Pulsed EPR Study of Spin-Coupled Radical Pairs in Photosynthetic

J. Phys. Chem. B 1997, 101, 1429-1436

1429

Pulsed EPR Study of Spin-Coupled Radical Pairs in Photosynthetic Reaction Centers: ••+ Measurement of the Distance Between P•+ 700 and A1 in Photosystem I and between P865 and Q•A in Bacterial Reaction Centers Robert Bittl* and Stephan G. Zech Max-Volmer-Institut fu¨ r Biophysikalische Chemie und Biochemie, Technische UniVersita¨ t Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany ReceiVed: July 24, 1996; In Final Form: October 28, 1996X

•The spin-polarized radical pairs P•+ 865QA in protonated and deuterated Zn-substituted reaction centers from •in plant two different mutants of the photosynthetic bacteria Rhodobacter sphaeroides and P•+ 700A1 photosystem I from Synechococcus elongatus are investigated by pulsed EPR spectroscopy. Spin-polarized radical pairs give rise to a characteristic out-of-phase electron spin echo. This echo shows a deep envelope modulation with a frequency governed by the spin-spin interaction. The known distance dependence of the magnetic dipolar interaction allows the determination of the distance between the cofactors carrying the unpaired electron spins. For the bacterial reaction centers this distance is known for the electronic ground state from crystal structures and is compared here with the distance of the radical pair spins, i.e. the charge-separated state. In photosystem I the location of the acceptor A1 is not known yet. A distance of 25.4 ( 0.3 Å between •P•+ 700 and A1 is obtained here and gives new structural information on photosystem I.

Introduction In photosynthetic reaction centers (RCs) radical pairs (RPs) occur as transient species in the course of light-induced electron transfer (ET). The first RP state within a RC after light excitation accessible to time-resolved EPR techniques is the state ••+ •P•+ 865QA in bacterial RCs (bRCs) and P700A1 in photosystem (PS) I. These states arise after ET from the primary donor P via an intermediate acceptor (bacteriopheophytin in bRCs, chlorophyll in PS I) to the primary quinone acceptor QA or A1. The information gained from time-resolved EPR studies on this state has been reviewed recently.1,2 The light-induced ET starts from a singlet state of the primary donor P. This spin state is conserved by the fast ET, and the ••+ •RP state P•+ 865QA or P700A1 is created virtually in a pure singlet state. This leads to an occupation of the energy levels of the two-spin system in an external magnetic field far from Boltzmann equilibrium. As a consequence, absorptive and emissive EPR signals are observed using the direct detection technique or the field-swept electron spin echo (ESE) method using a narrow excitation bandwidth. The superposition of absorptive and emissive signals together with the anisotropy of the magnetic interactions (g-tensors and dipolar interaction tensor) leads to a high sensitivity of the transient EPR spectra to the relative orientation of the cofactors involved.3 The experimental spectra can be analyzed in detail using the correlated coupled radical pair (CCRP) model.4,5 This model is adequate to describe well the transient EPR spectra in different frequency bands and the time evolution of the transient magnetization including quantum beats and transient nutations.6-10 As shown in theoretical studies,11,12 the CCRP model implies unusual properties of the RP in pulsed EPR experiments, with broad-band excitation. In a conventional two-pulse electron spin echo experiment employing a (π/2)-τ-π microwave (mw) pulse sequence with the B1 field of the mw pulse along the x-direction, the echo is detected at time T ) τ after the second pulse along the y-axis (in-phase). For a correlated coupled RP X

Abstract published in AdVance ACS Abstracts, January 15, 1997.

S1089-5647(96)02256-0 CCC: $14.00

at time T ) τ no in-phase echo signal occurs, but an out-ofphase echo along the x-axis does occur.11 Phase-shifted echos have been observed already in the past after light excitation of photosynthetic algae.13 However, in this work the phase shift has been interpreted as a result of the sequence of radical pair states in RCs. Recently, out-of-phase echos have been observed in Zn-substituted bRCs (Zn-bRCs) of Rhodobacter (Rb.) sphaeroides R-26.14 These authors could show that the time dependence of the out-of-phase echo is in agreement with the behavior predicted by the CCRP model. In this model the electron spin echo envelope modulation (ESEEM) of the out-of-phase echo amplitude is governed by the coupling between the two electron spins within the RP.11 The two different spin-spin couplings, the dipolar and the exchange coupling, can be obtained separately from an analysis of the echo modulation. Due to the r-3 dependence of the dipolar interaction on the spin-spin distance r within the RP, the observed dipolar coupling yields the distance between the electron spins of the RP. For the delocalized spins in the RPs ••+ •P•+ 865QA and P700A1 this distance is to a good approximation the distance between the center of the π-systems in the primary donor and the center of the carbonyl oxygens of the acceptor quinone. For bRCs this distance is known for the neutral ground state from X-ray crystallography and can be compared with the results of the EPR measurements. A discrepancy of about 2 Å between EPR experiments and the X-ray data has been found,14 with a shorter distance (larger coupling) in the EPR. In addition to this discrepancy in distance a rather large exchange interaction of more than 10 µT has been deduced.14 A value of this magnitude is not consistent with the interpretation of the transient EPR spectra in various frequency bands15 and is much larger than expected14 applying the empirical relationship between distance and ET rate in proteins16 to the distance dependence of the exchange interaction. In this study, we present pulsed EPR experiments on the •radical pair states P•+ 865QA in protonated and deuterated Znsubstituted bacterial RCs of Rb. sphaeroides R-26. The outof-phase echo modulation will be compared with that of © 1997 American Chemical Society

1430 J. Phys. Chem. B, Vol. 101, No. 8, 1997 protonated samples of the mutant Rb. sphaeroides HC(M266), also containing Zn2+. The dipolar and isotropic couplings are extracted from the experimental echo modulation by numerical simulation of their Fourier transforms. The results are discussed with respect to the X-ray structure and previously published pulsed EPR results.14 Furthermore, experiments on the RP •P•+ 700A1 in PS I are presented. In PS I the location of the quinone acceptor A1 (vitamin K1) is not unambiguously known from the present 4.5 Å X-ray structure yet.17 Therefore, a •determination of the P•+ distance by an independent 700A1 method is important. The distance obtained here should help to localize A1 in the electron density map17 of PS I. Materials and Methods The PS I has been isolated from the thermophillic cyanobacterium Synechococcus (S.) elongatus as reported.18 The sample used in this study contained 60% (v/v) glycerol. In bRCs 2+ ion a decoupling of Q•A from the paramagnetic nonheme Fe •is necessary for EPR experiments on QA . The decoupling can be achieved, for example, by substitution of the Fe2+ ion with diamagnetic Zn2+. The preparation of the Zn-bRC of Rb. sphaeroides R-26 has been described previously.19,20 An alternative to chemical Zn-substitution is the use of RCs from the mutant Rb. sphaeroides HC(M266).21 In this mutant the histidine (H) at position 266 in the M subunit, ligating the Fe2+ ion, is replaced by cysteine (C). Rb. sphaeroides HC(M266) incorporates Zn2+ instead of the nonheme Fe2+ ion if grown on a Zn2+-enriched medium.22 A detailed description of the preparation of Rb. sphaeroides HC(M266) and its characterization by EPR spectroscopy will be given elsewhere.23 All samples were filled into quartz tubes and frozen in the dark to 77 K. Due to the limited amount of the sample, the deuterated Zn-bRCs were filled in quartz tubes with 3 mm o.d. and 2 mm i.d.; for all other samples, tubes with 4 mm o.d. and 3 mm i.d. were used. All experiments described here have been performed at 150 K. For light excitation we used a Q-switched and frequency-doubled Nd:YAG laser (Spectra Physics GCR 130, λ ) 532 nm, ≈8 ns pulse width). The light energy at the sample was lower than 5 mJ per pulse. The EPR experiments have been performed on a BRUKER ESP 380 E X-band FT-EPR spectrometer with a ER 4118 X-MD-5W1 dielectric ring resonator and a helium flow cryostat (Oxford CF 935). For the two-pulse echo experiments the pulse lengths were set to 8 ns for the first and 16 ns for the second pulse. In our samples we found no ESE signal of stable radicals at 150 K. Therefore, it was not possible to calibrate the quadrature detection phase to a known in-phase echo signal as described previously.14 Instead we calibrated the phases with respect to the “cavity ringing” by cycling the phase of the excitation pulses. The maximum out-of-phase signal of the light-induced RP occurred at a TWT output damped by 1-2 dB from nominal 1 kW. Typically 512 points with an increment of 8 ns in τ were measured in quadrature detection mode. Depending on the concentration of the sample, up to 64 traces were accumulated to improve the signal/noise (S/N) ratio. The data analysis was performed on a IBM RS/6000 workstation. For the Fourier transforms (FTs) we used the Mathematica 2.2 standard packages. In contrast with previous experiments14 no zero-filling and no filtering of the data were performed in our studies. The numerical simulations of the FT signal were calculated using a program based on the simulation program for transient EPR spectra.6

Bittl and Zech Theory In the CCRP model for RPs the Zeeman interaction between the two electron spins S1, S2 and the external magnetic field B0, the hyperfine coupling Aij between the electron spin Si and the nucleus j with spin Ij, the exchange coupling J, and the anisotropic dipolar coupling D are considered. For a selected orientation of the RP with respect to the axis of B0, the Hamiltonian can be given by

AijSizIjz + ∑ i,j

H ) g1βB0S1z + g2βB0S2z + 3

/2d[(S1z + S2z)2 - 1/3(S1 + S2)2] + J(1/2 - 2S1S2) (1)

where g1 and g2 are the effective g-values of the two electron spins for this orientation and d ) 1/3D(3 cos2 θ - 1), with the angle θ between the dipolar axis Zdsconnecting the RP spinssand the axis of B0. In the basis of singlet (|S〉) and triplet (|T+〉, |T0〉, |T-〉) functions, the eigenvalues E1 to E4 and corresponding eigenfunctions |1〉 to |4〉 can be derived:

|1〉 ) |T+〉

E1 ) ω0 + d/2 E2 ) J - d/2 + Ω E3 ) J - d/2 - Ω

|2〉 ) cos R |S〉 + sin R |T0〉

(2)

|3〉 ) -sin R |S〉 + cos R |T0〉 |4〉 ) |T-〉

E4 ) -ω0 + d/2 where

∑j (A1j + A2j)mj

ω0 ) 1/2(g1 + g2)βB0 + 1/2

∑j (A1j - A2j)mj

∆ω ) 1/2(g1 - g2)βB0 + 1/2

(3)

Ω2 ) ∆ω2 + (J + d/2)2 sin (2R) ) ∆ω/Ω In eq 3, β is the Bohr magneton, mj is the projection of the spin state of nucleus j on the B0 axis, and R is the |S〉-|T0〉 mixing angle. The difference E2 - E3 ) 2Ω represents the frequency of the coherent singlet triplet oscillations observed as quantum beats in transient EPR studies.9,24 Using the density matrix formalism, an analytical expression for the echo intensity S(t,τ,T) detected at time T after a (π/2)xτ-πx mw pulse sequence following at time t after a laser pulse hν has been derived.11 As shown, an echo modulation arises as a function of τ, depending on the dipolar and the exchange coupling d and J, the zero quantum coherence frequency Ω, and the delay time t between laser and first mw pulse. In the narrow band excitation transient nutation experiments, the effects of the zero quantum coherence are visible only up to t ≈ 100 ns after the laser pulse in deuterated samples.9,24 In protonated Zn-substituted samples of Rb. sphaeroides R-26 a dependence of the shape of the out-of-phase echo on tsattributed to quantum beatsshas been found only for t e 40 ns.25 To simplify the analysis of our experiments, we used a large delay time t ≈ 800 ns between the laser pulse and the first mw pulse.14 Due to the large hyperfine interaction, the condition Ωt . 1 is then satisfied even in deuterated samples. In this case all terms including Ωt can be substituted by their average. Assuming a detection of the in-phase and out-of-phase signals Sy(τ) and Sx(τ), respectively, at T ) τ, the theoretical result11 reduces to

Pulsed EPR Study of Spin-Coupled Radical Pairs

Sx(τ) )

∆ω2(2J + d)2 4Ω4

J. Phys. Chem. B, Vol. 101, No. 8, 1997 1431

sin (Γτ)[1 - cos(2Ωτ)]

Sy(τ) ) 0

(4)

where Γ) E2 + E3 - E1 - E4 ) 2(J - d). Equation 4 implies that only an out-of-phase echo Sx is present, while the in-phase echo Sy vanishes at T ) τ. The out-of-phase echo modulation is governed by the term Γ, which depends only on the dipolar coupling d and the exchange coupling J, but is independent of any isotropic hyperfine interaction or g-factor differences. The evaluation of the spin-coupling parameters J and D is simplified by a sine Fourier transform (SFT) of Sx(τ). The SFT of eq 4 is given by

S˜ x(ν) )

∆ω2(2J + d)2 {δ(ν - Γ) - δ(ν + Γ) 4Ω4 1 /2[δ(ν - Γ - 2Ω) + δ(ν - Γ + 2Ω)] + 1

/2[δ(ν + Γ + 2Ω) + δ(ν + Γ - 2Ω)]} (5)

For each orientation of the RP with respect to the magnetic field six lines arise. The two lines at ν ) (Γ are dependent only on J and d, while the four lines at ν ) ((Γ ( 2Ω) also depend on ∆ω and, therefore, on the relative orientation of the two cofactors. Due to the unresolved hyperfine interactions of •P•+ and Q•A (or A1 ) these four lines aresfor protonated samplessabout 30 MHz wide. For a discussion of the main features of S˜ x(ν) we will focus on the two sharp lines at ν ) (Γ. Omitting all orientation dependent factors, e.g. all terms including Ω, eq 5 reduces to

S˜ x(ν) )

S˜ x(ν) ) δ(ν - Γ) - δ(ν + Γ) ≡ (δ(ν - Γ)

(6) 1

For a powder sample instead of these two sharp lines two distributions of frequencies arise, shown in Figure 1A. The intensities for the frequencies ν(θ) between ν| ) (2(J - 2/3D) for θ ) 0° and ν⊥ ) (2(J + 1/3D) for θ ) 90° are determined by the intensity pattern for d(θ). Figure 1B depicts the sum of the two intensity patterns. The prefactor ∆ω2(2J + d)2/(4Ω4) in eq 5, which is independent of Γ, contributes only a minor distortion of the intensity distribution. This is shown in Figure 1C, where all orientation dependent parameters (according to eq 5) were taken into account. For calculation of the SFT signals shown in Figure 1A-D, the g-tensor principal values have been taken from high-field cw-EPR results26,27 and the •28 and timeP•+ 865QA geometry as given by the X-ray structure 29 resolved high-field EPR. The most important parameter taken from the X-ray structure28 is the dipolar coupling D ) -0.12 mT. The exchange interaction has been set to J ) 0, consistent with simulations of transient EPR spectra.29 From the characteristic frequencies ν| and ν⊥ of the SFT pattern shown in Figure 1B,C, the spin-spin coupling parameters D and J can easily be calculated using the relations

D ) 1/2(ν⊥ - ν|) and J ) 1/6(2ν⊥ + ν|)

Figure 1. Simulation of sine Fourier transforms (SFTs) of the outof-phase ESEEM. (A) Tensorial distributions of the lines for +Γ (solid line) and -Γ (dashed line). A vanishing linewidth has been assumed (see eq 6). The tensor components ν| and ν⊥ are indicated by arrows. (B) Sum of the two single transitions shown in A. (C) SFT simulation according to eq 5, taking all orientation dependent parameters into account. (D) SFT simulation with a Lorentzian linewidth of ∆νL ) 0.5 MHz. The spin coupling parameters were set to D ) -0.12 mT and J ) 0.

(7)

For a comparison of the calculated SFT with that of the SFT from an experimental ESEEM trace it is necessary to account for the decay of the echo intensity with increasing τ due to the limited phase memory time. This is done here by substituting the δ-functions of eq 5 by Lorentz functions with half-linewidth ∆νL:

/2

[

{

∆ω2(2J + d)2

(∆νL2

4Ω4

∆νL2 + (ν - Γ)2

∆νL2

-

]}

∆νL2 + ∆νL2 + (ν - Γ - 2Ω)2 ∆νL2 + (ν - Γ ( 2Ω)2

(8)

Equation 8 has to be read as the sum taking once all terms with the upper signs and once the lower signs (see eqs 5 and 6). For Lorentzian lines an evaluation of J and D is no longer possible by simple inspection of the SFT. This can be seen in Figure 1D, which shows the simulation according to eq 8 with a linewidth of 0.5 MHz, as used in the simulation for the SFT for Zn-bRCs (see below). All other parameters are the same as in Figure 1C. As can be seen in Figure 1D, the maxima around ν⊥ are shifted to smaller values due to the linewidth. If this shift is not taken into account, the values for D and J obtained by analysis of the maxima and edges of Figure 1D are not precise. In our numerical simulations we included all six lines of S˜ x(ν) as in eq 8; however, the final results given below remain unchanged when omitting the four broad lines (see above), which depend on the orientational parameters of the RP. Results and Discussion In this study, pulsed EPR experiments on light-induced RPs in three different preparations of Zn-bRCs and in a PS I sample are presented. The theoretical study,11 briefly summarized above, predicts that the out-of-phase echo modulation is approximately independent from the hyperfine interaction in the RP. This has been tested14 by investigating Zn-bRCs containing 14N and 15N, respectively. Virtually no change of the ESEEM pattern was found for the two isotopes. Here, we investigate the effect of a 1H to 2H substitution in protonated and fully

1432 J. Phys. Chem. B, Vol. 101, No. 8, 1997 deuterated Zn-bRCs of Rb. sphaeroides R-26. Thereby, together with the previous results,14 all hyperfine couplings entering eqs 4 and 5 by the difference of resonance frequencies ∆ω or the zero quantum frequency Ω are modified. This provides a conclusive test whether the out-of-phase ESEEM can be used to determine the spin-spin couplings D and J. In all investigated samples neither an in-phase nor an outof-phase echo was found at a temperature of 150 K without exciting laser flashes or with the laser flash after the first mw pulse. A strong dependence of the echo shape in both quadrature detection channels on the delay time t between the laser flash and the first mw pulse for small t (t j 50 ns) was found, as predicted by the theoretical results11 and observed experimentally.25 This variation of the echo shape can be attributed11,25 to the coherent singlet-triplet oscillations of the RP and will not be discussed here. Instead we have chosen a large delay time t ≈ 800 ns where the echo shape is independent of variations in t. This simplifies the analysis of the observed ESEEM (see above). The echo shape is further influenced by the magnetic field position B0. In all our experiments B0 was set to the center of the transient EPR spectrum measured prior to the pulsed EPR experiments. The difference in the resonance frequency of the resonator in direct detection mode (critical coupling) and pulsed mode (strong overcoupling) has been taken into account. Qualitative Analysis of the Echo Modulation. The dependence of the echo amplitude as a function of the delay time τ between the two mw pulses is shown in Figure 2 for Zn-bRCs. The first points of the experimental ESEEM traces are masked by the dead time of the spectrometer (88 ns for protonated samples, 112 ns for the deuterated sample). As predicted in the theoretical studies11 and also observed experimentally,14 the out-of-phase echo (thick lines) shows a deep modulation as a function of τ, while the in-phase echo (thin lines) is about 1 order of magnitude smaller, but does not vanish completely. The most probable cause of the small in-phase echo is a slight misadjustment of the quadrature detection phases. A 5° misadjustment of the in-phase detection channel results in about 10% contamination through the out-of-phase signal. As can be seen in Figure 2, the echo modulation for the two protonated Zn-bRCs is virtually identical. Only a small difference in the decay of the echo amplitudes is found, while the modulation frequencies are the same. For the deuterated sample the relative echo amplitudes deviate from those of the protonated RCs, but the same main modulation frequencies as in the protonated samples are present. Within the poor S/N ratio of the deuterated sample the extrema in the ESEEM pattern occur at the same delay times τ. This shows that the prominent modulation of the out-of-phase echo is not affected by the hyperfine couplings of the nuclei of the RP, but can be related to the electron spin-spin interaction within the RP. The observation of identical modulation frequencies in all examined Zn-bRCs leads to the qualitative conclusion that the spin-spin coupling parameters within the RPs in these RCs are the same. Since an identical dipolar coupling leads to the same distance between the two spins, the distances between the oxidized •primary donor P•+ 865 and the reduced quinone acceptor QA in both protonated and the deuterated Zn-bRCs are equal. This is not surprising for the comparison of the protonated and deuterated RCs of Rb. sphaeroides R-26, where no structural differences are expected. However, this is important for the characterization of the mutant HC(M266) RCs. The amino acid residue M266 is in the vicinity of the QA binding pocket. The observation of an identical distance in Rb. sphaeroides R-26 and HC(M266) is consistent with the transient EPR spectra of

Bittl and Zech

Figure 2. Experimental out-of-phase (thick lines) and in-phase (thin •lines) echo modulation of P•+ 865QA in (A) protonated Zn-bRCs of Rb. sphaeroides R-26, (B) deuterated Zn-bRCs of Rb. sphaeroides R-26, and (C) protonated Zn-bRCs of Rb. sphaeroides HC(M266). Experimental conditions: T ) 150 K for all traces; (A) νmw ) 9.690 GHz, B0 ) 344.8 mT; (B) νmw ) 9.738 GHz, B0 ) 346.7 mT; (C) νmw ) 9.680 GHz, B0 ) 344.6 mT.

Figure 3. Comparison of the out-of-phase (thick lines) and in-phase •(thin lines) echo modulation of (A) P•+ 700A1 in PS I from S. elongatus •and (B) P•+ Q in protonated Zn-bRCs of Rb. sphaeroides R-26. 865 A Experimental conditions: T ) 150 K; (A) νmw ) 9.751 GHz, B0 ) 347.1 mT; (B) identical to Figure 2A. •30 and Q-band,23 where no P•+ 865QA measured in the X-band significant differences between the two bRCs could be found. A quantitative discussion of the cofactor distances will be given below. •Figure 3 shows the echo modulation of P•+ 700A1 in PS I from •+ •S. elongatus together with that of P865QA in Zn-bRCs of Rb. sphaeroides R-26. For PS I also a strong out-of-phase echo modulation as a function of τ is found. In Figure 3 clear deviations in the damping of the out-of-phase echo between •the two samples are visible. The echo for the RP P•+ 700A1 in •+ •PS I decays slower than for P865QA in Zn-bRCs at the same temperature. Such a difference in the out-of-phase ESEEM decay between PS I and Zn-bRCs has already been observed in a preliminary report.31 Here the decay of the modulation

Pulsed EPR Study of Spin-Coupled Radical Pairs

J. Phys. Chem. B, Vol. 101, No. 8, 1997 1433

Figure 4. Comparison of the out-of-phase echo modulation (thick lines) •for P•+ 865QA in Zn-bRCs of Rb. sphaeroides R-26 at different mw powers. The difference between the out-of-phase echo modulation at the indicated mw power compared with 1 dB (see Figure 2A) is shown as thin lines. Other experimental conditions are identical to Figure 2A.

for PS I is even slower than reported earlier.31 A possible factor causing this slowing of the decay for PS I is the use of the cryoprotectant glycerol for the sample in this study. Mobility has been envoked as the origin of the observed increase of the decay time with higher temperatures.14 Therefore, we attribute the different decay of the ESSEM of PS I to different mobilities of the protein complex in the water/glycerol glass compared with ice. More important, however, is a second clear difference between the ESEEM traces of the bRCs and PS I in Figure 3. The modulation frequency for the PS I sample is significantly higher than for the Zn-bRC. Besides the slower damping the modulation pattern observed here for PS I samples with a cryoprotectant is identical to the trace observed earlier31 for samples without glycerol. From the higher modulation frequency in the PS I sample, we conclude qualitatively that the ••+ •spin-spin coupling in P•+ 700A1 is stronger than in P865QA , and therefore the distance between the cofactors in PS I is shorter than the distance between the corresponding cofactors in bRCs. Influence of Different Pulse Angles. For a quantitative discussion, analysis of the Fourier transforms of the experimental time traces is necessary. For this purpose we will compare the Fourier transforms of the experiments with numerical simulations based on eq 8. Before this, we have to test the applicability of the theoretical study11 to our experiments. In that study, a (π/2)-π pulse sequence has been assumed. However, we have no experimental means to accurately measure the flip angles of our mw pulses. Therefore we have investigated the dependence of the ESEEM pattern on the flip angle in a (ζ/2)-ζ pulse sequence (second pulse flip angle ζ). To do so, we kept the 8 ns-16 ns pulse pattern and all other experimental conditions constant besides the mw power, as in Figure 2, where the maximum out-of-phase echo intensity occurred for a mw attenuation of 1 dB. Note that this mw power is similar to the power required for a 8 ns-16 ns ((π/2)-π) pulse sequence for a stable radical in our experimental setup. Figure 4A (thick line) shows the ESEEM after omitting the attenuation (0 dB). This increases the mw power by a factor 1.26 and, therefore, results in a increase of the flip angle ζ by a factor of 1.12. Damping the mw power by 4 dB, as shown in Figure 4B (thick line), results in a decrease of the flip angle by 29% compared with 1 dB (Figure 2A). The thin lines shown in Figure 4A,B indicate the residuals of the

Figure 5. Result of the dead time recovery algorithm (dashed lines) for the experimental out-of-phase echo modulations (solid lines) of •P•+ 865QA in protonated Zn-bRCs of (A) Rb. sphaeroides R-26, (B) Rb. •sphaeroides HC(M266), and (C) for P•+ 700A1 in PS I from S. elongatus.

ESEEM trace at the given mw powers with respect to the maximal echo intensity at 1 dB (Figure 2A), after normalization to equal intensity. The change of the mw attenuation from 4 to 0 dB corresponds to a 60% increase of the flip angle ζ. This drastic change in the flip angle does not result in a noticeable change of the ESEEM pattern, as documented by the very small residuals in Figure 4 (thin lines). This independence of the observed ESEEM pattern from the actual flip angle ζ allows us to use the theoretical model11 to analyze our data. Reconstruction of the Spectrometer Dead Time. The anaylysis of the experimental data is complicated by the minimal spectrometer dead time, obscuring the first 88 ns of the experimental ESEEM measurements. Due to the fast damping of the out-of-phase echo, this is an important part of the ESEEM trace. The loss of information in the first 88 ns results in distortions of the SFTs of the ESEEM signal. To recover the missing points, a linear function connecting the point at Sx(τ)0) ) 0 and the first experimental point has been used.14 The time interval of 88 ns, however, is too long for a linear approximation of the signal. The frequency domain of the signal ranges from 0 to approximately 4 MHz for bRCs (see below), i.e. ωmax ≈ 2.5 × 107 s-1. For this frequency a linear approximation is valid only for about 20 ns. With this in mind, we tried to recover the masked part of the ESEEM trace by application of different mathematical procedures. The best results were reached by the “maximum entropy method” (MEM).32 Figure 5 shows the result of the dead time recovery program using the MEM algorithm. As can be seen in Figure 5, the prediction shows a nonlinear behavior of the signal within the spectrometer dead time. Consistent with the theoretical study,11 the MEM algorithm results in an almost vanishing echo intensity for τ ) 0. Numerical Simulations. To evaluate the spin-spin coupling parameters D and J from the ESEEM traces, we have calculated the SFTs of the out-of-phase echo modulation. The small inphase part (shown in Figures 2 and 3 as thin lines) has thereby been neglected. The results of SFT can be seen in Figure 6.

1434 J. Phys. Chem. B, Vol. 101, No. 8, 1997

Bittl and Zech linewidth of ∆νL ) 0.50 ( 0.05 MHz has been found to give the best agreement in the damping between experiment and simulation. Using the parameters for D and J determined earlier14 left the decay of the calculated ESEEM practically unchanged. Therefore, we kept the linewidth parameter ∆νL fixed while searching for the optimum J and D values. The best agreement between the SFT of the experimental ESEEM trace and simulation was obtained for the protonated Rb. sphaeroides R-26 sample using

D ) -121 ( 4 µT and J ) 1.0 ( 0.5 µT

Figure 6. Sine Fourier transforms of the out-of-phase echo modulations (after dead time recovery; see Figure 5). Experimental (solid lines) and numerical simulations (dashed lines) for (A) protonated samples of Rb. sphaeroides R-26, (B) Rb. sphaeroides HC(M266), and (C) PS I from S. elongatus. Simulation parameters: (A and B) D ) -121 µT, J ) 1.0 µT, ∆νL ) 0.50 MHz; (C) D ) -170 µT, J ) 1.0 µT, ∆νL) 0.25 MHz.

The maximum intensity in the SFT signal reflects the main frequency of the ESEEM. Using different dead time reconstruction methods, these characteristic peaks have been found to be nearly unaffected. This is important becausesunder the assumption of a very small isotropic coupling, J ≈ 0sit is possible to extract the magnitude of the dipolar coupling D solely from this intense peak in the SFT. Figure 6 further shows that the SFT obtained from the ESEEM in Rb. sphaeroides R-26 and Rb. sphaeroides HC(M266) are almost indistinguishable, indicating that the distance •between P•+ 865 and QA in both species is identical. In contrast, clear deviations between the SFT of the PS I sample and the two Zn-bRCs are seen. In particular, the maximum intensity of the SFT (≈(2 MHz in Zn-bRCs) is shifted to higher values for PS I (≈(3 MHz). For PS I the ν| edge of the spectrum in Figure 6 is better resolved than in our preliminary report.31 The SFT of PS I compared with that of bRCs shows considerably less intensity for |ν| > |ν⊥| and the linewidth is smaller due to the slower decay of the ESEEM. In Figure 6, the dashed lines show numerical simulations of the SFT, which were calculated as outlined in the Theory section. As a starting point we used the magnetic and orientational parameters given for Figure 1C (see Theory section). As pointed out above, the simulation of the SFT is only weakly sensitive to the relative orientation of the RP but very sensitive to changes in the spin-spin coupling parameters D and J. This is in contrast to the simulation of transient EPR spectra, where the relative orientation of the cofactors is very important, while the dipolar coupling only influences the absolute amplitude of the transient spectrum.6 Reverse Fourier transform of the SFT of Figure 1C shows that even this wide frequency distribution results in a time signal with very weak damping. Therefore, we have taken the decay of the ESEEM into account by substituting the δ-functions by Lorentzian lines (see eq 8). For the protonated Zn-bRCs a

The error margin for D reflects the frequency resolution in the SFT. Since J is 2 orders of magnitude smaller than D, the relative error is large. The value of J derived here is below the frequency resolution of the SFT and has a vanishing influence on the frequencies of the characteristic features (ν⊥ and ν|). However, J influences the overall shape of the SFT. Due to the large similarities between the ESEEM of the three Zn-bRC samples (see Figure 2), the same values for D and J were obtained for the deuterated sample of Rb. sphaeroides R-26 and for Rb. sphaeroides HC(M266). From the point dipole model, the dipolar coupling as a function of the distance r between the two electron spins within the RP is given by D ) -2786r-3 mT‚Å3. The dipolar coupling derived from the echo modulation corresponds to a distance of 28.4 ( 0.3 Å between the centers of the spin densities on the cofactor radicals. The error margin given corresponds to the error in D and will be discussed below. The distance reported here is in very good agreement with the distance derived from X-ray structure by Ermler et al.,28 where the distance between the center of the Mg atoms of P865 and the center of the two carbonyl oxygen atoms of QA is 28.3 Å and yields D ) -123 µT. The dipolar coupling derived in our analysis is lower than the one obtained in an earlier study,14 while the experimental data seem to agree well. A possible explanation of this discrepancy might be the fast damping of the experimental ESEEM trace, which causes a broadening of the SFT, described by a Lorentzian linewidth in our simulation (see above). This line broadening results in a shift of the apparent tensor component ν⊥ for θ ) 90°. Therefore, it is not possible to assign the frequency with highest intensity in the SFT to ν⊥ (see Figure 1C,D). The broadening of the SFT results furthermore in a smoothing of the edge corresponding to ν| (see Figure 1C,D). This, in turn, renders it difficult to precisely locate ν|. Therefore it is not possible to deduce D and J values from the spectrum by inspection of the frequencies ν⊥ and ν|, but a simulation of the full SFT as peformed here is required. The temperature dependence of the damping of ESEEM and the resulting shift of the maximum in SFT might be a reason for the strong temperature dependence of D, discussed earlier.14 33 and the A•- g-tensor together For PS I, the P•+ 700 g-tensor 1 with the orientational parameters obtained from a recent W-band •study34 of the RP state P•+ 700A1 were used for the simulation. However, it is important to note that the relative orientations of the two cofactors as well as their precise g-tensors have no significant influence on the spin coupling derived from the SFT of the ESEEM (see Theory section). The distances reported here are, therefore, independent of the data provided by transient EPR. We first determined the value of ∆νL by adjusting the decay of calculated ESEEM traces to the experimental data. A value of ∆νL ) 0.25 ( 0.05 MHz has been found to give the best agreement. A linewidth consistent with this value is found in recent experiments on single crystals of PS I (unpublished results). Thereafter, D and J have been adjusted starting from D ) -4.5 MHz (D ) -0.16 mT) guided by the peak position

Pulsed EPR Study of Spin-Coupled Radical Pairs at (3 MHz in the SFT (Figure 6C) and J ) 1 µT as for ZnbRCs. The best fit for PS I was obtained using

D ) -170 ( 4 µT and J ) 1.0 ( 0.5 µT The error margins are determined as for bRCs. The agreement between the SFT of the experiment and the simulation is not as good as for the bRCs. This discrepancy is here slightly larger than in an earlier report,31 where a faster decay of the ESEEM was observed with a sample not containing glycerol. However, this discrepancy affects only the intensity ratio between the frequency νmax with maximum intensity and the frequencies above ν⊥ and can be related to imperfect reconstruction of the dead time. The characteristic frequencies νmax and ν| are reproduced well. Using the relation given above, D leads to the distance of 25.4 ( 0.3 Å between the centers of spin density on P•+ 700 and A•in PS I. As for the case of the bRC, the error margin 1 given corresponds to the error in D. Comparing the distances given for bRC and PS I implies an about 3 Å shorter distance between the centers of the unpaired ••+ •spin densities of P•+ 700A1 compared with P865QA . To judge whether this distance information can be translated to a difference in the cofactor location within the two RCs, the influence of possible changes in the spin density distribution over the redox active cofactors has to be discussed. The distance •of 28.3 Å for P•+ 865QA has been calculated from the X-ray structure assuming the center of the spin density on P•+ 865 located in the middle of the two magnesium atoms and the center of the spin density on Q•A in the middle of the carbonyl oxygen atoms. Locating the spin density on P•+ 865 fully on the L and the M side of the special pair, i.e. on the corresponding •Mg atoms, results in P•+ 865QA distances of 28.5 and 28.7 Å, respectively. Assuming that A1 is located in the electron transfer chain from P700 to FX, the line connecting P700 and A1 is expected to be roughly perpendicular to the membrane plane, as the line connecting P865 and QA. Therefore, an asymmetry of the spin density in P•+ 700 should cause no significant change •in the distance for P•+ 700A1 . As an example for an environmental effect of the protein on the spin density distribution on the acceptor radical, the two cases of QA and QB in bRC can be used. While for Q•B a symmetric binding with equal spin densities on the two carbonyl oxygens is found, Q•A shows an asymmetric binding situation with a ratio of 3:4 of the spin densities on the carbonyl oxygens.22 Using these oxygen spin densities the spin density center of Q•B is located exactly in the midpoint between the two carbonyl oxygens, while for Q•A the spin density center is shifted about 0.1 Å away from the midpoint. Therefore, the reported difference in the distances ••+ •cannot be caused by of P•+ 865QA compared with P700A1 different spin density distributions but reflects a difference in the geometric arrangement of the redox groups in the different proteins. •This about 3 Å shorter distance of P•+ 700A1 as compared with •+ •P865QA in bRCs is qualitatively consistent with the faster electron transfer to A1 than to QA. For the A1 reduction time constants between 21 and 50 ps have been measured,35-37 while the time constant for QA reduction is 250 ps.38 Using the empirical ET rate constant vs distance relation,16 a decrease in the distance by 3 Å would imply an increase in the ET rate by about 2 orders of magnitude. This is, however, not a discrepancy with our results since the rate for the A1 reduction is governed by the distance between A0 and A1 and not the distance •between P•+ 700 and A1 , as determined here. An electron

J. Phys. Chem. B, Vol. 101, No. 8, 1997 1435 •transfer rate constant that is directly related with the P•+ 865QA •+ •and the P700A1 distance is the recombination rate. Lowtemperature measurements of this rate show an about 2 orders ••+ •of magnitude faster decay of P•+ 700A1 than of P865QA with time 39 40,41 respectively, consistent constants of 200 µs and 25 ms, with the distance difference reported here.

Conclusion In our study we have investigated the dipolar and isotropic coupling of spin-coupled RPs in bRCs and in PS I. We have shown that it is possible to obtain the coupling parameters from a pulsed EPR experiment. In contrast to an earlier analysis for similar experiments on Zn-bRCs,14 we found a dipolar coupling that is in good agreement with the one derived from the X-ray structure.28 Additionally, the isotropic coupling obtained in our study is consistent with the transient EPR studies, where only small values of J (≈1 µT) are allowed to fit the experimental spectra.15 The comparison between the out-of-phase echo modulation of PS I and Zn-bRCs shows that in PS I a stronger dipolar coupling between the two spins is present. The numerical simulations of the Fourier transforms based on the CCRP model allowed the conclusion that the distance between the primary donor and the quinone acceptor is about 3 Å shorter in PS I than in bRCs. This information should help to localize the quinone acceptor A1 in the X-ray electron density map of PS I.17 •The knowledge of the P•+ 700A1 distance is further important for a comparison of quinone-substituted PS I preparations with the native system. In a transient EPR study,42 a drastically altered quinone orientation has been found if the native vitamin K1 is substituted by naphthoquinone. By measuring the •P•+ distance in such samples, it is possible to verify 700A1 whether the substituted quinones are bound at the native A1 site. Measurement of the out-of-phase ESEEM in single crystals of PS I will not only provide the distance between P•+ 700 and •A•but also allow the location of A in the protein. Experi1 1 ments along these lines are currently being performed in our laboratory. Acknowledgment. We thank E. C. Abresch (UC San Diego) for preparing the Zn-bRCs of Rb. sphaeroides R-26, J. P. Allen and J. C. Williams (ASU Tempe) for providing the mutant culture of Rb. sphaeroides HC(M266), A. T. Gardiner (TU Berlin) for the HC(M266) RC preparation, and E. Schlodder (TU Berlin) for the PS I sample. We are grateful to W. Lubitz (TU Berlin) for stimulating discussions and providing the experimental facilities. This work has been supported by DFG (SFB 312, TP A4) and NaFo¨G Berlin (graduate fellowship to S.G.Z.). Note Added in Proof: During publication of this paper we became aware of a recent similar study by Dzuba et al. on PS I from spinach. Dzuba et al. obtained values for D and J that are in excellent agreement with our results: Dzuba, S. A.; Hara, H.; Kawamori, A.; Iwaki, M.; Itoh, S.; Tsvetkov, Yu. D. Chem. Phys. Lett., in press. References and Notes (1) Snyder, S. W.; Thurnauer, M. C. In The Photosynthetic Reaction Center; Deisenhofer, J., Norris, J. R., Eds.; Academic Press, Inc.: San Diego, 1993; Vol. II, p 285. (2) Angerhofer, A.; Bittl, R. Photochem. Photobiol. 1996, 63, 11. (3) van der Est, A.; Stehlik, D. In The Reaction Center of Photosynthetic BacteriasStructure and Dynamics; Michel-Beyerle, M.-E., Ed.; Springer Verlag: Berlin, 1996; p 321.

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