted time constants of 190 ( 30 ps and 1.2 ( 0.4 ns. Because the data extend to only ∼3 ns, and this at best is about three lifetimes of the slower component, the value of the longer time constant is more uncertain than indicated by the error returned by a simple best fit. For example, fits in which the slow component was fixed at 1.5, 2.0, or 3.0 ns give visually good results with the value of the faster (190 ps) time constant only marginally changed. The use of time constants longer than about 4 ns for the slow component give poorer fits and unreasonable ∆A values (i.e., unreasonable spectra) at the asymptote of the decay. Thus, we estimate that the longer time constant has a value of 1-4 ns. Figure 6A is a plot of the preexponential values from the

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Figure 5. Decay of the anion-region transient absorption at 640-650 nm with the data during the instrument rise and during the P* decay removed. For both RCs, the fit with a function consisting of twoexponentials plus a constant (solid line), representing parallel decay of L-side P+I- and M-side P+BPhM-, is a much better fit of the data than the single-exponential fit (dashed line). The insets show the residuals.

Figure 6. Plots of the preexponential factors of the biexponential fits of the kinetics in the anion region.

dual-exponential fits to the anion-region data for the VDH mutant. The spectrum of the 190-ps component (filled triangles) shows broad absorption covering the entire region with two

5580 J. Phys. Chem. B, Vol. 105, No. 23, 2001 apparent maxima near 650 and 690 nm. The shape of this spectrum is similar to that of the raw spectrum observed shortly after the P* decay (35-ps spectrum in Figure 4A), reflecting the dominant contribution at early times of the state responsible for the fast (190-ps) decay component. The spectrum of this component is basically the same as that observed for the L-side intermediate P+I- (a mixture of P+BChlL- and P+β-) in simple L(M212)H RCs, and has the shape expected for BChl anions. Thus, the spectrum of the fast component to the anion-region absorption decay in the VDH mutant is assigned to the L-side intermediate P+I-. The preexponential-factor spectrum of the longer-lived component of the anion-region absorption decay shows a maximum near 640 nm (open circles in Figure 6A). This spectrum is the one expected for the BPhM anion based on studies in which the anion has been photochemically trapped.15 Note that the peak position is blue shifted to ∼640 nm from the 665-nm position of the BPhL anion in wt RCs.7b The small absorption near 710 nm can be assigned to the P+ component of P+BPhM- (based on P+ and P+QA- spectra in wt RCs7b,24). Further spectral support for the assignment of the 640-nm transient absorption and the longer-lived component of the anion-region decay to state P+BPhM- comes from studies of mutants in which a hydrogen bond to the ring-V keto group is added to BPhM25 or removed from BPhL,5c,17a thereby reversing the positions of the respective anion bands. It is thus clear that two spectrally and kinetically resolved anion-bearing states form from P* in the VDH mutant. The situation is similar to our previous findings for the G(M201)D/ L(M212)H and S(L178)K/G(M201)D/L(M212)H mutants.7b The two anion-bearing states result from parallel electron transfer to the L and M branches in this RC (Figure 2E). The state formed in higher yield is the L-side intermediate P+I-, which decays with a lifetime of 190 ps in the VDH RC. The state formed in lower yield is the M-side state P+BPhM-, which has a lifetime of 1-4 ns. Having identified these states from the QX and anion-region data, we can now return to the P-bleaching kinetics to fully map out the yields of all the states that form from P* as well as the yields for their decay pathways. The 830-850 nm P-bleaching data were fit to a sum of three exponentials plus a constant. Given the finding of a 1-4-ns component to the anion-region decay (associated with decay of P+BPhM-), and the small amplitude of this slow component in the P bleaching decay, the time constant (but not the preexponential value) of this component was fixed at 2 ns. The fits to the 830-850-nm data returned time constants for the other two components of 10 ( 2 ps and 200 ( 30 ps (Figure 3B inset). These values are in excellent agreement with the time constants found for the P* lifetime (determined from the stimulated emission decay kinetics) and the P+I- lifetime (the faster of the two components in the anion region), respectively. Thus, in addition to the formation of P+I- and P+BPhM(deduced from the anion-region and QX data), P* decay also returns a fraction of the RCs to the ground state. The yield of this ground-state recovery process is estimated to be 5% from the preexponential value of the 10-ps component of the P-bleaching decay. In other words, the magnitude of this preexponential factor corresponds to 5% of the initial magnitude of P bleaching. The preexponential value of the 2-ns component is 12% of the initial magnitude of P bleaching. This latter value reflects the yield of P+BPhM- and is in excellent agreement with the yield determined from the integrated magnitude of the BPhM QX bleaching. This leaves 83% as the yield of electron transfer to the L-side, resulting in P+I- formation. These yields

Roberts et al. are summarized in Figure 2E. The transient state lifetimes are given in Table 1. The middle time constant (200 ( 30 ps) determined from the P-bleaching decay kinetics is in excellent agreement with the 190-ps time constant found in the anion region and assigned to the lifetime of P+I- on the L-side. The fact that this kinetic component is found in the P-bleaching kinetics means that P+Idecays in part by return to the ground state, with the remaining fraction giving the long-lived state P+QA-. The yield of the latter state is determined to be 38% from the ratio of the asymptote of the fit to the P bleaching decay (i.e., the ∆A∞ value) to the initial amplitude of P bleaching immediately following excitation (i.e., the bleaching due to P* formation). The fit utilized for this analysis had the longest component fixed at 2 ns. This time constant, associated with charge recombination of P+BPhM-, may actually be as long as 4 ns as described above. If so, several percent of the estimated long-time P bleaching is actually associated with this M-side state. Thus, the P+QA- yield could be as low as about 35% (or as high as about 41% if the P+BPhM- lifetime is about 1 ns). Using the value of 38% for the yield of P+QA- and an initial 83% yield of P+I-, it is straightforward to calculate that P+I- decay is partitioned 46% (38/83) to electron transfer to QA and 54% to charge recombination to the ground state. From these relative yields and a P+Ilifetime of 190 ps, we estimate rate constants of (415 ps)-1 for P+I- f P+QA- and (350 ps)-1 for P+I- f ground state, respectively (Figure 2E). The VDDH Mutant. The second RC investigated here, VDDH, incorporates two Asp mutations: G(M201)D near the BChlL and F(L121)D near β. The P* lifetime determined from a single-exponential fit of the stimulated emission decay kinetics is 19 ( 3 ps. Representative data at 900-910 nm and fit are shown in the main panel of Figure 3D. As is the case for the VDH mutant, the decay of P bleaching between 830 and 850 nm requires fitting to the sum of three exponentials plus a constant to be consistent with the results in other spectral regions (Figure 3D inset) The data in both the QX and anion regions also are qualitatively similar to that described above for the VDH mutant. However, the 530-nm BPhM QX bleaching for the VDDH mutant is somewhat larger (Figure 4B). To estimate the yield of P+BPhM-, we again compared the integrated magnitudes of the 530-nm bleaching observed in the transient spectra of the VDDH mutant to those of the G(M201)D/L(M212)H and S(L178)K/G(M201)D/L(M212)H mutants. The spectra that were compared were again acquired at delay times equivalent to three to four P* lifetimes (as appropriate to each RC) after being normalized to the same initial P* concentration via the initial magnitude of 600-nm (or 850-nm) P bleaching. This analysis gives an 18% yield of electron transfer to BPhM in the VDDH mutant, compared to 12% for the VDH RC. Electron transfer to give P+BPhM- also is evident in the spectra and kinetics in the anion region (Figures 4B and 5B). In analyzing the kinetic data, for simplicity we again deleted the points before ∼60 ps from the data. Representative data at 640-650 nm and an associated monoexponential fit (dashed line) and biexponential fit (solid line) are shown in Figure 5B. As is the case for VDH RCs, the singe-exponential fit is clearly inadequate. One time constant returned from the biexponential fits is 200 ( 30 ps, which can be ascribed to the lifetime of P+I- on the L-side. The fits return a value of 1-2 ns for the slower component. Again, the true value could be longer than that obtained from a best fit due to the fact that the longest time of the measurements is only ∼3 ns. Fits with the time constant of the slow component fixed at values up to about 4

Photosynthetic Reaction Centers with Multiple Mutations ns (all other parameters free) are acceptable. Thus, we place the P+BPhM- lifetime at 1-4 ns in the VDDH RC and, like the VDH mutant, specifically relate this time constant to charge recombination P+BPhM- f ground state (probably involving P+BChlL- or P*).7b Two additional points are noteworthy. First, the preexponential-factor spectrum for this slower component of the dual-exponential fits (time constant fixed at 2 ns) has a peak near 640 nm (Figure 6B, open circles). As described above for the VDH mutant, this position is consistent with the presence of the BPhM anion (i.e., formation of state P+BPhM-). Second, the preexponential factor for the P+BPhM- component measured at 640-650 nm in the VDDH RC is ∼1.5-times larger than that for the VDH RC when the data sets are normalized to the same initial P* concentration (equal magnitude initial P bleaching at 840-850 nm). This ratio agrees with the P+BPhM- yields of 12% and 18% in the VDH and VDDH mutants, respectively, as derived from the 530-nm QX bleachings. As in the case of the VDH mutant, we fit the P bleaching decay kinetics between 830 and 850 nm to the sum of three exponentials plus a constant, holding one time constant fixed at 2 ns (for the decay of P+BPhM-). These fits returned values of 20 ( 4 ps and 230 ( 40 ps for the two other time constants. These values are in good agreement with the P* lifetime of 19 ( 1 ps determined via stimulated-emission decay and the P+Ilifetime of 200 ( 30 ps determined in the anion region (Table 1). Hence, the transient-state lifetimes determined in all spectral regions are in excellent agreement. Analysis of the preexponential values of the P-bleaching decay components for the VDDH RC gives a 15% yield for P* f ground state (from the 20-ps component) and an 18% yield of P+BPhM- formation from P*. The latter is determined (as described above for the VDH mutant) from the long (∼2 ns) component, since P+BPhM- decays solely by charge recombination to the ground state. The 18% P+BPhM- yield determined in this manner agrees with the value deduced from the QX and anion regions. Thus, the yield of P* f P+I- on the L branch is 67%. However, the magnitude of the long-time 850-nm P bleaching (determined by the asymptote of the fit when the long component is fixed at 2 ns) is smaller, namely about 48% of the initial bleaching found for state P*. As in the case of the triple mutant, this residual P-bleaching value can be assigned primarily to the long-lived state P+QA-. Again, given the uncertainty in the P+BPhM- lifetime (1-4 ns), it is possible that this state makes a small contribution to the long-time P bleaching, which would reduce the P+QA- yield to ∼44%. If an absolute P+QA- yield of 48% is used, then the P+I- decay is partitioned 72% (48/67) electron transfer to give P+QA- and 28% charge recombination to the ground state (Figure 2F). These relative yields together with the 200-ps P+I- lifetime give rate constants of (715 ps)-1 for P+I- f ground state and (275 ps)-1 for P+I- f P+QA-. Discussion In previous studies, we have established a framework for understanding the primary electron-transfer reactions in a variety of RCs with mutations near the bacteriochlorophyll (BChl) and bacteriopheophytin (BPh) intermediary electron carriers.5-7 A central aspect of the model is that the mutations can significantly affect the free energies of the associated charge-separated states. The reorganization energies for electron transfers involving these cofactors may also change to some extent depending on the mutation,5b-6b but the photochemistry together with resonance Raman21,22,26 and some X-ray27 data indicate that structural effects are of small consequence compared to the changes in

J. Phys. Chem. B, Vol. 105, No. 23, 2001 5581 energetics. That the mutations primarily affect the energetics of electron transfer is supported by the fact that the mutated/ modified RCs cluster into groups that have similar electrontransfer properties despite having very different changes in the nature or environments of the cofactors. Prime examples are the class of RCs that display the so-called “beta” type photochemistry, namely branching at P+I- (as in Figure 2B). These RCs include such diverse members as the L(M212)H, F(L121)D, and L(F97)V/F(L121)H mutants and the RCs where plant pheophytin (Pheo) is substituted for BPhL. The key point is that while these RCs differ widely in what pigment and/or surrounding amino acids are in the native BPhL pocket, they have a common effect on the primary electron-transfer reactions that can be understood in terms of common and rational changes in energetics. Furthermore, either complementary or offsetting effects are observed as appropriate when mutations are combined.5b,c,7 In the present studies, we have extended this picture to two new multiple mutants, F(L97)V/F(L121)D/L(M212)H (VDH) and F(L97)V/F(L121)D/G(M201)D/L(M212)H (VDDH). The changes that are observed in the primary events in these mutants are here again qualitatively consistent with the additive effects of the individual mutations. Although a goal of suppressing electron transfer to P+β- was not achieved, the substantial changes in the L-side events suggest that this objective may be attainable in the future. Our findings further demonstrate that the relative free energies of the L-side states contribute substantially to the directionality of initial charge separation to the L versus M sides, as well as to the balance between charge separation versus charge recombination on the L side. These points and directions for future studies on mutants of this genre are outlined in the following discussion. The VDH Mutant. Based on our work on the simple F(L121)D mutant, the introduction of Asp L121 near β should raise P+β- higher in free energy than in the L(M212)H RC. This should put P+β- well above P+BChlL-, and we believe this assertion is borne out by the experimental results presented here. Our earlier estimates for the changes in free energy of the states induced by the individual mutations in fact would predict that P+β- would move high enough in free energy to be above P* (see below). However, the combined findings on the VDH and VDDH RCs suggest that this has not occurred, or that if P+β- has moved above P* the spacing is very small. Despite this uncertainty, it is clear that F(L121)D and L(M212)H do have complementary effects on the state energies and photochemistry in the VDH mutant. In particular, the increased free energy of P+β- puts this state significantly above P+BChlLand results in increased weighting of the latter state in P+I-. This in turn enhances charge recombination so that it competes even more favorably with forward electron transfer to QA than in either single mutant. Specifically, the branching percentages for electron transfer to QA versus charge recombination to the ground state are, respectively, 78% versus 22% in F(L121)D RCs and 74% versus 26% in L(M212)H RCs, compared to 46% versus 54% in the VDH mutant (Figures 2B,C,E). Thus, the relative importance of the two pathways is reversed in the VDH mutant. This altered branching ratio for decay of P+I- accounts for most of the substantial reduction in the P+QA- yield (overall 38%) in the VDH RC compared to 70% in the single L(M212)H mutant. Electron transfer to the M side and P* internal conversion to the ground state also contribute to the reduced P+QA- in both these mutants, but by relatively small amounts. Comparing the VDH and L(M212)H mutants, P* decay gives

5582 J. Phys. Chem. B, Vol. 105, No. 23, 2001 electron transfer to the M-side to form P+BPhM- in 12% and 6% yield, respectively. Similarly, P* internal conversion to the ground state occurs in 5% yield in VDH RC and at most a few percent in the L(M212H) mutant. A similar comparison exists between the photochemistry in the VDH and F(L121)D mutants. A small reduction in the yield of electron transfer to the L side and a proportionate increase in the M-side yield in the VDH RC, like the changes in the P+I- decay branching ratio, can be rationalized in terms of the free-energy increase of P+β- relative to the single mutants. This shift is most readily understood in terms of a decreased contribution of the one-step process P* f P+β-, which uses P+BChlL- as a superexchange mediator. A change in the P* lifetime as a consequence of a change in the free energy of P+β- is not compatible with the simple twostep chemical-intermediate mechanism P* f P+BChlL- f P+β-. That these two mechanisms operate in parallel, or that some modified mechanism involving strong mixing between P+BChlL- and P+β- (or P+BPhL-) must apply to a variety of RCs has been discussed previously.3,5b,6b,7b,9,19 A small reduction in the rate of electron transfer to the L side in the VDH RC is reflected in the slightly longer P* lifetime of 10 ( 2 ps, compared to 6 ( 1 and 8 ( 1 ps in the F(L121)D and L(M212)H RCs, respectively. Whatever the detailed mechanism(s) of initial electron transfer to the L-side, a common conclusion from most recent work by a number of groups on a variety of RCs is that P+BChlL- lies below P*.3-10 This free-energy position underpins the ability of P+BChlL- to thermally or quantum-mechanically mix with states such as P+β- or P+BPhL- when they are raised in free energy. This mixing results in what we have referred to as “betatype” photochemistry, namely a branched decay of P+I- with charge recombination competing effectively with electron transfer to QA (see above). The increased electron density on BChlL in P+I- is closer to the hole on P+ than if the electron carrier were solely BPhL or β, thereby increasing the effective electronic coupling for the process. Similarly, electron transfer to QA is slowed because of the larger distance (and reduced effective coupling) involved. As discussed above, a variety of mutants and RCs in which BPhL is replaced with a different pigment (or have amino acid changes near BPhL) exhibit this behavior. An interesting aspect of this, and one that has not been widely discussed, is that these RCs divide into two sub-groups distinguished by whether P+BPhL- (or P+β- or P+Pheo-) lies below P+BChlL- or vice versa. Interestingly, the simple Rb. capsulatus L(M212)H mutant may sit on the dividing line with P+β- essentially isoenergetic with P+BChlL-. This view is based on the finding that the same (∼70%) fraction of P+I- decays to P+QA- at both 285 and 77 K.6b In comparison, the Rb. capsulatus F(L121)D mutant has a slightly higher ∼78% P+QA- yield at room temperature, and which increases to ∼88% at 77 K.6b This suggests that P+BPhLlies slightly below P+BChlL- in this mutant. Contrasting to both of these situations, we place the Rb. sphaeroides L(M214)H mutant in the other sub-group with P+β- higher in free energy than P+BChlL-. This mutant has a lower P+QA- yield (∼60%) at 285 K and that decreases to 50% at 77 K and to 25% at 5 K.5b Pushing P+β- still further above P+BChlL- via removal of the hydrogen bond to ring-V keto group of β in the Rb. sphaeroides E(L104)V/L(M214)H double mutant further reduces the P+QA- yield to 27% at 285 K and to 18% at 77 K.5b We place the Rb. capsulatus VDH mutant studied here into this second group based on the very low P+QA- yield of 38% at 285.28

Roberts et al. The VDDH Mutant. The quadruple mutant F(L97)V/ F(L121)D/G(M201)D/L(M212)H extends our work on the G(M201)D/L(M212)H RC.7a Our previous work on the latter mutant has indicated that Asp M201 near ring V of BChlL raises the free energy of P+BChlL-, possibly putting this state above P* (Figure 2D). In the VDDH mutant Asp L121 is placed near ring-V of β to raise the free energy of P+β- as well (Figure 2F). The destabilization of P+β- in the VDDH mutant causes two effects on the primary electron-transfer reactions relative to those in the G(M201)D/(L(M212)H RC. First, the free-energy upshift of P+β- by Asp L121 complements the destabilization of P+BChlL- by Asp M201 and slightly slows electron transfer to the L-side. This lengthens the P* lifetime (to 19 ps versus 15 ps in G(M201D/L(M212H) RCs) and increases the yield of electron transfer to the M side forming P+BPhM- (18% versus 15% yield).29 The slower L-side transfer can be ascribed to a reduced contribution of the one-step mechanism due to the increased free energy of P+β-; this compounds the substantially reduced L-side rate (probably primarily due to a reduced two-step contribution) that has already occurred in G(M201)D/L(M212)H RCs.7a The second result of the destabilization of P+β- by Asp L121 in the VDDH mutant compensates or couteracts the free-energy upshift of P+BChlL- by Asp M201. A higher free energy for P+β- restores the small spacing between these two states that was present in the L(M212)H single mutant and hence restores the branching ratio at P+I- decay to a value close to that found in the L(M212)H mutant. Thus, along the series of RCs illustrated in Figures 2A,B,D, and F, the following trend occurs. P+BPhL- lies well below P+BChlL- in wt by some 150-200 meV. P+β- is raised very close to P+BChlL- in L(M212)H, with the two states possibly isoenergetic. P+BChlL- is pushed well above P+β- (and probably above P*) in the G(M201)D/L(M212)H mutant. And finally, P+β- is raised and again very close to P+BChlL- in the VDDH RC. As dictated by these changes in the free energies of the states, the nature of P+I- changes along this series of RCs from being purely P+BPhL-, to a mixture of P+β- and P+BChlL-, to largely P+β-, to again a mixture of P+β- and P+BChlL-. The consequence is oscillation of the value of the branching ratio for P+I- for forward electron transfer and charge recombination as P+β- and P+BChlL- are alternately destabilized. These branching ratios are ∼100/0 in wt, ∼70/30 in L(M212)H, g90/e10 in G(M201)D/L(M212)H, and ∼72/28 in VDDH. Thus, this series of mutants, culminating in the VDDH RC studied here, gives strong support to the general conclusions we have drawn concerning (1) the effects of the mutations in shifting the free energies of the charge-separated states, and (2) the central role that the free energy gap between L-side chargeseparated states has on the competition between charge separation and charge recombination along the L-side and the consequent effect on the P+QA- yield. There are two additional interesting aspects of these comparisons. First, the essentially identical branching ratio for the VDDH and L(M212)H mutants implies that, all other things being equal, Asp L121 and Asp M201 destabilize P+β- and P+BChlL-, respectively, by a comparable amount.30 Second, while the initial yield of electron transfer to the L-side is comparable in the VDDH and G(M201)D/L(M212)H mutants (67 and 70%, respectively), the difference in the free-energy gap significantly affects the nature and decay properties of P+Iand results in a much larger difference in the overall P+QAyields (48% versus >60%) (Figures 2F and D). The latter finding is consistent with Asp L121 raising P+β- significantly

Photosynthetic Reaction Centers with Multiple Mutations but probably not so much as to put this state very much above P*, if at all. If the latter were the case, we might have expected to see even lower initial electron transfer to the L side and/or an even lower yield of P+QA-. Hence we place P+β- very close to P* in the VDDH mutant, as in Figure 2F.28 These two conclusions supplement our previous estimates (from modeling the kinetic data at room and low temperature) for the effects of the mutations on the free-energies of the charge-separated states in L(M212)H, F(L121)D, G(M201)D/ L(M212)H and related RCs.5b,6b,7 Thus, in combination we now have the following factors that must be simultaneously satisfied among these RCs: (1) Asp L121 and Asp M201 have similar effects on the free energies of P+β- and P+BChlL-, respectively, (2) the combination of L(M212)H and F(L121)D puts P+βclose to P*, (3) the L(M212)H mutation alone puts P+β- close to P+BChlL-, (4) to a first approximation, the L(M212)H mutation alone should put P+β- higher than P+BPhL- in wt by roughly the 160-280 mV difference in redox potentials of BChl versus BPh in vitro,31 and (5) the effect of Asp L121 on P+BPhL- (i.e., in the F(L121)D RC) is smaller than the effect of replacing BPhL by β (i.e., in the L(M212)H RC) but larger than the estimated 50-80 meV effect32 of removing the ringV-keto hydrogen bond to BPhL (e.g., in the E(L104)L RC).5b,17 The baseline upon which the effects of the various mutations rest is the estimated free energies of the states in wt RCs, namely that P+BChlL- and (relaxed) P+BPhL- lie below P* by 0.050.08 eV4b,5b,6b,11e and 0.25-0.28 eV, respectively.11,12a It should be noted that each of the above estimates has associated with it certain assumptions and experimental limitations, and that even small (tens of meV) deviations in (free) energy may have important consequences on electron transfer given the small spacings between P*, P+BChlL-, and P+BPhL- in wt RCs and the even smaller spacings between the states in many of the mutants.33 Taking these factors and uncertainties into account, we estimate that the M(L212)H mutation places P+β- higher in free energy than P+BPhL- in wt by 150-200 meV and that Asp L121 and Asp M201 destabilize P+β- (or P+BPhL-) and P+BChlL-, respectively, by 100-150 meV. We note that the most reasonable assessment of the results on the VDH and VDDH RCs studied here, which combine multiple mutations, is obtained if (1) the effect of L(M212)H is nearer the lower end of the range (150 meV or so), (2) Asp L121 upshifts P+βby 100 meV or so (with a somewhat a larger effect on P+BPhLin the F(L121)D single mutant), and (3) P+BChlL- lies below P* by at least 0.08 eV (if not ∼0.1 eV) and the native P+BPhLis below P* by ∼0.25 eV. These estimates provide a selfconsistent view of the energetics in mutant and wt RCs that is compatible with previous work and the studies presented here. One of the things we would have liked to have happened in the VDH mutant is for P+β- to have been pushed significantly enough above P* in free energy, thereby preventing P+β- from forming. With P+BChlL- still below P*, this would have given the cleanest opportunity to date to directly observe and characterize this charge-separated state. Similarly, it was hoped that both P+β- and P+BChlL- would have been pushed above P* in the VDDH mutant, effectively shutting off L-side electrontransfer altogether. Comparison of the M-side yield in such an RC with the yields in other mutants would have given further insight into the relative contributions to directionality of the energetics versus electronic couplings on the two sides of the RC. Neither of these scenarios was realized in the VDDH and VDH RCs, although we believe that P+β- is indeed very close to P* in these mutants. We are continuing our effort here by

J. Phys. Chem. B, Vol. 105, No. 23, 2001 5583 adding the L(E104)V mutation (initially in the VDH mutant) to further destabilize P+β- in hope that the above-mentioned goals will be achieved. Similarly, one could add the F(L121)D mutation to the Rb. sphaeroides L(M214)H or E(L104)V/ L(M214)H mutants. Although these ultimate goals were not met in the present studies, the results have demonstrated unifying consistency in the interpretations and conclusions drawn from a series of single and double mutants. Our work suggests that the free-energy gap between P+BChlL- and P* is 80-100 meV, which is slightly larger than previous estimates. We have again seen in detail how this free-energy gap affects directionality of transfer to the L versus M branches, and similarly how the free-energy gap between P+BChlL- and P+BPhL- (or P+β-, etc.) dictates the competition between electron transfer to QA versus chargerecombination, and thus the overall yield of P+QA-. These same principles recently have been applied, and will continue to be used, to enhance the yield of electron transfer to the M side7,16a and further along the M-side chain to produce P+QB-.34 The combined results should help to achieve an understanding of the electron-transfer events and their underlying mechanisms on both sides of the RC as a unified whole. Acknowledgment. This work was supported by Grant MCB0077187 from the National Science Foundation. References and Notes (1) (a) The Photosynthetic Reaction Center; Deisenhofer, J., Norris, J. R., Eds.; Academic: San Diego, 1993; Vol. II. (b) Anoxygenic Photosynthetic Bacteria; Blankenship, R. E., Madigan, M. T., Bauer, C. E., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1993. (c) The Reaction Center of Photosynthetic Bacteria; Michel-Beyerle, M. E., Ed.; Springer: Berlin-Heidelberg, 1995. (2) (a) Ermler, U.; Fritzsch, G.; Buchanan, S.; Michel, H. Structure 1994, 2, 925-936. (b) Deisenhofer, J.; Epp, O.; Sinning, I.; Michel, H. J. Mol. Biol. 1995, 246, 429-457. (c) Yeates, T. O.; Komiya, H.; Chirino, A.; Rees, D. C.; Allen, J. P.; Feher, G. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 7993-7997. (d) El-Kabbani, O.; Chang, C.-H.; Tiede, D.; Norris, J.; Schiffer, M. Biochemistry 1991, 30, 5361-5369. (3) (a) Finkele, U.; Lauterwasser, C.; Zinth, W. Biochemistry 1990, 29, 8517-8521. (b) Nagarajan, V.; Parson, W. W.; Gaul, D.; Schenck, C. C. Biochemistry 1993, 32, 12324-12336. (c) Jia, Y.; DiMagno, 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, 13180-13191. (d) 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, 7256-7268. (4) (a) Shkurapatov, A. Y.; Shuvalov, V. A. FEBS Lett. 1993, 322, 168-172. (b) Schmidt, S.; Arlt, T.; Hamm, P.; Huber, H.; Nagele, T.; Wachtveitl, J.; Meyer, M.; Scheer, H.; Zinth, W. Chem. Phys. Lett. 1994, 223, 116-120. (c) Huber, H.; Meyer, M.; Nagel, T.; Hartl, I.; Scheer, H.; Zinth, W.; Wachtveitl, J. Chem. Phys. 1995, 197, 297-305. (d) Kennis, J. T. M.; Shkuropatov, A. Y.; van Stokkum, I. H. M.; Gast, P.; Hoff, A. J.; Shuvalov, V. A.; Aartsma, T. J. Biochemistry 1997, 36, 16231-16238. (5) (a) Kirmaier, C.; Gaul, D.; DeBey, R.; Holten, D.; Schenck, C. C. Science 1991, 251, 922-927. (b) Kirmaier, C.; Laporte, L.; Schenck, C. C.; Holten, D. J. Phys. Chem. 1995, 99, 8910-8917. (c) Kirmaier, C.; Laporte, L.; Schenck, C. C.; Holten, D. J. Phys. Chem. 1995, 99, 8903-8909. (6) (a) Heller, B. A.; Holten, D.; Kirmaier, C. Biochemistry 1995, 34, 5294-5302. (b) Heller, B. A.; Holten, D.; Kirmaier, C. Biochemistry 1996, 35, 15418-15427. (7) (a) Heller, B. A.; Holten, D.; Kirmaier, C. Science 1995, 269, 940945. (b) Kirmaier, C.; Weems, D.; Holten, D. Biochemistry 1999, 38, 11516-11530. (8) Arlt, T.; Dohse, B.; Schmidt, S.; Wachtveitl, J.; Laussermair, E.; Zinth, W.; Oesterhelt, D. Biochemistry 1996, 35, 9235-9244. (9) (a) Bixon, M.; Jortner, J.; Michel-Beyerle, M. E. Biochim. Biophys. Acta 1991, 1056, 301-315. (b) Bixon, M.; Jortner, J.; Michel-Beyerle, M. E. Chem. Phys. 1995, 197, 389-404. (10) (a) Parson, W. W.; Chu, Z.-T.; Warshel, A. Biochim. Biophys. Acta 1990, 1017, 251-272. (b) Alden, R. G.; Parson, W. W.; Chu, Z. T.; Warshel, A. J. Am. Chem. Soc. 1995, 117, 12284. (11) (a) Goldstein, R. A.; Boxer, S. G. Biochim. Biophys. Acta 1989, 977, 70-77. (b) Chidsey, C. E. D.; Takiff, L.; Goldstein, R. A.; Boxer, S. G. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 6850-6854. (c) Ogrodnik, A.;

5584 J. Phys. Chem. B, Vol. 105, No. 23, 2001 Volk, M.; Letterer, R.; Feick, R.; Michel-Beyerle, M. E. Biochim. Biophys. Acta 1988, 936, 361-371. (d) Ogrodnik, A.; Keupp, W.; Volk, M.; Aumeier, G.; Michel-Beyerle, M. E. J. Phys. Chem. 1994, 98, 3432-3439. (e) Volk, M.; Aumeier, G.; Langenbacher, T.; Feick, R.; Ogrodnik, A.; MichelBeyerle, M. E. J. Phys. Chem. B 1998, 102, 735-751. (12) (a) Woodbury, N. W. T.; Parson, W. W. Biochim. Biophys. Acta 1984, 767, 345-361. (b) Peloquin, J. M.; Williams, J. C.; Lin, X.; Alden, R. G.; Taguchi, A. K. W.; Allen, J. P.; Woodbury, N. W. Biochemistry 1994, 33, 8089-8100. (c) Holzwarth, A. R.; Mhller, M. G. Biochemistry 1996, 35, 11820-11831. (13) Gunner, M. In Current Top. Bioenergetics 1991, 16, 319. (14) Thompson, M. A.; Zerner, M. C.; Fajer, J. J. Am. Chem. Soc. 1991, 113, 8210-8215. (d) Marchi, M.; Gehlen, J. N.; Chandler, D.; Newton, M. Science 1994, 263, 499-502. (e) Gunner, M. R.; Nicholls, A.; Honig, B. J. Phys. Chem. 1996, 100, 4277-4291. (f) Blomberg, M. R. A.; Siegbahn, P. E. M.; Babcock, G. T. J. Am. Chem. Soc. 1998, 120, 8812-8824. (15) (a) Kellogg, E. C.; Kolaczkowski, S.; Wasiewlewski, M. R.; Tiede, D. M. Photosynth. Res. 1989, 22, 47-59. (b) Robert, B.; Tiede, D. M.; Lutz, D. M. FEBS Lett. 1985, 183, 326-330. (16) (a) Katilius, E.; Turanchik, T.; Lin, S.; Taguchi, A. K. W.; Woodbury, N. W. J. Phys. Chem. B 1999, 103, 7386-7389. (b) Lin, S.; Xiao, W.; Eastman, J. E.; Taguchi, A. K. W.; Woodbury, N. W. Biochemistry 1996, 35, 3187-3196. (17) (a) Bylina, E. J.; Kirmaier, C.; McDowell, L. M.; Holten, D.; Youvan, D. C. Nature 1988, 336, 182-184. (b) (a) Kirmaier, C.; Holten, D.; Bylina, E. J.; Youvan, D. C. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 7562-7566. (c) McDowell, L. M.; Gaul, D.; Kirmaier, C.; Holten, D.; Schenck, C. C. Biochemistry 1991, 30, 8315-8322. (18) (a) Allen, J. P.; Williams, J. C. J. Bionergetics and Biomembranes 1995, 27, 275-283. (b) Williams, J. C.; Alden, R. H.; Murchison, H. A.; Peloquin, J. M.; Woodbury, N. W.; Allen, J. P. Biochemistry 1992, 31, 11029-11037. (c) (a) Murchason, H. A.; Alden, R. G.; Allen, J. P.; Peloquin, J. M.; Taguchi, A. K. W.; Woodbury, N. W.; Williams, J. C. Biochemistry 1993, 32, 3498-3505. (d) Allen, J. P.; Artz, K.; Lin, X.; Williams, J. C.; Ivanicich, A.; Albouy, D.; Mattioli, T. A.; Fetsh, A.; Kuhn, M.; Lubitz, W. Biochemistry 1996, 35, 6612. (19) (a) Woodbury, N. W.; Lin, S.; Lin, X.; Peloquin, J. M.; Taguchi, A. K. W.; Williams, J. C.; Allen, J. P. Chem. Phys. 1995, 405, 421. (b) Lin, S.; Taguchi, A. K. W.; Woodbury, N. W. Excitation Wavelength. J. Phys. Chem. 1996, 42, 17067-17078. (20) Goldsmith, J. O.; King, B.; Boxer, S. G. Biochemistry 1996, 35, 2421-2428. (21) Cua, A.; Kirmaier, C.; Holten, D.; Bocian, D. F. Biochemistry 1998, 37, 6394-6401. (22) (a) This shift could occur by several mechanisms depending on the ionization state of Asp M201. Resonance Raman studies are most consistent with the residue being ionized at room temperature.22b (b) Czarnecki, K.; Kirmaier, C.; Holten, D.; Bocian, D. F. J. Phys. Chem. A 1999, 2235-2246. (23) (a) Bylina, E. J.; Youvan, D. C. Z. Naturforsch. 1987, C 42, 769774. (b) Bylina, E. J.; Youvan, D. C. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 7226-7230. (c) Bylina, E. J.; Jovine, R. V. M.; Youvan, D. C. Bio/ Technology 1989, 7, 69. (24) Parson, W. W.; Cogdell, R. J. Biochim. Biophys. Acta 1975, 416, 105-149.

Roberts et al. (25) Incorporation of AspM131 or GluM131 near ring V of BPhM causes a small red shift in the Qx band in the ground-state absorption spectrum and a substantial red shift in the anion band of state P+BPhM-. A manuscript is in preparation on this work. (26) Czarnecki, K.; Cua, A..; Kirmaier, C.; Holten, D.; Bocian, D. F. Biospectroscopy 1999, 5, 346-357. (27) Chirino, A. J.; Lous, E. J.; Huber, M.; Allen, J. P.; Schenck, C. C.; Paddock, M.; Feher, G.; Rees, D. C. Biochemistry 1994, 33, 4584. (28) In this regard, one might expect that low-temperature measurements on the VDDH mutant would be quite informative. Unfortunately, we have found that the effects of Asp at M201 on both the primary events and the resonance Raman characteristics of BChlL are reduced at low temperature.22b Whether this occurs via a change in the ionization state of this residue or a change in other types of interactions with the chromophore is not clear at present. In any case, at low temperature the photochemistry of G(M201)D/L(M212)H RC becomes more similar to that of M(L212)H RCs (i.e., as if Asp M201 were not present), and one would expect a similar effect in the VDDH mutant. This would make lowtemperatures studies on this mutant aimed at further elucidating the relative free energies of the states not particularly useful. (29) In both the VDDH and G(M201)D/L(M212)H RCs, about 15% of the P* decay also occurs by charge recombination to the ground state (Figure 2). (30) Both residues are near ring V of their respective pigments, but Asp L121 lies over the face of β (or BPhL) while Asp M201 lies more edge on with respect to BChlL and should lie somewhat closer. Both residues upshift the ring-V keto vibrations of the respective chromophores (by different amounts). The Raman and electron-transfer data are both consistent with the Asp residues being ionized, although the effects of the residues to increase the free energies of the respective charge-separated states do not require this situation.6,7,21,22 (31) (a) Fajer, J.; Borg, D. C.; Forman, A.; Dolphin, D.; Felton, R. H. J. Am. Chem. Soc. 1973, 95, 42739. (b) Fajer, J.; Brune, D. C.; Davis, M. S.; Forman, A.; Spaulding, L. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 49564960. (32) Michel-Beyerle, M. E.; Plato, M.; Deisenhofer, J.; Michel, H.; Bixon, M.; Jortner, J. Biochim. Biophys. Acta 1988, 932, 52-70. Similar 50-100 mV shifts in the redox potential of P have been found upon addition or removal of single hydrogen bonds.18 (33) (a) These factors include: (i) the use of equilibrium kinetic models that may not include all the possible relevant states or processes, (ii) changes in temperature (used in Arrhenious analyses) certainly affect more than simply kT,5b,22 (iii) the mixing between the charge-separated states may not simply be thermal in character (dictated by free energy gaps) but also quantum mechanical in nature (dictated by energy gaps) and thus that small entropic contributions (which are temperature dependent) may be important,5b,6b (iv) there are likely both dynamic relaxation events12 and static distributions33b,3c,11e that may affect the applicable (free) energy gaps between the states. (b) Kirmaier, C.; Holten, D. Proc. Natl. Acad. Sci. U.S.A. 1990, 97, 3522-3556. (34) Laible, P. D.; Kirmaier, C.; Holten, D.; Tiede, D. M.; Schiffer, M.; Hanson, D. K. In Photosynthesis: Mechanisms and Effects; Garab, G., Ed.; Kluwer: Dordrecht, The Netherlands, 1998.