Y(M208)F Mutant of

The combination of the Phe(L181) f Tyr and Tyr(M208) f Phe amino acid substitutions in reaction centers. (RCs) of Rhodobacter capsulatus yields an RC ...
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J. Phys. Chem. B 2004, 108, 11827-11832

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B-Side Electron Transfer To Form P+HB- in Reaction Centers from the F(L181)Y/ Y(M208)F Mutant of Rhodobacter capsulatus Christine Kirmaier,*,† Philip D. Laible,‡ Deborah K. Hanson,‡ and Dewey Holten† Department of Chemistry, Washington UniVersity, St. Louis, Missouri 63130, and Biosciences DiVision, Argonne National Laboratory, Argonne, Illinois 60439 ReceiVed: April 2, 2004; In Final Form: May 21, 2004

The combination of the Phe(L181) f Tyr and Tyr(M208) f Phe amino acid substitutions in reaction centers (RCs) of Rhodobacter capsulatus yields an RC in which charge separation to the B-side bacteriopheophytin (HB) occurs in about 15% yield. This yield is determined from analysis of the relative bleachings of the QX bands of HA and HB at 542 and 527 nm, respectively, and comparison to simulations. These results are presented along with comparison to previous work on this “YF” mutant and other mutant RCs that produce electron transfer to the B-side of the RC.

Introduction The bacterial photosynthetic reaction center (RC) is a membrane-bound pigment-protein complex with remarkable pseudo-C2 symmetry.1-4 The L and M polypeptides of the RC each have five mirror-image membrane-spanning helices housing the arrangement of bacteriochlorophyll (B), bacteriopheophytin (H), and quinone (Q) moieties shown in Figure 1A. Despite the gross C2 symmetry of the RC protein and cofactors, it is functionally asymmetric with electron transfer unidirectional to the so-called A-side. In wild-type RCs, photoexcitation of the primary electron donor (P), a bacteriochlorophyll dimer, to its lowest excited singlet state (P*) initiates charge separation exclusively to one of the two nominally identical sets of electron acceptors, forming P+HA- in ∼3 ps. This initial charge separation is facilitated by BA in parallel mechanisms involving P+BA- as a discrete and virtual intermediate. Subsequent P+HAf P+QA- electron transfer occurs with an ∼200 ps time constant, yielding P+QA- with an overall quantum yield of ∼1 (Figure 1B). In recent years this asymmetric functionality has been broken via site-directed mutagenesis producing RCs that undergo B-side electron transfer to give either P+HB- or P+φB(where φB denotes a bacteriopheophytin that replaces BB) in yields as high as 35%.5-13 Charge-separated states on the B-side of the RC also have been reported under conditions of high pulse energy or short wavelength excitation with native complexes.14,15 Recently it was shown that P+HB- supports electron transfer to QB, with the rate more than 10-fold slower than P+HA- f P+QA- electron transfer.6,16 Manipulation of the free energies of the P+BA- and P+BBstates has been a key to unlocking the activity of the B-side cofactors. Unidirectional versus bidirectional electron transfer may depend additionally on differential electronic matrix elements on the two sides of the RC. Among the sites where amino acid substitutions have yielded B-side electron transfer are the C2-symmetry-related-residues M208Tyr and L181Phe, which are highly conserved in bacterial RCs including those of Rhodobacter (Rb.) capsulatus, Rb. sphaeroides, and Blastochlo* To whom correspondence should be addressed. E-mail: kirmaier@ wuchem.wustl.edu. † Washington University. ‡ Argonne National Laboratory.

Figure 1. (A) Arrangement of the RC cofactors as determined by X-ray structures of RCs from Blastochloris Viridis and Rb. sphaeroides. (B) Schematic energy level diagram and pathway of charge separation for wild-type RCs.

ris Viridis.17 Reversing these amino acids, (i.e., swapping L181 to Tyr and M208 to Phe) in combination with mutation of the native Leu residue at M212 to His (the “YFH” mutant) results in an ∼30% yield of P+HB-.7 The YFH RC is a direct offshoot of early theoretical work that had indicated the amino acids at M208 and L181 have significant effects on the free energies of P+BA- and P+BB-, respectively, and hence on the RC photochemistry.18-20 In the early 1990s, a large family of RCs with single and combination mutations at these two residues was studied with the main focus of investigating the role of BA in initial charge separation.21-34 Among those earlier studied RCs was the YF double mutant, having the swap mentioned above but lacking the additional Leu f His mutation at M212.23,28,31 In this study, we have reinvestigated the YF mutant of Rb. capsulatus specifically with an eye toward assaying B-side electron transfer. As noted, earlier work on this mutant did not focus on B-side electron transfer, and in light of the YFH mutant, with its 30% yield of P+HB-, it is useful to examine the YF mutant again for several reasons. A primary task is to assess how difficult it is to detect B-side electron transfer spectroscopically when electron transfer to HA is expected to occur in the major fraction of the RCs. The principal assay for monitoring HA reduction versus HB reduction is the relative bleachings of the QX bands of these molecules at ∼542 and ∼527 nm, respectively. When HA is replaced with a bacteriochlorophyll as a result of the His-M212 mutation

10.1021/jp0485441 CCC: $27.50 © 2004 American Chemical Society Published on Web 07/10/2004

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(attendant QX band near 600 nm), the 520-550 nm region provides a clean spectroscopic window to assay HB bleaching. This is not the case for the YF mutant. The issue of detectability is relevant to other RC mutants that retain HA. A second, more fundamental impetus for reexamining the photochemistry of the YF mutant is to assess whether and how the results add in a consistent way to the small knowledge base that ultimately provides the core understanding of the origins of unidirectionality (contributions of the free energy relationships of the states, electronic matrix elements, and rate constants for P* decay pathways). Along these lines, the studies herein test a 17% yield of P+HB- in the YF mutant predicted from work on the YFH mutant.7 Materials and Methods Construction of the Rb. capsulatus YF mutant was described previously.23,33 The preexisting YF mutant L and M genes were subcloned into expression vectors that append a polyhistidine tag to the C-terminus of the M subunit.35 Two expression vectors were used: one yields cells that express both light harvesting I (LHI) and RC complexes and the other carries a mutation that prevents formation of LHI. RCs were isolated from LHI- cells using the detergent Deriphat 160-C.36 RCs were isolated from LHI-containing cells by using the standard detergent LDAO (N,N-dimethyldodecylamine-N-oxide). Further details of the plasmids, strains, and RC isolation protocols are described elsewhere.36 We made no attempt to either incorporate or assay QB occupancy or activity in the samples studied here. On the basis of previous works, we expect that the Deriphat-prepared RCs will have a fully occupied QB site, and that in the LDAOprepared RCs the QB site will be partially occupied.36 The QB occupancy will affect the value and amplitude of long-time (nanosecond) components associated with P+HB- decay in our kinetic data, but these are beyond the scope of this work. Here we focus exclusively on the P* decay kinetics and relative yields of initial charge separation to the A- and B-sides of the YF mutant RC. The time-resolved absorption measurements were carried out on an apparatus that utilizes ∼130 fs excitation and white-light probe flashes. Samples (2.5-3 mL volume of 25-35 µM RCs in buffers indicated below) were held in an ice-cooled reservoir and flowed through a 2-mm path length cell. This arrangement maintained a sample temperature of ∼10 °C. Further details of the instrumentation and data analysis procedures can be found elsewhere.37,38 Samples consisted of RCs in 10 mM Tris, pH 7.8, containing either 0.05% LDAO or 0.1% Deriphat 160-C. Results Excitation of LDAO-isolated YF RCs with a 130 fs, 590 nm flash produces the excited singlet state of the primary donor, P* (0.5 ps spectrum in Figure 2 inset). The transient absorption difference spectrum of P* displays bleaching of the 850 nm ground-state absorption band of P and concomitant stimulated emission from P* on the long-wavelength side of the P bleach. The spectra in Figure 2 indicate that at all wavelengths throughout this 840-950 nm spectral region there is a contribution from a process such as P+HA- f P+QA- electron transfer (or decay of P+HB-) after P* decay. This is evidenced by the differences in the spectra at 35 ps and 3 ns. Consequently, there is virtually no wavelength in this region of the spectrum at which the kinetic profiles are truly fit by a single exponential function due simply to P* stimulated emission decay or P bleaching decay. At wavelengths past ∼900 nm, P* stimulated emission dominates the spectrum and the amplitude of a second com-

Figure 2. Stimulated emission decay for the YF mutant. The circles are averaged data between 900 and 910 nm, and the solid line is a fit to the instrument function plus two exponentials plus a constant. The inset shows transient absorption difference spectra acquired at the times indicated following a 130 fs, 590 nm excitation flash.

ponent is very small. Fitting the data to either one exponential or to the sum of two exponentials, plus the instrument response plus a constant, gives 8 ( 1 ps for the P* lifetime. A representative data set and dual-exponential fit are shown in the main part of Figure 2. As is often the case for the P* stimulated emission data of RCs, there is a general trend that the fit time constant varies with probe wavelength, tending toward a shorter P* lifetime at longer wavelengths (e.g., ∼6 ps at 940 nm). The time constant of the second component is much longer (at least hundreds of picoseconds) and cannot be determined with accuracy from the data owing to its very small amplitude in this spectral region. Our ∼8 ps value for the P* lifetime compares well with the P* lifetimes reported in earlier works on the Rb. capsulatus YF mutant: ∼6 ps, weighted average of a two-component fit;23 ∼9 ps, weighted average of a two-component fit;28 and ∼12 ps, weighted average of a twocomponent fit on membrane-bound samples of YF mutant RCs.31 The spectra in Figure 2 (inset) also show that there is decay of P bleaching during the ∼3 ns time course of the experiments. This ground-state recovery takes place both during the decay of P* and on a longer time scale (most easily observed at ∼850 nm). We will describe below evidence for formation of P+HB-, which is known to have a lifetime on the order of several nanoseconds and to decay in part by charge recombination to the ground state even when QB is present. The spectral changes that take place between 35 ps and 3 ns can be associated with this process. The amplitude here is small and the data are restricted to 3 ns, making unreliable a fit to determine the P+HBlifetime. (See ref 6 for a detailed discussion of this topic.) Thus the data throughout the P bleaching region, ∼840-900 nm, were fit to the instrument response plus two exponentials plus a constant, with the second component, for P+HB- decay, held fixed at values between 1 and 4 ns. Whether the fixed value was 1 or 4 ns mattered little in determining, again, an average P* lifetime of ∼8 ps. On the blue side of the P bleaching (e.g., 830-850 nm), little contribution from P* stimulated emission is expected. Hence the amplitude of the 8 ps component here is a measure of the yield of P* f ground state. From the preexponential factors of the fits, we estimate this yield to be 5-10%. Essentially the same results were obtained with Deriphat-isolated YF RCs except that the main P bleaching is at ∼865 nm (in accordance

B-Side Electron Transfer in Mutant Reaction Centers

Figure 3. Comparison of the BPh QX and anion region transient absorption spectra for YF and wild-type RCs isolated using LDAO (A) or Deriphat 160-C (B). The spectral pairs were acquired using 130 fs flashes at 850 nm and are normalized to the same initial P* concentration. For each RC the spectrum shown was acquired at a delay time following excitation that corresponds to about 4 times the 1/e value of the respective P* lifetime. See text for further details.

with the ground-state absorption spectrum), the P* lifetime is 14 ( 2 ps, and the yield of direct P* decay to the ground state may be slightly larger, 10-15%. Figure 3 shows the data that are the crux of this study to determine whether electron transfer takes place from P* to the B-side H acceptor in YF RCs. Figure 3A compares spectra taken in the QX and anion region for YF and wild-type RCs (both prepared using LDAO) at 30 and 12 ps, respectively. For Deriphat-isolated RCs (Figure 3B), the spectra were acquired at longer times, in accord with the longer P* lifetimes for RCs isolated with this detergent (∼14 ps for YF and ∼6 ps for wild type). In all cases, the delay times of compared spectra correspond to roughly 4 times the 1/e value of the respective P* lifetimes and reflect a comparison of the maximal concentration of the product(s) of P* decay. Further, the spectra in Figure 3 were acquired on samples that had identical ground-state absorption at 850 (LDAO RCs) or 865 nm (Deriphat) and gave pairwise identical magnitudes of P bleaching in the initial P* spectra. In other words, the pairwise spectral comparisons are quantitative for the same initial P* concentration, as we have described previously.5,7,8 The results are very similar for the LDAO and Deriphat RCs. In considering the LDAO-isolated RCs first, it can be seen from Figure 3A that the bleaching of the QX band of HA at ∼542 nm is clearly much smaller, about 30% smaller at the maximum of the bleaching, for the YF mutant compared to wild type. Let us take at face value the apparent 30% reduction of HA QX bleaching as reflecting an ∼70% yield of P+HA- from P* in the YF mutant. Although the QX bleaching profile is clearly asymmetrically broadened on the blue side of the YF spectrum compared to the wild-type spectrum, it is not readily apparent that concomitant bleaching of the QX band of HB at ∼527 nm occurs with an amplitude that is 30% that of the wild-type 542 nm bleaching. We have already determined above that P* decay

J. Phys. Chem. B, Vol. 108, No. 31, 2004 11829 in this mutant is accompanied by ∼10% direct return (internal conversion) to the ground state. This leaves then, in the simplest analysis, a yield of ∼20% for P+HB-. In the anion region (620-700 nm), we consider whether the observed spectra are consistent with this scenario. The HA and HB anion absorption bands are known to be very broad (4050 nm full width at half-maximum, as in the spectra for the wild-type RC in Figure 3) with peaks in the difference spectra near ∼665 and ∼645 nm, respectively. With ∼10% of P* decaying directly to the ground state, and 90% the expected combined yield of P+HA- and P+HB-, we should find an integrated 620-700 nm absorption that is reduced in magnitude by only ∼10% compared to wild type (assuming the anions of HA and HB have equal oscillator strength). Qualitatively, this is seen in the spectral comparisons in Figure 3A. In addition, the amplitude at 665 nm (HA-) is reduced and there is a slight increased anion absorption near 630-650 nm (expected for HB-) for the YF mutant compared to wild-type RCs. Essentially the same results can be seen for the comparison of wild-type and YF RCs isolated using Deriphat 160-C (Figure 3B). Here the amplitude of reduction of the QX bleaching of HA is perhaps a little larger (30-35%) and the QX bleaching profile is broadened even more asymmetrically on the blue side. However, there is also potentially a slightly larger yield of initial P* decay to the ground state (estimated to be 10-15%). Figure 4 (bottom panel) shows our efforts to simulate quantitatively, for direct comparison with our experimental YF spectrum, the QX bleaching profile expected for 10%, 20%, and 30% yield of P+HB- plus 80%, 70%, and 60% yield, respectively, of P+HA-. (The remaining 10% of P* decay is simulated to occur via return to the ground state.) To perform these simulations of the QX bleachings, we used a basis spectrum for 100% yield of P+HA- derived from the experimental data from wild-type RCs (e.g., the wild-type spectrum in Figure 3A), and a basis spectrum for 30% yield of P+HB- derived from experimental data from the YFH mutant (taken from ref 7). These basis spectra are shown in Figure 4 (top panel). In the YFH RC, the P+HB- QX bleaching is not distorted from simultaneous HA reduction because the HA pigment is replaced with a bacteriochlorophyll (β). The comparison of the experimental YF data to the simulations is consistent with a yield of P+HB- that is between 10% and 20%. This agrees well with the simpler analysis presented above as a starting point. With yields this small (∼10% ground-state return and 10-20% P+HB-) and known and unknown electrochromic shifts on top of the bona fide QX bleachings of HA and HB, our best estimate is that there is a 15 ( 5% yield of P+HB- in both LDAO- and Deriphat-isolated RCs of the YF mutant. Figure 5 shows kinetic data averaged over the interval 536546 nm, monitoring the appearance and decay of HA bleaching in LDAO-isolated YF RCs. The solid line is a fit to the instrument response plus two exponentials plus a constant, with fit values of 8.2 ( 0.4 ps and 225 ( 17 ps. These time constants are assigned to the P* lifetime and the P+HA- lifetime (dominated by electron transfer to QA), respectively. Fits throughout the 640-700 nm region return the same values within experimental error. We expect there to be deviation from exponentiality for the ∼225 ps component at wavelengths where HB- contributes strongly (e.g., 640-650 nm), reflecting the fewnanosecond lifetime of P+HB-. However, we did not attempt to acquire a large kinetic data set and focus on resolving this lifetime, since the yield of P+HB- is so low and because determining its lifetime is very difficult from picosecond measurementsswell beyond the scope of this work.

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Figure 5. Appearance and decay of the QX bleaching averaged between 536 and 546 nm for YF RCs isolated with LDAO. The inset focuses on the first 100 ps of the kinetic profile. The data (circles) were acquired using ∼130 fs excitation flashes at 850 nm. The solid lines are a fit to the instrument response plus two exponentials plus a constant, giving the P* lifetime and the lifetime (major component) of P+HA- given in the text.

Figure 4. Experimental and simulated QX bleaching spectra for different combined yields of P+HA- (denoted A) and P+HB- (denoted B) in LDAO-prepared RC samples. The top panel shows transient difference spectra that were used as the basis for the generation of the simulated transient difference spectra in the bottom panel. The solid spectrum in the top panel was acquired immediately following P* decay (i.e., taken at ∼12 ps) for wild-type RCs and corresponds to the transient spectrum associated with 100% yield of state P+HA-. Relative to the same initial P* concentration, the dashed spectrum in the top panel corresponds to 30% yield of state P+HB-, as determined in the YFH mutant RC (ref 7). The direct experimental YFH spectrum acquired immediately following P* decay is known to be a mixture of 30% P+HB- and 60% P+β-. To obtain the spectrum corresponding to solely 30% P+HB- that is shown in the top panel (dashed line), we subtracted (from the direct experimental YFH transient spectrum) a 60% contribution of the known P+ absorption and assumed that the species β- does not contribute appreciably to the absorption changes between 510 and 570 nm. The absorption due to P+ in the 510-570 nm region is essentially “flat” (i.e., nearly constant ∆A, as is seen, for example, in the spectrum of P+QA- in wild-type RCs or the spectrum of RCs in which P is oxidized with potassium ferricyanide). Hence subtracting the P+ absorption merely subtracts a small positive “baseline” that has no effect on the shape or amplitude of the QX bleaching of HB. The bottom panel shows the simulated transient difference spectra that result from linear combinations of the two basis spectra in the top panel for the three mixtures of P+HA- and P+HB- indicated (dashed and dotted lines) in comparison to the experimental YF data (solid line).

Discussion We undertook this study to investigate whether electron transfer takes place to the B-side of the RC in the YF mutant, with the expectation from work on the YFH mutant that it does. We have provided evidence here, by means of a controlled comparison to wild-type RCs, that in the YF mutant (for RCs isolated with LDAO) P* decays to give P+HB- in ∼15% yield and P+HA- in ∼75% yield, with the remaining ∼10% of P* decaying by internal conversion to the ground state. For RCs isolated with Deriphat 160-C, the yields of both ground-state recovery and electron transfer to the B-side may be slightly larger with a concomitant small reduction in the yield of P+HA-. We adopt the simple branching scheme shown in Figure 6 for use in comparing our results on the YF mutant to those reported previously for RCs that have mutations at L181 and M208. (All of the following discussion compares work on

Figure 6. Simple kinetic model used to calculate effective rates of electron transfer to the A and B sides of the RC (kPA and kPB, respectively) and return to the ground state (kPG). See text for discussion details.

LDAO-isolated Rb. capsulatus RCs, except as noted.) This simple model utilizes the apparent (effective) rate constants kPB, kPA, and kPG for P* f P+HB-, P* f P+HA-, and P* f ground state, respectively, which can be calculated from the measured P* lifetime and the yields of the three product states. The resultant values are given in Table 1 for the YF mutant, calculated from the results reported here. The other values in the table have been taken from previous studies. A time constant for P* f ground state of 1/kPG ∼ 100 ps is a fairly consistent result from this small set of mutants. This is about a factor of 2 smaller than the P* lifetime measured in the Rb. capsulatus DLL mutant in which no electron transfer takes place.39 In principle, the inherent P* lifetime (i.e., 1/kPG) should be relatively invariant; however, this point is largely unexplored. The YFH and YF mutants provide two independent measurements of the rate of B-side charge separation for the case of a BPh acceptor and a Tyr residue at L181. The agreement between the results is excellent, with calculated time constants of ∼37 and ∼53 ps, respectively, for YFH and YF RCs. These are well within experimental error of each other when one takes into account the error bar on the P* lifetimes and the error in the estimated yields of the products of P* decay. The calculated ∼11 ps time constant for the A-side charge separation in the YF mutant cannot be compared to any of the other values of kPA for mutants listed in Table 1, since in all the other cases the mutants have a bacteriochlorophyll (β) in place of HA. However, this 11 ps time constant can be used to predict a P* lifetime for the single TyrM208 f Phe mutant of ∼9 ps. This is the value calculated from 1/(kPG + kPA + kPB), with kPG ∼ 1 × 1010 (∼100 ps time constant as discussed above), with kPB also ∼1 × 1010 (∼100 ps time constant determined from the H and DH mutants, both having the native Phe at L181), and with kPA ) 8.75 × 1010 (11 ps time constant from the YF mutant,

B-Side Electron Transfer in Mutant Reaction Centers

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TABLE 1: P* Lifetimes, Decay Products, and Associated Apparent Rate Constantsa % yield RC

P* lifetime (ps)

ground state

P+I- b

P+HB-

wt H YFH YF DH KDH

4.3 ( 0.3 8.5 ( 0.8 11 ( 2 8(2 15 ( 2 15 ( 2

ndc nd 10 10 15 15

100 93 60 75 70 62

nd 7 30 15 15 23

(kPG)-1 (ps)

(kPA)-1 (ps)

(kPB)-1 (ps)

110 80 100 100

4.3 9.1 18 11 21 24

121 37 53 100 65

a All data were taken at ∼285 K. The measured yields of the states have an error of (5%. The values for RCs of the wild type and the H, DH, and KDH mutants are those reported in refs 5, 7, and 8. b P+I- denotes the A-side intermediate acceptor state, which is P+HA- in wild-type and YF RCs. In all the other mutants listed, P+I- is an admixture of P+BA- and P+β-.5 c Not detected.

where HA is present and there is a Phe at M208). It is interesting that each of the values going into this (overly) simple calculation comes from different mutants, yet the ∼9 ps P* lifetime calculated for the single “F” mutant (TyrM208 f Phe) is in excellent agreement with the experimental value. Specifically, Jia et al. reported a 9.3 ps component with ∼80% amplitude in their two-exponential fit to the P* lifetime in the F mutant of Rb. capsulatus.28 The ∼(11 ps)-1 rate constant for A-side charge separation in RCs with a Phe at M208 is roughly 3-fold longer than the ∼(4 ps)-1 rate constant for electron transfer to the A-side in wild type, where Tyr is present at M208. If the same relative effect holds on the B-side, this would say that the B-side rate of electron transfer in wild-type RCs is in the neighborhood of (120-150 ps)-1, based on (40-50 ps)-1 for kPB with a Tyr at L181. No doubt this is far too simple a comparison to make, but an interesting one for discussion purposes nonetheless because the value of kPB for wild-type RCs (blank in Table 1) is not known except by construction of this type of comparison. Previous workers have suggested that there is roughly a 30-fold (or larger) difference in the rates of charge separation to the A- and B-sides in wild-type RCs.40,41 A 30-fold difference would correspond to a B-side rate constant of ∼(120 ps)-1. These two estimates, coming from completely independent means, match very well. Additionally, these estimates match those of kPB in the H and DH mutants, in which the B-side of the RC is native.7,8 Clearly, having a Tyr at M208 provides a competitive advantage of charge separation to the A-side, but also it is clear that charge separation to the A-side will still far dominate with a Phe (or perhaps an aliphatic amino acid) at this position. Underscoring this, M208 is a leucine in RCs from Chloroflexus aurantiacus. As a final point, we can calculate that the A- and B-side yields of charge separation expected in the Rb. capsulatus F mutant (Phe at M208) are ∼93% and ∼6%, respectively. The yield of P+HB- in YF RCs that we report here is in excellent agreement with the previous prediction.7 The challenges of spectroscopically assaying the B-side electron transfer yield under conditions for which absorption changes associated with P+HA- dominate are portrayed. The simulations indicate that not until 20 to 30% yield P+HB- is reached will HB bleaching at ∼527 nm begin to be distinctly resolved in transient spectra at room temperature so long as HA is present and electron transfer to the A-side dominates. On the other hand, what is more readily apparent and easily measured from the transient absorption difference spectra is a reduction of HA bleaching magnitude in a controlled quantitative comparison to wild-type RCs. These results provide benchmark information for use in continuing studies of mutants that provide a means of exploring electron transfer to the B-side, and particularly for further studies aimed at investigating P+HB- f P+QB- electron transfer. Many

of these studies may utilize the detergent Deriphat 160-C and RCs expressed in the LHI- strain, since purification of RCs with this mild detergent provides the simplest manner in which to obtain RCs that have full occupancy of QB. To that end, we have shown that use of either LDAO or Deriphat to isolate YF RCs results in about the same yield of P+HB- formation. A point worth noting with regard to future studies is that analogous mutations made in RCs of Rb. sphaeroides have been shown to result in about half the yield of B-side electron transfer as found in Rb. capsulatus.35 We expect this trend to continue for the YF mutant of Rb. sphaeroides. Acknowledgment. This work was supported by Grant MCB0314588 from the National Science Foundation (D.H. and C.K.) and the U.S. Department of Energy, Office of Biological and Environmental Research, under Contract No. W-31-109-ENG38 (P.D.L. and D.K.H.). References and Notes (1) Deisenhofer, J.; Epp, O.; Miki, K.; Huber, R.; Michel, H. Nature 1985, 318, 618-624. (2) Allen, J. P.; Feher, G.; Yeates, T. O.; Komiya, H.; Rees, D. C. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5730-5734. (3) Chang, C.-H.; El-Kabbani, O.; Tiede, D. M.; Norris, J. R.; Schiffer, M. Proc. Natl. Acad. Sci. U.S.A. 1991, 30, 5352-5360. (4) Ermler, U.; Fritsch, G.; Buchanan, S. K.; Michel, H. Structure 1994, 2, 925-936. (5) Heller, B. A.; Holten, D.; Kirmaier, C. Science 1995, 269, 940945. (6) Kirmaier, C.; Laible, P. D.; Hanson, D. K.; Holten, D. Biochemistry 2003, 42, 2016-2024. (7) Kirmaier, C.; He, C.; Holten, D. Biochemistry 2001, 40, 1213212139. (8) Kirmaier, C.; Weems, D.; Holten, D. Biochemistry 1999, 38, 11516-11530. (9) Katilius, E.; Turanchik, T.; Lin, S.; Taguchi, A. K. W.; Woodbury, N. W. J. Phys. Chem. B 1999, 103, 7386-7389. (10) Katilius, E.; Babendure, J. L.; Katiliene, Z.; Lin, S.; Taguchi, A. K.; Woodbury, N. W. J. Phys. Chem. B 2003, 107, 12029-12034. (11) Haffa, A. L. M.; Lin, S.; Williams, J. C.; Bowen, B. P.; Taguchi, A. K. W.; Allen, J. P.; Woodbury, N. W. J. Phys. Chem. B 2004, 108, 4-7. (12) de Boer, A. L.; Neerken, S.; de Wijn, R.; Permentier, H. P.; Gast, P.; Vijgenboom, E.; Hoff, A. J. Biochemistry 2002, 41, 3081-3088. (13) de Boer, A. L.; Neerken, S.; de Wijn, R.; Permentier, H. P.; Gast, P.; Vijgenboom, E.; Hoff, A. J. Photosynth. Res. 2002, 71, 221-239. (14) Lin, S.; Jackson, J. A.; Taguchi, A. K. W.; Woodbury, N. W. J. Phys. Chem. B 1999, 103, 4757-4763. (15) Haffa, A. L. M.; Lin, S.; Williams, J. C.; Taguchi, A. K. W.; Allen, J. P.; Woodbury, N. W. J. Phys. Chem. B 2003, 107, 12503-12510. (16) Laible, P. D.; Kirmaier, C.; Holten, D.; Tiede, D. M.; Schiffer, M.; Hanson, D. K. In Photosynthesis: Mechanisms and Effects; Garab, G., Ed.; Kluwer Academic Publishers: Dordrecht, 1998; pp 849-852. (17) Tiede, D. M.; Budil, D. E.; Tang, J.; El-Kabbani, O.; Norris, J. R.; Chang, C.-H.; Schiffer, M. In The Photosynthetic Bacterial Reaction Center; Breton, J., Vermeglio, A., Eds.; Plenum: New York, 1988; pp 13-20. (18) Parson, W. W.; Chu, Z. T.; Warshel, A. Biochim. Biophys. Acta 1990, 1017, 251-272. (19) Alden, R. G.; Parson, W. W.; Chu, Z. T.; Warshel, A. J. Phys. Chem. 1996, 100, 16761-16770.

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