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ESEEM study of spin-spin interactions in spin-polarised P+QA− pairs in the photosynthetic purple bacterium Rhodobacter sphaeroides R26
28 April
1995
CHEWCAL PHYSICS LETTERS ELSEVIER
Chemical
Physics Letters
236 (1995)
595-602
ESEEM study of spin-spin interactions in spin-polarised P’Q, pairs in the photosynthetic purple bacterium Rhodobacter sphaeroides R26 Sergei A. Dzuba ‘, Peter Gast, Arnold J. Hoff * Department
of Biophysics,
Huygens
Laboratory,
Received
5 January
Leiden
University,
P.O. Box 9504,230O
1995; in final form 27 February
RA Leiden,
The Netherlands
1995
Abstract Electron spin echo envelope modulation (ESEEM) of the transient spin-polarised PfQA pairs has been studied at 20 and 220 K. The observed strong out-of-phase modulation is interpreted as resulting from electron-electron dipolar and exchange interactions. Fourier-transformed echo envelopes are consistent with the theory developed recently by Tang, Thumauer and Norris and allow us to obtain readily the values of dipolar and exchange couplings. The results are insensitive to 15N enrichment.
1. Introduction
Charge separation in photosynthetic reaction centers CRC) leads to formation of radical pairs. The primary light-induced reactions in RCs of photosynthetic bacteria are: (i) The electronically excited state of the primary donor P, a bacteriochlorophyll dimer, produces the primary radical pair P’I-, where I denotes a bacteriopheophytin molecule. This reaction occurs within = 3 ps. (ii> Dark electron transfer within 200 ps from I- to QA, the primary acceptor quinone, creates the secondary radical pair P’Q,. Finally, (iii) within 200 bs the electron reaches Qn, the secondary acceptor quinone.
* Corresponding author. ’ On leave from Institute of Chemical Kinetics and Combustion, Russian Academy of Sciences, 630090 Novosibirsk, Russian Federation. 0009-2614/95/$09.50
8 1995 Elsevier
SSDIOOO9-2614(95)00259-6
Science
B.V. All rights reserved
The study of spin-spin interactions between radicals in the pairs, which include magnetic dipole-dipole and spin-exchange interactions, could reveal important structural information. The dipole-dipole interaction reflects mutual radical positions in the pair, while the exchange interaction is related to the overlap of electronic wavefunctions, which correlates with the rate of electron transfer. As the radical pairs mentioned above are formed shortly after the light flash, they are spin polarised. The method of electron spin polarisation (ESP) spectroscopy is effective in the determination of dipolar and exchange couplings [l-6]. For RCs in which Q, is not reduced, the results of these measurements refer to the P+Q- pairs, because the PfIpairs decay very fast. It has been known for a long time that spin-spin interactions in radical pairs result in so-called electron spin echo envelope modulation (ESEEM) [7]. The theory developed in Ref. [7] was applied to
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et al. /Chemical
Physics Letters
experiments on stable radical pairs and biradicals in solids [7] and on biradicals in solutions [8]. The modulation phenomenon manifests itself as an oscillation of the in-phase echo signal (i.e. along the Y axis of the rotating frame if the microwave field is along the X axis). For species in thermal equilibrium the out-of-phase signal does not appear. Recently, Tang et al. [9] presented a theory of ESEEM for spin-polarised radical pairs. Their results predict that in this case an out-of-phase modulation exists while the in-phase signal is completely absent. An important condition for the observation of modulations induced by spin-spin interactions is full excitation of both radicals in the pair by the microwave pulse. The EPR spectrum of the P’Q, pair has a characteristic width of = 1 mT [5]. Therefore the amplitude B, of the microwave pulses should be of the same order or higher. Note that only the amplitude of the second pulse is important for the full excitation condition used in the above theories. The role of the first pulse is to create the initial transverse magnetization, and it may be of arbitrary amplitude. Another interaction that may induce the modulation phenomenon is the electron-nuclear hyperfine interaction (hfi) [lo]. For P+ and Q, radicals the main source of nuclear modulations is coupling with nitrogen nuclei [lo]. In the present work we study ESEEM induced by spin-spin interactions between P+ and Qi in the photosynthetic RC of the purple bacterium Rhodobacter sphaeroides R26, in which the native non-heme iron was replaced by the diamagnetic Zn2+ ion. Removing the magnetic coupling between the Fe2+ ion and Q, in this way allows the observation of ESP of the P’Q, radical pair at cryogenic temperatures [3,4,11]. Because the envelope modulation is known to be shallower for hfi with ’ N nuclei [lo], we studied the “N-enriched sample as well.
2. Experimental RCs of Rb. sphaeroides R26 were isolated as described in Ref. [12]. “N-containing cultures were grown with “NI-I,OH (99% enriched, Isotec Inc.,
236 (1995)
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USA) as the nitrogen source. The Fe2+ ion was removed according to Refs. [6,13] and replaced by Zn2+ as described in Ref. [14]. A typical EPR sample contained about 60% to 70% (v/v> glycerol. The final RC concentration was about 50 pM. The samples were frozen in the dark to 77 K and stored at this temperature. As a light source for sample irradiation we used a Continuum Surelite I laser pumping an optical parameter oscillator (OPO) that consisted of a non-linear /3-BaB,O, crystal. The idler wavelength was selected by a filter and adjusted to 860 nm. The pulsewidth was less than 5 ns, and output power was about 300 mJ/pulse. The repetition frequency of laser flashes was 10 Hz or lower, depending on the chemical lifetime of the radical pair. A Bruker ESP 380 FT EPR electron spin echo spectrometer was used, employing a dielectric cavity (Bruker ER 4118 X-MD-51 inside a liquid helium Oxford Instruments CF 935 cryostat. The cavity Q value was adjusted to provide a spectrometer dead time of = 100 ns. To acquire the two-pulse echo signal, two microwave pulse-former units were employed, providing a common pulse sequence 90” -T180”. The amplitude of the first pulse was set to half the value of that of the second pulse, while the pulse length for both pulses was the same and equal to 16 or 24 ns. This implies that the B, value of the second pulse is 1.1 or 0.74 mT, respectively. The former option (pulse lengths of 16 ns) required the maximal output microwave power of the device. The phases of both channels were adjusted to the same value. The spectrometer was triggered externally by the laser. A sweep of r comprised 150 points with a 24 ns increment (Nyquist frequency 20.8 MHz) or 200 points with a 16 ns increment (Nyquist frequency 31.25 MHz). The sweep was repeated lo-20 times for accumulating the signal, which was acquired using a Bruker Integrator ESP-380-1078 IN. To eliminate a small amount of background signal the experiment was performed twice with the magnetic field set for the second time far from resonance, and the results subtracted. Data treatment and Fourier transformation was performed using standard Bruker software (ESP380e, version 3.04). Computer simulations were performed on a personal computer.
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S.A. Dzuba et al. /Chemical Physics Letters 236 (1995) 595-602
3. Results The sample was studied at different temperatures between 20 and 220 K. At 20 K a small admixture of a stable signal of unknown nature has been found (probably it was a small amount of P’ Q, pairs). This signal was used to set the true phase for quadrature detection (only the in-phase signal is expected for spins in thermal equilibrium). The light flash produced a transient signal two orders of magnitude higher than the background signal. The intensity of the transient signal was found to be temperature-independent. Its amplitude decreased exponentially when the time delay t after the laser pulse was increased. The echo signal was measured as a function of the time delay r between two microwave echo-forming pulses. The results are shown in Fig. 1 for temperatures of 20 and 220 K. The data for 20 K shows that for the spin-polarised signal the echo is mainly out of phase. Note that increasing the temperature from 20 to 220 K produces a remarkable drift of the cavity resonance frequency, which results in a phase drift for signal detection. Because of this we could not set the phase properly for 220 K. We therefore set the phase such that for 220 K the same ratio of the in-phase and out-of-phase signals at starting T point was observed as for 20 K (see Fig. 1). Fig. 1 shows that the echo decays are strongly modulated. The echo intensity passes several times from positive to negative values. Note that for ESEEM induced by hfi with magnetic nuclei only a shallow modulation is usually observed [lo]. The decays shown in Fig. 1 were acquired for a delay t = 500 ns between the first microwave pulse and the laser flash. Within the experimental accuracy the same data were obtained when t was set to 1500 or 2500 ns. According to the theory developed by Tang et al. [9] the echo signal for the spin-polarised pairs is equal to zero when the time delay T = 0. This property is of essential help in overcoming the dead time problem. Indeed, because the starting point for the echo envelope is known, one may restore with good accuracy the initial part of this envelope that is obscured during the dead time. We used a simple linear function connecting the starting point (7 = 0) and the first point obtained in measurements. An
1, 220 K, “N
.,,;_,....,,..... ..._.__._.._.__..,............,.............,.............................................
0
1
2
3 7 W)
Fig. 1. Two-pulse echo signal as a function of the time separation T between microwave pulses at hvo different temperatures for RCs with natural-abundant nitrogen and for those “N-enriched. The solid lines represent the out-of-phase signal, the dotted lines the in-phase one. The dashed line (in the middle) shows the example of the linear-function approximation of the signal dependence in the dead-time region (see text). The data for 220 K were obtained 20 min after the sample was warmed up from low temperatures (cf. Fig. 3).
example of such a linear function is shown by the dashed line in Fig. 1, middle. The time delay r was taken as the difference between the beginning of the second pulse of the spectrometer’s pulse controller and the end of the first one, from which one more pulse length was subtracted. The latter subtraction was done because the microwave pulses are not exactly rectangular and the microwave oscillations continue after the controller pulse is switched off. Before Fourier transformation (IT) the highfrequency noise was reduced with a two-order polynomial filter function, and the data set was zero
S.A. Dzuba et al. /Chemical Physics Letters 236 (1995) 595-602
I-
-12
-8
-4
0
Frequency
4
8
12
not depend on the laser repetition frequency, which was varied between 1 and 10 Hz. Annealing at 220 K for 70 min, however, produced such a dependence. The signal intensity at 10 Hz then became much lower than before annealing; decreasing the repetition frequency to 2 Hz restored the signal amplitude. This means that through annealing, the chemical lifetime became longer than 100 ms and at 10 Hz the system failed to return to the initial state before the next laser flash. The resulting spectra in the frequency domain were found to be independent of the B, value, which in different measurements was set either to 0.74 or 1.1 mT (see Section 2). It shows that the condition of full excitation is fulfilled to good accuracy. Also, the results did not depend on the sampling r increment (see Section 2).
(MHz)
Fig. 2. The sine lT of the out-of- hase traces in Fig. 1 and of analogous data obtained for the IPN-enriched sample at 20 K. Before FT the experimental data in the dead-time region were approximated as shown in Fig. 1, filtered and zero-filled. The arrows show the positions of peaks associated with the nuclei-induced ESEEM (see text for details).
4. Computer
simulations
The spin Hamiltonian given by z=
PB,IIlSl+
PB,g,S2
+ S,CAzjIj filled to increase the resolution in the frequency domain. The small amount of in-phase signal (see Fig. 1) was discarded. A sine FT was performed to compare the experimental results with theory (see Section 4). The results of FT are shown in Fig. 2 for temperatures of 20 and 220 K. The frequency spectrum is much more resolved for 220 than for 20 K. Resolution is also enhanced by 15N enrichment. The spectral shape at 220 K was found to be time-dependent, as shown in Fig. 3 for the 15N-enriched sample. Just after the sample has been warmed up to 220 K, the spectrum consists of broad lines. Annealing for 20 min at this temperature results in a remarkable increase of resolution. Further annealing at this temperature again leads to line broadening. The latter effect was found to be completely reversible: when the sample was annealed at 240 K for 1 h the spectrum resolution increased again. The annealing at 220 K was also found to produce a remarkable increase of the radical pair lifetime after the laser flash. Normally the signal intensity did
+.q;
- 2s,
of the P’Q, + SlCAljIj
+ 3D(3cos28s,).
radical pair is
l)(S,2 - 3s’)
(1) Here, p is the Bohr magneton, B, is the magnetic field, g1 and g2 are the g tensors for P+ and Q,, S, and S, are their electron spin operators, A, j and A, j are their hfi tensors with the jth nucleus, Ij are nuclear spin operators, D represents the dipolar coupling, 8 is the angle between the line connecting two species and the direction of the external magnetic field, S = S, + S,, and J is the value of the spin-exchange interaction. Because the radicals in the pair are far apart, the asymmetry of the dipolar tensor is small and is neglected by the present theories of ESEEM induced by electron spin-spin interactions [7,9] (see also Section 5). As the g-tensor anisotropy for P+ and Q, is small, the non-secular contributions to the electron Zeeman interactions are also neglected; e.g., the term j3BogIS, in (1) is replaced by B, glz,Sz (B, is along the 2 axis of the laboratory frame). In the theories developed so far, only isotropic interactions with surrounding nuclei are taken into account. This l
599
Sd. Dzuba et al. / Chemical Physics Letters 236 (1995) 595402
means that, for example, the S,A,,Zj terms in Hamiltonian (1) are replaced by SIZAljrrZr. Anisotropic hfi and the nuclear Zeeman and quadrupole interactions give rise to the ESEEM due to magnetic nuclei. For spin-polarised radical pairs that are originally formed in a singlet state, Tang et al. [9] obtained an analytical expression for the out-of-phase primary echo. In the convenient notation of Ref. [7] it has the form E,(T)
= (A w2B2/R4) sin AT[l - cos(R~)],
(220 K + 70 min)
(220 K + 20 min)
(2)
where A= - ~D(3cos28B = 3D(3cos28-
1) + 25, 1) + 25,
-12
-8
-4
0
4
8
12
R2=Am2+B2, and Aw is the difference of resonance frequencies for two spins in the absence of dipolar and exchange interactions: Au=
h,z-
g2zz)PBll
+ c(
Aljrr
-A2jzz)mj*
Frequency
(MHz)
Fig. 3. Dependence of IT spectra at 220 K on the time after the sample was warmed from low temperatures. (a) The IT spectrum at 20 K given for comparison; (h) just after the temperature stabilisation; (c) 20 min later; Cd) 70 min later. The 15N-enriched sample is used.
(3)
Here mj denotes the projection of the jth nuclear spin onto the 2 axis for a particular nuclear spin state. In (2) we neglected terms proportional to sin Rt and cos Rt (t is the time delay after the laser pulse, see Ref. [9]). These terms should average to zero because of the large EPR linewidth (i.e. Rt >> 1 in our case). This is supported by the experimental observations mentioned in the previous section, namely that the echo signal does not depend on time t.
For comparing with experiment we must perform a FT of expression (2) and average over all different space and spectral positions of the two radicals forming the pair. Space positions are determined by the distance between the two molecules and their mutual orientation. The general idea of our calculations is to leave the distance between P+ and Q; as a free parameter (in other words, the D value is free), which is to be adjusted from comparison with experiment, while using the crystal structure known from X-ray studies of RCs of Rb. sphaeroids R26 [15,16] for the mutual orientations. Note that the
results were found to be only slightly sensitive to orientation (see below). In our calculations we adopted the molecular framework used by Fiichsle et al. [5]. All sets of data used in our calculations are also found in Ref. [5]. They are: (1) The Euler angles specifying the orientation of the g tensor of Pf obtained by 95 GHz (W band) EPR measurements on a single crystal of R26 RCs [17]. (Four data sets are given in Refs. [5,17]; according to recent studies only the set II gives the correct ESP pattern as observed at W band 1181. For checking the sensitivity of the calculations to mutual orientation, we have used all four data sets in our calculations.) (2) the Euler angles specifying the orientation of the g tensor of Q, with respect to the coordinate frame used, which can be obtained from the structure of the quinone molecule together with the RC X-ray data. (3) The angles of the dipole-dipole axis, which was found from the X-ray studies. (4) The principal values of the g tensor of P+ 1171. And (5) the principal values of the g tensor of Q, 1191.
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S.A. D.&a et al. /Chemical Physics Letters 236 (1995) 595402
The spectral positions were calculated using the principal values of the g tensor for both species and the orientation of the magnetic field with respect to these axes. For example, in Eq. (3) gl,, is substituted by !?I*, = g,xx sin%, cos$t + g,,, sin28, sin2q, +
g1zz
c-%
7
(4)
and grzz are the principal values where glxx9 glyy9 of the corresponding g tensor, and the angles e1 and ‘pl are the usual polar angles determining the orientation of the magnetic field with respect to the principal axes of the g tensor. These angles are to be found using the Euler angles specifying the orientation of the g tensor orientation with respect to the coordinate frame used. Neighbouring nuclei contribute to the line positions also. This is taken into account by introducing an individual Gaussian lineshape with peak-to-peak half-linewidth equal to 0.32 mT [5]. In our computations we used a sine Fourier transformation of Eq. (2). The results are shown in Fig. 4.
In addition to the D value, one more free parameter in these computations is the .7 value. Both these parameters influence the positions of the two pronounced peaks seen in the FT spectra (Figs. 2-41, and the total spectral width. These peaks are induced by singularities occurring for the perpendicular orientation of the line connecting the two species with respect to the magnetic field (0 = 90” 1. The spectral edges correspond to the parallel orientation (0 = 0). These spectral positions are determined by the simple relations f, = k($D-t2J) f,,= *(-;D+23).
(5)
The frequencies fl and f,, are given in Fig. 4 for the left part of the spectrum. Eq. (5) provides a simple and reliable way to obtain the D and J values from comparison with experiment. Note that mutual space orientations of both species does not influence the f, and fi, values. All four data sets for the Euler angles specifying the orientation of the g tensor of P+ [5,17] resulted in very similar lineshapes in the frequency domain. 5. Discussion
-12
-8
-4
0
Frequency
4
8
12
(MHz)
Fig. 4. The sine FT of theoretical expression (2) averaged over space and spectral positions. The free parameters in these simulations, the D and J values, were set to - 156 and 11 ~.LT, respectively (cf. Fig. 2, top). For the left part of the spectrum, the frequencies fl and f,, are indicated (see text).
Comparison of the experimental (Figs. 2 and 3) and the computed (Fig. 4) frequency spectra shows that the computations generally reproduce the spectral shape. Some differences, however, may be noticed, which may have two obvious reasons: (i) in the theory the transverse relaxation has not been taken into account, and (ii) the ESEEM due to nuclear hfi will affect the spectra. All three factors influencing the ESEEM - electron spin-spin interaction, transverse relaxation and nuclear hfi - may be considered independent, and therefore their influence in the time domain must be multiplicative. In the frequency domain this means the convolution of three lineshapes. If for example the transverse relaxation is exponential, it will result in a Lore&an lineshape in the frequency domain, which will broaden the experimental lines. The rather good agreement between theory and experiment stated above allows to conclude that spin-spin interactions dominate, and that the other two factors may be considered as higher-order corrections.
S.A. Dzuba
et al. /Chemical
Physics Letters
The frequency spectra of nuclei-induced primary ESEEM for P+-860 are,p in Refs. [20-221. In Refs. [21,22] data for N-enriched RCs are presented. In all cases the spectra contain a pronounced maximum near 2.6-2.9 MHz. One can see in Figs. 2 and 3 four additional small peaks, which in Fig. 2 are indicated by arrows. They are located by k2.9 MHz away from the two main peaks and, most probably, are induced by nitrogen nuclear ESEEM. The spectra obtained for i5N-enriched samples are more resolved than those with natural 14N abundance. This can be explained readily by the following two factors. First, the frequency spectrum for “N nuclear modulation is more resolved than that for 14N nuclei [21,22]; and, secondly, the “N modulation is shallower than the 14N modulation, therefore its total influence on the data should be smaller. One more difference between the data sets for 14N and 15N RCs is that the outer shoulders for the 15N-enriched sample are apparently smaller than for i4N RCs - see Fig. 2. It is however difficult to discuss this difference, because for high frequencies the actual time-domain data for small r (within the dead time) are important. As stated above, one can determine the D and J values from the experimental spectra, using the spectral positions of the f, and f,, frequencies - see (5). Model calculations, in which the spectrum in Fig. 4 was convoluted with a Lorentzian lineshape (data not given), showed that it is necessary to adjust these frequencies to the maximal slopes of corresponding line shoulders. For the spectra in Fig. 3 we obtained the following values: (a) D = - 175 + 8 p,T, J = 16 If: 4 IJT; (b) D= -162+5 pT, J=11+2 p,T; (c) D= -156f4 pT, J=11*2 p,T; (d) D= -157&5 pT, J = 9 f 2 pT. Note that X-ray data obtained for single crystals at room temperature gives the centerto-ce$er distance between the two species as r = 208.1A [15], resulting in D = - 123 p,T, and r = 29.2 A [16], resulting in D = - 106 pT. We may conclude that the temperature increase and the annealing cause an increase of the distance in the pair and a decrease of the exchange interaction. This result is consistent with the increase in the lifetime of the pair during sample annealing (see Section 3). The spectral transformations are reversible (see Section 3).
236 (1995)
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601
The experimental data in Figs. 2 and 3 show that at low temperatures the spectra are broader than at high temperatures. It is not likely that this difference is induced by the transverse relaxation. Indeed, at low temperatures this relaxation most probably should be slower and therefore lead to a reverse effect. A possible cause of the temperature-dependent line broadening is a distribution of the D and/or J values. The data in Fig. 3 suggest that an increase in temperature leads to a sharpening of this distribution in the beginning of thermal annealing at constant temperature (220 K). Later this distribution becomes wider again. A cause of concern in the interpretation of our results is the possible deviation from uniaxiality of the dipolar tensor coupling P+ and Q, due to spatial delocalization of the electron spin density at both radicals. To evaluate this non-axiality, we considered the spin to be delocalised at four points for Pf and at two points for Q,. These points were the centers of the pyrrole rings II and IV of both BChl a molecules constituting the dimer P [23], to each of which we assigned 0.25 partial spin density, and for Q, the carbonyl oxygen nearest to the Fe2+ ion and the carbon atom of the opposite carbonyl function, each of which obtained 0.5 partial spin density. Using the crystal coordinates for Rb. sphaeroides R26 [15,16], we evaluated the orientation dependence of the dipolar tensor and found that for our model tensor E = 0.030.
The flash-induced P’Q, radical pair system has been studied also by ESP spectroscopy in Ref. [6], where the J value obtained was J = 1 pT. In this work we find a value for J that is an order of magnitude larger. Two possible reasons may be put forward for this disagreement. First, the experimental uncertainty in the determination of the J value, in conjunction with the = 3% non-axiality of the dipolar coupling, are not small. Secondly, if, as our results suggest, the J values are distributed, then this distribution could manifest itself in a different way for the different experimental techniques. In Ref. [6] the magnitude of J was discussed in light of the distance between P+ and Q,. Applying a similar distance relationship for J as in Ref. [24], the experimental value of J in Ref. [6] was found to be a factor of about 6 larger than the calculated value. Our present results suggest that the deviation be-
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Physics
tween the value of J calculated with the distance relationship of Ref. [24] and the experimental J may be even larger, calling for a more refined theoretical evaluation of exchange interactions in the RCs. Acknowledgements We thank Mr. A.H.M. de Wit for growing the bacteria, Ms. S.J. Jansen for help with preparing the RCs, and Mr. J.S. van den Brink for sharing his results on ESP spectroscopy of reaction centers. This research was supported by the Netherlands Foundation for Chemical Research (SON), financed by the Netherlands Organisation for Scientific Research (NWO). SAD acknowledges a travel grant of NW0 in the framework of grant No. 713-036 for Netherlands-Russian scientific collaboration, and is indebted to Dr. O.G. Poluektov for assistance in starting these experiments and for numerous helpful discussions. References [l] L.T. Muus, P.W. Atkins, K.A. McLauchhm and J.B. Pedersen, eds., Chemically induced magnetic polarization (Reidel, Dordrecht, 1977). [2] A.J. Hoff, Quart. Rev. Biophys. 17 (1984) 153. [3] P.J. Hore, D.A. Hunter, C.D. M&e and A.J. Hoff, Chem. Phys. Letters 137 (1987) 495. [4] D. Stehlik, C.H. Bock and J. Petersen, J. Phys. Chem. 93 (1989) 1612. [5] G. Fiichsle, R. Bittl, A. van der Est, W. Lubitz and D. Stehlik, Biochim. Biophys. Acta 1142 (1993) 23.
Letters
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[6] J.S. van den Brink, R.J. Hulsebosch, P. Gast, P.J. Hore and A.J. Hoff, Biochemistry 33 (1994) 13668. [7] V.F. Yudanov, K.M. Salikhov, G.M. Zhidomirov and Yu.D. Tsvetkov, Teor. Eksper. Khim. 5 (1969) 663. [8] A.D. Milov and Yu.D. Tsvetkov, Doll. AN SSSR 288 (1986) 924. [9] J. Tang, MC. Thumauer and J.R. Norris, Chem. Phys. Letters 219 (1994) 283. [lo] S.A. Dikanov and Yu.D. Tsvetkov, Electron spin echo modulation @SEEM) spectroscopy (CRC Press, Boca Raton, 1992). [ll] A.J. Hoff, P. Gast and J.C. Romijn, FEBS Letters 73 (1977) 185. [12] G. Feher and M.Y. Okamura, in: The photosynthetic bacteria, eds. R.K. Clayton and W.R. Sistrom (Plenum Press, New York, 1978). [13] D.M. Tiede and P.L. Dutton, Biochim. Biophys. Acta 637 (1981) 278. [14] R.J. Debus, G. Feher and M.Y. Okamura, Biochemistry 25 (1986) 2276. [15] H. Komiya, T.O. Yeates, DC. Rees, J.P. Allen and G. Feher, Proc. Natl. Acad. Sci. US 85 (1988) 9012. 1161 C.-H. Chang, 0. El-Kabbani, D. Tiede, J. Norris and M. Schiffer, Biochemistry 30 (1991) 5352. [17] R. Ktette, J.T. Toning, M. Plato, B. Bijnigk, W. Lubitz and K. Mobius, J. Phys. Chem. 97 (1993) 2015. [lS] T.F. Prisner, A. van der Est, R. Bittl, W. Lubitz, D. Stehlik and K. Mobius, Chem. Phys. (1995), in press. [19] 0. Burghaus, Ph.D. Thesis, Free University Berlin (1991). [20] LH. Davis, P. Heathcote, D.J. MacLachlan and M.C.W. Evans, Biochim. Biophys. Acta 1143 (1993) 183. [21] A.J. Hoff, A. de Groot, S.A. Dikanov, A.V. Astashkin and Yu.D. Tsvetkov, Cbem. Phys. Letters 118 (1985) 40. [22] A. de Groot, A.J. Hoff, R. de Beer and H. Scheer, Chem. Phys. Letters 113 (1985) 286. [23] M. Plato, E. Tr;inMe, W. Lubitz, F. Lendzian and K. Mdbius, Chem. Phys. 107 (1986) 185. [24] C.C. Moser and P.L. Dutton, Biochim. Biophys. Acta 1101 (1992) 171.
1995
CHEWCAL PHYSICS LETTERS ELSEVIER
Chemical
Physics Letters
236 (1995)
595-602
ESEEM study of spin-spin interactions in spin-polarised P’Q, pairs in the photosynthetic purple bacterium Rhodobacter sphaeroides R26 Sergei A. Dzuba ‘, Peter Gast, Arnold J. Hoff * Department
of Biophysics,
Huygens
Laboratory,
Received
5 January
Leiden
University,
P.O. Box 9504,230O
1995; in final form 27 February
RA Leiden,
The Netherlands
1995
Abstract Electron spin echo envelope modulation (ESEEM) of the transient spin-polarised PfQA pairs has been studied at 20 and 220 K. The observed strong out-of-phase modulation is interpreted as resulting from electron-electron dipolar and exchange interactions. Fourier-transformed echo envelopes are consistent with the theory developed recently by Tang, Thumauer and Norris and allow us to obtain readily the values of dipolar and exchange couplings. The results are insensitive to 15N enrichment.
1. Introduction
Charge separation in photosynthetic reaction centers CRC) leads to formation of radical pairs. The primary light-induced reactions in RCs of photosynthetic bacteria are: (i) The electronically excited state of the primary donor P, a bacteriochlorophyll dimer, produces the primary radical pair P’I-, where I denotes a bacteriopheophytin molecule. This reaction occurs within = 3 ps. (ii> Dark electron transfer within 200 ps from I- to QA, the primary acceptor quinone, creates the secondary radical pair P’Q,. Finally, (iii) within 200 bs the electron reaches Qn, the secondary acceptor quinone.
* Corresponding author. ’ On leave from Institute of Chemical Kinetics and Combustion, Russian Academy of Sciences, 630090 Novosibirsk, Russian Federation. 0009-2614/95/$09.50
8 1995 Elsevier
SSDIOOO9-2614(95)00259-6
Science
B.V. All rights reserved
The study of spin-spin interactions between radicals in the pairs, which include magnetic dipole-dipole and spin-exchange interactions, could reveal important structural information. The dipole-dipole interaction reflects mutual radical positions in the pair, while the exchange interaction is related to the overlap of electronic wavefunctions, which correlates with the rate of electron transfer. As the radical pairs mentioned above are formed shortly after the light flash, they are spin polarised. The method of electron spin polarisation (ESP) spectroscopy is effective in the determination of dipolar and exchange couplings [l-6]. For RCs in which Q, is not reduced, the results of these measurements refer to the P+Q- pairs, because the PfIpairs decay very fast. It has been known for a long time that spin-spin interactions in radical pairs result in so-called electron spin echo envelope modulation (ESEEM) [7]. The theory developed in Ref. [7] was applied to
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SA. Dzuba
et al. /Chemical
Physics Letters
experiments on stable radical pairs and biradicals in solids [7] and on biradicals in solutions [8]. The modulation phenomenon manifests itself as an oscillation of the in-phase echo signal (i.e. along the Y axis of the rotating frame if the microwave field is along the X axis). For species in thermal equilibrium the out-of-phase signal does not appear. Recently, Tang et al. [9] presented a theory of ESEEM for spin-polarised radical pairs. Their results predict that in this case an out-of-phase modulation exists while the in-phase signal is completely absent. An important condition for the observation of modulations induced by spin-spin interactions is full excitation of both radicals in the pair by the microwave pulse. The EPR spectrum of the P’Q, pair has a characteristic width of = 1 mT [5]. Therefore the amplitude B, of the microwave pulses should be of the same order or higher. Note that only the amplitude of the second pulse is important for the full excitation condition used in the above theories. The role of the first pulse is to create the initial transverse magnetization, and it may be of arbitrary amplitude. Another interaction that may induce the modulation phenomenon is the electron-nuclear hyperfine interaction (hfi) [lo]. For P+ and Q, radicals the main source of nuclear modulations is coupling with nitrogen nuclei [lo]. In the present work we study ESEEM induced by spin-spin interactions between P+ and Qi in the photosynthetic RC of the purple bacterium Rhodobacter sphaeroides R26, in which the native non-heme iron was replaced by the diamagnetic Zn2+ ion. Removing the magnetic coupling between the Fe2+ ion and Q, in this way allows the observation of ESP of the P’Q, radical pair at cryogenic temperatures [3,4,11]. Because the envelope modulation is known to be shallower for hfi with ’ N nuclei [lo], we studied the “N-enriched sample as well.
2. Experimental RCs of Rb. sphaeroides R26 were isolated as described in Ref. [12]. “N-containing cultures were grown with “NI-I,OH (99% enriched, Isotec Inc.,
236 (1995)
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USA) as the nitrogen source. The Fe2+ ion was removed according to Refs. [6,13] and replaced by Zn2+ as described in Ref. [14]. A typical EPR sample contained about 60% to 70% (v/v> glycerol. The final RC concentration was about 50 pM. The samples were frozen in the dark to 77 K and stored at this temperature. As a light source for sample irradiation we used a Continuum Surelite I laser pumping an optical parameter oscillator (OPO) that consisted of a non-linear /3-BaB,O, crystal. The idler wavelength was selected by a filter and adjusted to 860 nm. The pulsewidth was less than 5 ns, and output power was about 300 mJ/pulse. The repetition frequency of laser flashes was 10 Hz or lower, depending on the chemical lifetime of the radical pair. A Bruker ESP 380 FT EPR electron spin echo spectrometer was used, employing a dielectric cavity (Bruker ER 4118 X-MD-51 inside a liquid helium Oxford Instruments CF 935 cryostat. The cavity Q value was adjusted to provide a spectrometer dead time of = 100 ns. To acquire the two-pulse echo signal, two microwave pulse-former units were employed, providing a common pulse sequence 90” -T180”. The amplitude of the first pulse was set to half the value of that of the second pulse, while the pulse length for both pulses was the same and equal to 16 or 24 ns. This implies that the B, value of the second pulse is 1.1 or 0.74 mT, respectively. The former option (pulse lengths of 16 ns) required the maximal output microwave power of the device. The phases of both channels were adjusted to the same value. The spectrometer was triggered externally by the laser. A sweep of r comprised 150 points with a 24 ns increment (Nyquist frequency 20.8 MHz) or 200 points with a 16 ns increment (Nyquist frequency 31.25 MHz). The sweep was repeated lo-20 times for accumulating the signal, which was acquired using a Bruker Integrator ESP-380-1078 IN. To eliminate a small amount of background signal the experiment was performed twice with the magnetic field set for the second time far from resonance, and the results subtracted. Data treatment and Fourier transformation was performed using standard Bruker software (ESP380e, version 3.04). Computer simulations were performed on a personal computer.
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3. Results The sample was studied at different temperatures between 20 and 220 K. At 20 K a small admixture of a stable signal of unknown nature has been found (probably it was a small amount of P’ Q, pairs). This signal was used to set the true phase for quadrature detection (only the in-phase signal is expected for spins in thermal equilibrium). The light flash produced a transient signal two orders of magnitude higher than the background signal. The intensity of the transient signal was found to be temperature-independent. Its amplitude decreased exponentially when the time delay t after the laser pulse was increased. The echo signal was measured as a function of the time delay r between two microwave echo-forming pulses. The results are shown in Fig. 1 for temperatures of 20 and 220 K. The data for 20 K shows that for the spin-polarised signal the echo is mainly out of phase. Note that increasing the temperature from 20 to 220 K produces a remarkable drift of the cavity resonance frequency, which results in a phase drift for signal detection. Because of this we could not set the phase properly for 220 K. We therefore set the phase such that for 220 K the same ratio of the in-phase and out-of-phase signals at starting T point was observed as for 20 K (see Fig. 1). Fig. 1 shows that the echo decays are strongly modulated. The echo intensity passes several times from positive to negative values. Note that for ESEEM induced by hfi with magnetic nuclei only a shallow modulation is usually observed [lo]. The decays shown in Fig. 1 were acquired for a delay t = 500 ns between the first microwave pulse and the laser flash. Within the experimental accuracy the same data were obtained when t was set to 1500 or 2500 ns. According to the theory developed by Tang et al. [9] the echo signal for the spin-polarised pairs is equal to zero when the time delay T = 0. This property is of essential help in overcoming the dead time problem. Indeed, because the starting point for the echo envelope is known, one may restore with good accuracy the initial part of this envelope that is obscured during the dead time. We used a simple linear function connecting the starting point (7 = 0) and the first point obtained in measurements. An
1, 220 K, “N
.,,;_,....,,..... ..._.__._.._.__..,............,.............,.............................................
0
1
2
3 7 W)
Fig. 1. Two-pulse echo signal as a function of the time separation T between microwave pulses at hvo different temperatures for RCs with natural-abundant nitrogen and for those “N-enriched. The solid lines represent the out-of-phase signal, the dotted lines the in-phase one. The dashed line (in the middle) shows the example of the linear-function approximation of the signal dependence in the dead-time region (see text). The data for 220 K were obtained 20 min after the sample was warmed up from low temperatures (cf. Fig. 3).
example of such a linear function is shown by the dashed line in Fig. 1, middle. The time delay r was taken as the difference between the beginning of the second pulse of the spectrometer’s pulse controller and the end of the first one, from which one more pulse length was subtracted. The latter subtraction was done because the microwave pulses are not exactly rectangular and the microwave oscillations continue after the controller pulse is switched off. Before Fourier transformation (IT) the highfrequency noise was reduced with a two-order polynomial filter function, and the data set was zero
S.A. Dzuba et al. /Chemical Physics Letters 236 (1995) 595-602
I-
-12
-8
-4
0
Frequency
4
8
12
not depend on the laser repetition frequency, which was varied between 1 and 10 Hz. Annealing at 220 K for 70 min, however, produced such a dependence. The signal intensity at 10 Hz then became much lower than before annealing; decreasing the repetition frequency to 2 Hz restored the signal amplitude. This means that through annealing, the chemical lifetime became longer than 100 ms and at 10 Hz the system failed to return to the initial state before the next laser flash. The resulting spectra in the frequency domain were found to be independent of the B, value, which in different measurements was set either to 0.74 or 1.1 mT (see Section 2). It shows that the condition of full excitation is fulfilled to good accuracy. Also, the results did not depend on the sampling r increment (see Section 2).
(MHz)
Fig. 2. The sine lT of the out-of- hase traces in Fig. 1 and of analogous data obtained for the IPN-enriched sample at 20 K. Before FT the experimental data in the dead-time region were approximated as shown in Fig. 1, filtered and zero-filled. The arrows show the positions of peaks associated with the nuclei-induced ESEEM (see text for details).
4. Computer
simulations
The spin Hamiltonian given by z=
PB,IIlSl+
PB,g,S2
+ S,CAzjIj filled to increase the resolution in the frequency domain. The small amount of in-phase signal (see Fig. 1) was discarded. A sine FT was performed to compare the experimental results with theory (see Section 4). The results of FT are shown in Fig. 2 for temperatures of 20 and 220 K. The frequency spectrum is much more resolved for 220 than for 20 K. Resolution is also enhanced by 15N enrichment. The spectral shape at 220 K was found to be time-dependent, as shown in Fig. 3 for the 15N-enriched sample. Just after the sample has been warmed up to 220 K, the spectrum consists of broad lines. Annealing for 20 min at this temperature results in a remarkable increase of resolution. Further annealing at this temperature again leads to line broadening. The latter effect was found to be completely reversible: when the sample was annealed at 240 K for 1 h the spectrum resolution increased again. The annealing at 220 K was also found to produce a remarkable increase of the radical pair lifetime after the laser flash. Normally the signal intensity did
+.q;
- 2s,
of the P’Q, + SlCAljIj
+ 3D(3cos28s,).
radical pair is
l)(S,2 - 3s’)
(1) Here, p is the Bohr magneton, B, is the magnetic field, g1 and g2 are the g tensors for P+ and Q,, S, and S, are their electron spin operators, A, j and A, j are their hfi tensors with the jth nucleus, Ij are nuclear spin operators, D represents the dipolar coupling, 8 is the angle between the line connecting two species and the direction of the external magnetic field, S = S, + S,, and J is the value of the spin-exchange interaction. Because the radicals in the pair are far apart, the asymmetry of the dipolar tensor is small and is neglected by the present theories of ESEEM induced by electron spin-spin interactions [7,9] (see also Section 5). As the g-tensor anisotropy for P+ and Q, is small, the non-secular contributions to the electron Zeeman interactions are also neglected; e.g., the term j3BogIS, in (1) is replaced by B, glz,Sz (B, is along the 2 axis of the laboratory frame). In the theories developed so far, only isotropic interactions with surrounding nuclei are taken into account. This l
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means that, for example, the S,A,,Zj terms in Hamiltonian (1) are replaced by SIZAljrrZr. Anisotropic hfi and the nuclear Zeeman and quadrupole interactions give rise to the ESEEM due to magnetic nuclei. For spin-polarised radical pairs that are originally formed in a singlet state, Tang et al. [9] obtained an analytical expression for the out-of-phase primary echo. In the convenient notation of Ref. [7] it has the form E,(T)
= (A w2B2/R4) sin AT[l - cos(R~)],
(220 K + 70 min)
(220 K + 20 min)
(2)
where A= - ~D(3cos28B = 3D(3cos28-
1) + 25, 1) + 25,
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-8
-4
0
4
8
12
R2=Am2+B2, and Aw is the difference of resonance frequencies for two spins in the absence of dipolar and exchange interactions: Au=
h,z-
g2zz)PBll
+ c(
Aljrr
-A2jzz)mj*
Frequency
(MHz)
Fig. 3. Dependence of IT spectra at 220 K on the time after the sample was warmed from low temperatures. (a) The IT spectrum at 20 K given for comparison; (h) just after the temperature stabilisation; (c) 20 min later; Cd) 70 min later. The 15N-enriched sample is used.
(3)
Here mj denotes the projection of the jth nuclear spin onto the 2 axis for a particular nuclear spin state. In (2) we neglected terms proportional to sin Rt and cos Rt (t is the time delay after the laser pulse, see Ref. [9]). These terms should average to zero because of the large EPR linewidth (i.e. Rt >> 1 in our case). This is supported by the experimental observations mentioned in the previous section, namely that the echo signal does not depend on time t.
For comparing with experiment we must perform a FT of expression (2) and average over all different space and spectral positions of the two radicals forming the pair. Space positions are determined by the distance between the two molecules and their mutual orientation. The general idea of our calculations is to leave the distance between P+ and Q; as a free parameter (in other words, the D value is free), which is to be adjusted from comparison with experiment, while using the crystal structure known from X-ray studies of RCs of Rb. sphaeroids R26 [15,16] for the mutual orientations. Note that the
results were found to be only slightly sensitive to orientation (see below). In our calculations we adopted the molecular framework used by Fiichsle et al. [5]. All sets of data used in our calculations are also found in Ref. [5]. They are: (1) The Euler angles specifying the orientation of the g tensor of Pf obtained by 95 GHz (W band) EPR measurements on a single crystal of R26 RCs [17]. (Four data sets are given in Refs. [5,17]; according to recent studies only the set II gives the correct ESP pattern as observed at W band 1181. For checking the sensitivity of the calculations to mutual orientation, we have used all four data sets in our calculations.) (2) the Euler angles specifying the orientation of the g tensor of Q, with respect to the coordinate frame used, which can be obtained from the structure of the quinone molecule together with the RC X-ray data. (3) The angles of the dipole-dipole axis, which was found from the X-ray studies. (4) The principal values of the g tensor of P+ 1171. And (5) the principal values of the g tensor of Q, 1191.
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The spectral positions were calculated using the principal values of the g tensor for both species and the orientation of the magnetic field with respect to these axes. For example, in Eq. (3) gl,, is substituted by !?I*, = g,xx sin%, cos$t + g,,, sin28, sin2q, +
g1zz
c-%
7
(4)
and grzz are the principal values where glxx9 glyy9 of the corresponding g tensor, and the angles e1 and ‘pl are the usual polar angles determining the orientation of the magnetic field with respect to the principal axes of the g tensor. These angles are to be found using the Euler angles specifying the orientation of the g tensor orientation with respect to the coordinate frame used. Neighbouring nuclei contribute to the line positions also. This is taken into account by introducing an individual Gaussian lineshape with peak-to-peak half-linewidth equal to 0.32 mT [5]. In our computations we used a sine Fourier transformation of Eq. (2). The results are shown in Fig. 4.
In addition to the D value, one more free parameter in these computations is the .7 value. Both these parameters influence the positions of the two pronounced peaks seen in the FT spectra (Figs. 2-41, and the total spectral width. These peaks are induced by singularities occurring for the perpendicular orientation of the line connecting the two species with respect to the magnetic field (0 = 90” 1. The spectral edges correspond to the parallel orientation (0 = 0). These spectral positions are determined by the simple relations f, = k($D-t2J) f,,= *(-;D+23).
(5)
The frequencies fl and f,, are given in Fig. 4 for the left part of the spectrum. Eq. (5) provides a simple and reliable way to obtain the D and J values from comparison with experiment. Note that mutual space orientations of both species does not influence the f, and fi, values. All four data sets for the Euler angles specifying the orientation of the g tensor of P+ [5,17] resulted in very similar lineshapes in the frequency domain. 5. Discussion
-12
-8
-4
0
Frequency
4
8
12
(MHz)
Fig. 4. The sine FT of theoretical expression (2) averaged over space and spectral positions. The free parameters in these simulations, the D and J values, were set to - 156 and 11 ~.LT, respectively (cf. Fig. 2, top). For the left part of the spectrum, the frequencies fl and f,, are indicated (see text).
Comparison of the experimental (Figs. 2 and 3) and the computed (Fig. 4) frequency spectra shows that the computations generally reproduce the spectral shape. Some differences, however, may be noticed, which may have two obvious reasons: (i) in the theory the transverse relaxation has not been taken into account, and (ii) the ESEEM due to nuclear hfi will affect the spectra. All three factors influencing the ESEEM - electron spin-spin interaction, transverse relaxation and nuclear hfi - may be considered independent, and therefore their influence in the time domain must be multiplicative. In the frequency domain this means the convolution of three lineshapes. If for example the transverse relaxation is exponential, it will result in a Lore&an lineshape in the frequency domain, which will broaden the experimental lines. The rather good agreement between theory and experiment stated above allows to conclude that spin-spin interactions dominate, and that the other two factors may be considered as higher-order corrections.
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et al. /Chemical
Physics Letters
The frequency spectra of nuclei-induced primary ESEEM for P+-860 are,p in Refs. [20-221. In Refs. [21,22] data for N-enriched RCs are presented. In all cases the spectra contain a pronounced maximum near 2.6-2.9 MHz. One can see in Figs. 2 and 3 four additional small peaks, which in Fig. 2 are indicated by arrows. They are located by k2.9 MHz away from the two main peaks and, most probably, are induced by nitrogen nuclear ESEEM. The spectra obtained for i5N-enriched samples are more resolved than those with natural 14N abundance. This can be explained readily by the following two factors. First, the frequency spectrum for “N nuclear modulation is more resolved than that for 14N nuclei [21,22]; and, secondly, the “N modulation is shallower than the 14N modulation, therefore its total influence on the data should be smaller. One more difference between the data sets for 14N and 15N RCs is that the outer shoulders for the 15N-enriched sample are apparently smaller than for i4N RCs - see Fig. 2. It is however difficult to discuss this difference, because for high frequencies the actual time-domain data for small r (within the dead time) are important. As stated above, one can determine the D and J values from the experimental spectra, using the spectral positions of the f, and f,, frequencies - see (5). Model calculations, in which the spectrum in Fig. 4 was convoluted with a Lorentzian lineshape (data not given), showed that it is necessary to adjust these frequencies to the maximal slopes of corresponding line shoulders. For the spectra in Fig. 3 we obtained the following values: (a) D = - 175 + 8 p,T, J = 16 If: 4 IJT; (b) D= -162+5 pT, J=11+2 p,T; (c) D= -156f4 pT, J=11*2 p,T; (d) D= -157&5 pT, J = 9 f 2 pT. Note that X-ray data obtained for single crystals at room temperature gives the centerto-ce$er distance between the two species as r = 208.1A [15], resulting in D = - 123 p,T, and r = 29.2 A [16], resulting in D = - 106 pT. We may conclude that the temperature increase and the annealing cause an increase of the distance in the pair and a decrease of the exchange interaction. This result is consistent with the increase in the lifetime of the pair during sample annealing (see Section 3). The spectral transformations are reversible (see Section 3).
236 (1995)
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The experimental data in Figs. 2 and 3 show that at low temperatures the spectra are broader than at high temperatures. It is not likely that this difference is induced by the transverse relaxation. Indeed, at low temperatures this relaxation most probably should be slower and therefore lead to a reverse effect. A possible cause of the temperature-dependent line broadening is a distribution of the D and/or J values. The data in Fig. 3 suggest that an increase in temperature leads to a sharpening of this distribution in the beginning of thermal annealing at constant temperature (220 K). Later this distribution becomes wider again. A cause of concern in the interpretation of our results is the possible deviation from uniaxiality of the dipolar tensor coupling P+ and Q, due to spatial delocalization of the electron spin density at both radicals. To evaluate this non-axiality, we considered the spin to be delocalised at four points for Pf and at two points for Q,. These points were the centers of the pyrrole rings II and IV of both BChl a molecules constituting the dimer P [23], to each of which we assigned 0.25 partial spin density, and for Q, the carbonyl oxygen nearest to the Fe2+ ion and the carbon atom of the opposite carbonyl function, each of which obtained 0.5 partial spin density. Using the crystal coordinates for Rb. sphaeroides R26 [15,16], we evaluated the orientation dependence of the dipolar tensor and found that for our model tensor E = 0.030.
The flash-induced P’Q, radical pair system has been studied also by ESP spectroscopy in Ref. [6], where the J value obtained was J = 1 pT. In this work we find a value for J that is an order of magnitude larger. Two possible reasons may be put forward for this disagreement. First, the experimental uncertainty in the determination of the J value, in conjunction with the = 3% non-axiality of the dipolar coupling, are not small. Secondly, if, as our results suggest, the J values are distributed, then this distribution could manifest itself in a different way for the different experimental techniques. In Ref. [6] the magnitude of J was discussed in light of the distance between P+ and Q,. Applying a similar distance relationship for J as in Ref. [24], the experimental value of J in Ref. [6] was found to be a factor of about 6 larger than the calculated value. Our present results suggest that the deviation be-
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tween the value of J calculated with the distance relationship of Ref. [24] and the experimental J may be even larger, calling for a more refined theoretical evaluation of exchange interactions in the RCs. Acknowledgements We thank Mr. A.H.M. de Wit for growing the bacteria, Ms. S.J. Jansen for help with preparing the RCs, and Mr. J.S. van den Brink for sharing his results on ESP spectroscopy of reaction centers. This research was supported by the Netherlands Foundation for Chemical Research (SON), financed by the Netherlands Organisation for Scientific Research (NWO). SAD acknowledges a travel grant of NW0 in the framework of grant No. 713-036 for Netherlands-Russian scientific collaboration, and is indebted to Dr. O.G. Poluektov for assistance in starting these experiments and for numerous helpful discussions. References [l] L.T. Muus, P.W. Atkins, K.A. McLauchhm and J.B. Pedersen, eds., Chemically induced magnetic polarization (Reidel, Dordrecht, 1977). [2] A.J. Hoff, Quart. Rev. Biophys. 17 (1984) 153. [3] P.J. Hore, D.A. Hunter, C.D. M&e and A.J. Hoff, Chem. Phys. Letters 137 (1987) 495. [4] D. Stehlik, C.H. Bock and J. Petersen, J. Phys. Chem. 93 (1989) 1612. [5] G. Fiichsle, R. Bittl, A. van der Est, W. Lubitz and D. Stehlik, Biochim. Biophys. Acta 1142 (1993) 23.
Letters
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[6] J.S. van den Brink, R.J. Hulsebosch, P. Gast, P.J. Hore and A.J. Hoff, Biochemistry 33 (1994) 13668. [7] V.F. Yudanov, K.M. Salikhov, G.M. Zhidomirov and Yu.D. Tsvetkov, Teor. Eksper. Khim. 5 (1969) 663. [8] A.D. Milov and Yu.D. Tsvetkov, Doll. AN SSSR 288 (1986) 924. [9] J. Tang, MC. Thumauer and J.R. Norris, Chem. Phys. Letters 219 (1994) 283. [lo] S.A. Dikanov and Yu.D. Tsvetkov, Electron spin echo modulation @SEEM) spectroscopy (CRC Press, Boca Raton, 1992). [ll] A.J. Hoff, P. Gast and J.C. Romijn, FEBS Letters 73 (1977) 185. [12] G. Feher and M.Y. Okamura, in: The photosynthetic bacteria, eds. R.K. Clayton and W.R. Sistrom (Plenum Press, New York, 1978). [13] D.M. Tiede and P.L. Dutton, Biochim. Biophys. Acta 637 (1981) 278. [14] R.J. Debus, G. Feher and M.Y. Okamura, Biochemistry 25 (1986) 2276. [15] H. Komiya, T.O. Yeates, DC. Rees, J.P. Allen and G. Feher, Proc. Natl. Acad. Sci. US 85 (1988) 9012. 1161 C.-H. Chang, 0. El-Kabbani, D. Tiede, J. Norris and M. Schiffer, Biochemistry 30 (1991) 5352. [17] R. Ktette, J.T. Toning, M. Plato, B. Bijnigk, W. Lubitz and K. Mobius, J. Phys. Chem. 97 (1993) 2015. [lS] T.F. Prisner, A. van der Est, R. Bittl, W. Lubitz, D. Stehlik and K. Mobius, Chem. Phys. (1995), in press. [19] 0. Burghaus, Ph.D. Thesis, Free University Berlin (1991). [20] LH. Davis, P. Heathcote, D.J. MacLachlan and M.C.W. Evans, Biochim. Biophys. Acta 1143 (1993) 183. [21] A.J. Hoff, A. de Groot, S.A. Dikanov, A.V. Astashkin and Yu.D. Tsvetkov, Cbem. Phys. Letters 118 (1985) 40. [22] A. de Groot, A.J. Hoff, R. de Beer and H. Scheer, Chem. Phys. Letters 113 (1985) 286. [23] M. Plato, E. Tr;inMe, W. Lubitz, F. Lendzian and K. Mdbius, Chem. Phys. 107 (1986) 185. [24] C.C. Moser and P.L. Dutton, Biochim. Biophys. Acta 1101 (1992) 171.