1612
J . Phys. Chem. 1989, 93, 1612-1619
Anisotropic Electron Spin Polarization of Correlated Spin Pairs in Photosynthetic Reaction Centers Dietmar Stehlik,* Christian H. Bock, and Jan Petersent FB Physik, Freie Universitat Berlin, Arnimallee 14, D - 1000 Berlin 33, West Germany (Received: June 13, 1988)
Transient electron spin polarization (ESP) spectra of deuteriated photosystem I (PS I) preparations at early times after light excitation (from 50 ns to a few microseconds) and at higher spectral resolution (24 GHz) provide evidence for a resolved g anisotropy of a quinone type secondary acceptor A,- and an anisotropic ESP mechanism. For bacterial reaction centers where the structure is known, transient ESP spectra are available after removal of the non-heme iron at 9 GHz. At this lower level of resolution ESP patterns of PS I and bacterial reaction centers are similar, indicating analogy of the relevant structural and mechanistic properties. Conventional ESP mechanisms are explored as well as the recently proposed concept of direct EPR transitions during the lifetime of the P + , d I - radical ion pair whose spins are magnetically inequivalent due to their specific g tensors and are coupled by dipolar spin-spin coupling. The latter mechanism is found to dominate the observed transient EPR data in photosynthetic reaction centers. Most of the essential parameters needed for the simulations can be obtained from independent experimental information and related to the known 3D structure of the bacterial reaction center. With the identification of the relevant ESP mechanism the experimental spectrum renders direct information on the nature of the coupled spin pair as well as on the magnetic interactions including their anisotropic parts and their respective orientation. Since the dipolar principal axis zd (connecting the molecular centers of the paired radical ions) represents a fixed axis in the reaction center structure and the g tensor principal axes are related to the molecular axes, the orientation of the charge (spin)-carrying units with respect to the reaction center structure is obtained for the charge separated state.
1. Introduction One of the important phenomena in the time-resolved electron paramagnetic resonance (EPR) spectroscopy of photosynthetic reaction centers is electron spin polarization (ESP) following light excitation of the primary donor P (for a review see ref 1). ESP has been observed in the triplet state PT following charge recombination as well as via the first EPR-detectable charge-separated state P'IQ-. Both phenomena have been explained as a consequence of the radical pair mechanism (RPM) operating in the primary light-induced radical pair P+I- involving a transient intermediary acceptor I. In this paper we deal with ESP observed in the P'IQ- state, Le., under normal conditions of forward electron transfer. Numerous reports have demonstrated a polarized EPR spectrum of untreated photosystem I (PS I) in various plant preparations and whole algae as well as of bacterial reaction centers (BRC)after decoupling, removal or substitution of the non-heme iron or reconstitution of the native quinone.' In all cases the X-band (9 GHz) spectra show ESP with a characteristic emission/absorption/emission (E/A/E) pattern. Its early interpretation did not seem to require the consideration of anisotropy in the g tensors of the involved radical ions or in the interaction entering the RPM. However, ESP spectra taken at higher frequencies showed more resolved ~ t r u c t u r e . ~ -The ~ known g anisotropy of the quinone (Q-) acceptor has also been confirmed in Q-band (35 GHz) spectra of bacterial reaction center^.^ Q-band ESP spectra have been reported also in PS I particles4by using light modulation. Together with the ESP ~ p e c t r ain ~ .the ~ K band they provide evidence for the quinone nature of the first stable PS I acceptor. It should be noted that within the limited resolution of protonated material the ESP patterns of bacterial and PS I reaction centers show no significant differen~e.~ Similarly, the better resolved K-band ESP spectrum of perdeuteriated PS I particle^^,^ as well as the Q-band spectrum with light modulation4 are virtually identical, indicating a common origin of the respective ESP patterns. As a consequence, the analysis of ESP spectra will be based on a resolved g anisotropy of a Q- contribution. This in turn is associated with an anisotropic spin polarization mechanism. On the basis of the conventional radical pair mechanism a preliminary account of such a concept has been considered *To whom correspondence should be addressed. 'Present address: Department of Chemistry. Michigan State University, East Lansing, MI 48824-1322.
0022-3654/89/2093-1612$01.50/0
Recently, a different ESP scheme has been realized to be of importance if the spins of a light-induced correlated radical pair A are observed while they are still coupled to each preliminary analysis with this concept has been applied to the X-band ESP spectra of bacterial reaction centers,12but a definite conclusion has not been reached concerning the mechanism and interaction parameters involved. The predominant questions to be asked in this situation are as follows: (i) Is it possible to determine the relevant ESP mechanism from the available experimental data? (ii) Can the number of open parameters needed to fit the experimental ESP patterns be sufficiently reduced, e.g., by relying on independently determined structural and coupling parameters, to deduce conformational or functional information about active reaction centers? In this paper, we argue that given best time and spectral resolution presently available (at K-band (24 GHz) or higher frequencies) both questions are likely to be answered conclusively.
2. Kinetics and ESP Mechanism We first review the relevant information available concerning the nature and properties of the specific radical ions generated by the light-induced primary charge separation and detectable by EPR. Essential information comes from kinetics and more recently from the molecular structure of specific bacterial reaction centers (RCs) . I 4 - l 6 ( 1 ) Hoff, A . J . Q. Reu. Biophys. 1984, 7 , 153. (2) Furrer, R.; Thurnauer, M . C. FEBS Letr. 1983, 153, 399. ( 3 ) Gast, P.; Swarthoff, T.; EbskamD. F. C. R.: Hoff. A. J. Biochim. Biophys. Acta 1983, 722, 163. Gast, P. Thesis, University of Leiden, 1983. (4) Thurnauer, M. C.; Gast, P. Photobiochem. Photobiophys. 1985,9, 29. ( 5 ) (a) Gast; P.; Thurnauer, M . C.; Petersen, J.; Stehlik, D. Photosynth. Res. 1987, 14, 15. (b) Petersen, J. Thesis, Free University Berlin, 1986. (6) Thurnauer, M. C.; Gast, P.; Petersen, J.; Stehlik, D. Prog. Photosynth. Res. 1987, I , 237. (7) Stehlik, D.; Petersen, J.; Thurnauer, M. C. Proc. Congr. Amp. Roma 1986, 316. ( 8 ) Hore, P. J.; Watson, E. T.; Petersen, J . B.; Hoff, A. J. Biochim. Biophys. Acta 1986, 849, IO. (9) Broadhurst, R. W.; Hoff, A. J.; Hore, P. J . Biochim. Biophys. Acra 1986,852, 106. (IO) Buckley, C. D.; Hunter, D. A.; Hore, P. J.; McLauchlan, K. A. Chem. Phys. Lett. 1987, 135, 307. ( I I ) Closs, G. L.; Forbes, M . D. E.; Norris, J. R. J . Phys. Chem. 1987. 91, 3592. (12) Hore, P. J.; Hunter, D. A.; McKie, C. D.; Hoff, A . J. Chem. Phys. Lett. 1987, 137, 495. ( 1 3 ) C~OSS, G. L.: Forbes, M. D. E. J . A m . Chem. SOC.1987, 109, 6185.
@ 1989 American Chemical Society
The Journal of Physical Chemistry, Vol. 93, No. 4, 1989
ESP of Photosynthetic Reaction Centers
TABLE I: Dipolar Interaction Parameters in BRC Updated from Ref 22 for the P+I- and P+Q- Radical Pairs directional cosines of zd Z pair D," mT X Y polar coord. angles a,@ P+I-0.521 (E N 0) 0.5989 0.6612 0.438 1 .01. = 41.7'. . ar. = 64' P+Q-
( E N 0)
-0.119
a D(P'I-)/D(P+Q-)
0.8646
0.4171
> J ' / 2 d ( E ) , Le., the larger spectral separation is governed by the g shift between the radicals, i.e., R q. The sign of the smaller energy splitting is assumed to be J - d(() > 0. For negligible J coupling and D < 0 as expected for a pair of distant spins, J - d(() -d([) > 0 is fulfilled for orientations with angle f < 54'. For the known relation of the isotropic g factors gp < gQ and an initial singlet population the polarized stick spectrum is obtained as shown in Scheme 11. Note that as in the usual EPR spectrum the energy scale is replaced by a magnetic field sweep at constant microwave frequency. Emissive (E) signals are shown with negative and absorptive (A) signals with positive intensity. One important point omitted in setting up the populations in Table I1 is the time evolution of the eigenstates in the P'Q- pair. In fact, starting from an initial singlet population the spin pair is generated in a coherent superposition of eigenstates, and the time development of the full density matrix has to be considered. For the present purpose we concentrate on the diagonal elements, although coherent phenomena associated with the offdiagonal elements may provide useful additional information. The time development of triplet character in the eigenstates 2 and 3 (Scheme I) will be proportional to
+
-
Hz). Singlet-triplet coupling is then limited to the middle triplet Zeeman level To, while the outer Zeeman levels T, and T- remain eigenstates. The spin-spin interactions in the coupled radical pair add the following energies to the singlet and triplet states: Es = +J
g,P
-
state 2:
1 - ( q 2 / Q 2sin2 ) at
state 3:
( q 2 / Q 2sin2 ) Rt
(9a)
Only if the frequency Q involved in the singlet-triplet mixing is large compared to the inverse lifetime and the EPR observation time are we allowed to replace sin2 Qt by its time average, being as assumed in setting up Table 11. On the other hand, the observation of initial oscillatory behavior as given in eq 9 would represent an excellent criterion for the ESP mechanism outlined here. Since the P+Q- pair is not generated directly from the P* singlet state but via the P+I- intermediate, the singlet precursor character might be admixed with some triplet character. However, the corresponding mixing frequency (P'I-) will be small compared to the inverse P+I- lifetime (200 ps)-I in the untreated RCs. Therefore, any triplet admixture can be considered negligible when the P+Q- pair is generated. 6. Conventional CIDEP-Type ESP Scheme. Finally, we consider the polarization pattern expected from the conventional CIDEP scheme.@ As outlined before this may be observable when the electron transfer has proceeded to an acceptor A,, with n > 1, where the radical ions P+ and A; are not longer coupled. The polarization will result from the pair where S-T mixing occurs predominantly. As argued before, this will be the P'Q- pair if subsequent pairs can be considered uncoupled. Note that this would not be the case for BRCs because the electron transfer QA Qe takes place parallel to the membrane into a site related by rotation around the overall C2 axis.16 Hence, the dipolar coupling between P+ and QB- remains unchanged compared to that of the P+QA-pair. In PS I the electron is expected to move from the P+700A,- pair to ironsulfur centers. It is not yet known whether these are sufficiently distant from the P+700center to be considered magnetically uncoupled. If this is the case, the well-known ESP
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ESP of Photosynthetic Reaction Centers
The Journal of Physical Chemistry, Vol. 93, No. 4, 1989 1615
line-shape functions1Vs become applicable. The ESP-generating pair will now be the P+A1- instead of the P+Ao- pair and EPR transitions are observed after the P+AI- decay in the uncoupled radicals P+ and A,,-. The ESP will be determined by the same spin-density matrix used to set up eq 9, but the observables will now be the individual Zeeman magnetizations of P+ and A; given by the expectation value of the respective S, operators. For the typical situation of a field sweep mode at constant microwave frequency the ESP line-shape function is given as a function of the magnetic field
EPR signal
~ : 1 3
+
P ( B ) = CAEsT[(Bpo- BAlo)Lp+(B) ( B - Bpo)Lp+(B)(BPo - BA,o)LA,(B)l (10) This first and second term represent the so called "net" and "multiplet" effect of P+. Only the "net" effect of AI- remains after electron transfer to A,,- and is given in the third term. Lp+(B)and LA"-(@are the EPR line-shape functions of the corresponding radical ions. B: = hvMw/(Mggj) is the resonance field of the radical ion j = P or A, at a given microwave frequency vMW. The proportionality constant C i s positive in case of a singlet precursor to the ESP-generating radical pair (here P+A,-). From eq 3 we obtain
q3 a 0J + (3 cos2 ([ - 1)) The sign of the ESP is readily evaluated. For a singlet precursor we get for the last term in (10) sgn PA,(B) = sgn
AEST
sgn (BA,' - Bpo)
(12)
-
We can illustrate the sign analysis for the same conditions used to construct the stick spectrum of Scheme 11. We choose J 0, D < 0, and an angle between magnetic field and dipolar axis zd, [ < 54'. According to eq 11 we get AEST d ( t ) < 0. With gp < gAl, i.e., (BA,O - B t ) < 0, eq 12 renders positive sign, Le., absorptive (A) polarization for A; and correspondingly emissive (E) net polarization for P+. 7. Powder Average. Although we will be able to assume that the individual reaction centers have fixed orientations with respect to the magnetic field during the detection time, their orientations in the whole sample will have a statistical distribution unless oriented samples are used. In the former case the obtained ESP patterns of Scheme I1 or eq 10 will have to be averaged over all orientations. This can be done following standard "powder-pattern" procedures. Here one is free to use a suitable reference frame of axes to which the other anisotropic interactions will have to be transformed. When the principal axis system of the tensorjQ is taken as reference system, the vectors of the magnetic field B and of the dipolar axis may be characterized by polar coordinates 0, p and aQ,/3Q,respectively. Convenient analytic expressions exist for powder averages as a function of these polar coordinates (see, e.g., ref 1, 8, and 9).
-
861 I
Figure 1. PS I transient EPR data set including the full spectral and kinetic information at room temperature. Freeze-dried perdeuteriated Synechococcus lividus algae as in ref 2 were dissolved in D20with a concentration of 7 X lo-' M Chl. The time scale runs from 1 ps before to 4 ps after the laser flash (Nd-YAG laser, second harmonic at 532 nm, width 6 ns, repetition rate 10 Hz); magnetic field scan range 4 mT; microwave frequency 24.234 GHz; microwave power 2 mW. Each transient at one of 161 field positions spaced 0.025 mT represents an average of 1024 events. Transient sampling rate 100 MS/s. In this figure the information is condensed so that the lines are spaced 0.08 mT and 100 ns, respectively. Note the EPR signal is monitored in direct detection perpendicular to the phase of the BI microwave field. Positive sign represents absorptive (A) and negative sign emissive (E) signals. TABLE III: Parameter Set Needed in ESP Pattern Simulation physical meaning notation no.
b tensor P+
2 tensor Q-
gxm gyy,
line width P', Qexchange integral dipolar coupling scaling factor
J D ( E assumed to be 0)
2 3 up to 4 2 1 1
C
1
gll, g,
gz,
and tensor orientations AB(P+);AB(Q-)
resolution up to several microseconds as limited by the spin relaxation time, a single ESP pattern is observed. Apparent spectral changes at early times are due to initial broadening associated with fast decaying off-resonance contribution^.^^^^^ (ii) The ESP pattern stays essentially unchanged when observed at low temperature in frozen s o l ~ t i o n .Therefore, ~ the room-temperature pattern also represents a rigid powder pattern. (iii) The latter conclusion is confirmed by the observation of transient spin nutations with high microwave power levels at room t e m p e r a t ~ r e . ~ ~ (iv) No Boltzmann signal or other spectra (such as in ref 35) can be observed at longer times. Therefore, with the given signalto-noise level the initial polarization is very high. In accordance with the arguments given in section 2 the observed ESP pattern can be assigned only to direct EPR transitions in the P+AI- pair, even though no oscillatory behavior due to the frequency 0 (eq 7) has been observed. With the anisotropy of the g tensors 0 would have a wide distribution of values, with the 3. Interpretation of Experimental ESP Patterns averge expected to be 2 lo8 s-l. Only oriented samples would offer a certain chance to detect Q oscillations at the present time 1. Experimental ESP Pattern of PS I . Most transient EPR resalution. spectra of photosynthetic reaction centers have been obtained in 2. Independent Information on Magnetic Interaction Paramthe X band (9 GHz). As anisotropic interactions are realized to eters. To distinguish different ESP mechanisms, we have to keep be important, higher spectral resolution is essential. The most in mind that all recent simulations of ESP patterns8p9J2render resolved transient EPR spectra have been observed for PS I of deuteriated whole algae (S.lividus) in the K band (24 G H Z ) . ~ , ~ , ~ acceptable results in spite of the fact that very different and incompatible parameter sets have been used. The problem resides They agree well with those obtained in the Q band (35 GHz) with in the limited experimental information compared to the large light m ~ d u l a t i o n . ~ Recently, we repeated the early transient number of unknown parameters. We will adopt a more cautious K-band spectra with the same freeze-dried algae material used strategy and analyze only the effect of specific parameter variations in ref 2 but with considerably improved time resolution and on the simulation result rather than aim at a final fit. Furthersensitivity. A typical result is presented in Figure 1 with a plot more, as independent experimental information on more and more against both the magnetic field and time axis. A detailed description of the experimental results is given el~ewhere.'~ Essential conclusions from these results are as follows: (i) In (28) Furrer, R.; Fujara, F.; Lange, C.; Stehlik, D.; Vieth, H.-M.; Vollthe whole time range from 50 ns, Le., the instrumental time mann, W. Chem. Phys. Let?. 1980, 75, 332.
1616 The Journal of Physical Chemistry, Vol. 93, No. 4, 1989
parameters becomes available, simulation with a strongly reduced number of open parameters will be more promising. The parameters needed in ESP pattern simulations are collected in Table 111. We summarize the independent information on these parameters and comment on their relevance. Even though the identification of A, as a quinone molecule is not fully confirmed yet, compiling e v i d e n ~ e ~gives ~ J ~support 9 ~ ~ to this working hypothesis adopted here. The principal values of the 2 tensor are well e ~ t a b l i s h e d ~ ”for ~ ’ Q- in BRCs: g,, = 2.0067; gyy= 2.0056; g,, = 2.0024 (13) The x,y,z axes have been found to coincide with the long and short (along the C O bond) in-plane and the out-of-plane axes, res p e ~ t i v e l y .From ~ ~ (1 3) we obtain g, = 2.0049. g,,,, however, has been shown to vary significantly between different quinone molecules.32 A variation of the order of 10 units in the last decimal place is quite conceivable. A crude evaluation of the principal g values from the downfield E and A feature in Figure 1 would indicate such a shift to smaller g values as compared to (13). However, it remains open whether these g shifts are the consequence of a reduced g anisotropy or of an overall shift of gi,. Initially an isotropic g(P+) = 2.0026 is assumed for the donor. The line width of the EPR signal P+7wfor deuteriated S.lividus algae has been determined to be AB(P+) = 0.27 mT.5b For protonated algae the width is 0.79 mT, in both cases with a nearly Gaussian line shape. The line width of the individual spin packets in the Q- powder spectrum is compatible with a value of 0.20 mT for deuteriated quin0ne.j A wide range of parameters J and D for the magnetic interactions between the different radical ions in RCs exists in the literature. With the advent of the BRC structure the situation has changed, and electron-transfer matrix e l e m e n t ~ and ’ ~ the dipolar t e n ~ o rcan ~ ~be, ~calculated ~ rather accurately. For the P+Q- pair the exchange integral J(P+Q-) is probably very small, while the dipolar tensor parameters have been collected in Table I. Again the application to PS I is questionable but certainly provides the best guess. Summarizing, we can state that for nearly all parameters listed in Table 111 some independent experimental information is available. Therefore, variation is meaningful only in restricted ranges. Parameters of particular interest are the respective orientation of the g tensor principal axes with respect to the dipolar axis zd(P+Q-),which can be considered as a fixed axis representing the overall RC structure. 3. Simulation of ESP Patterns. Starting with the parameters just discussed, we now investigate how specific parameter variations influence the ESP patterns simulated with powder averages of basic stick spectra as given in Scheme I1 and Table 11. Comparison is done with the experimental spectrum (Figure 1). We emphasize once more that the same spectrum is seen starting within the present instrumental time constant of -50 ns extending to several microseconds as limited by the room-temperature spin-lattice relaxation. The experimental spectrum after the fast decay of initial broadening effects ( t 2 100 ns) is shown in Figure 2a. Given the narrow spectral range of the P+ signal, the low-field E/A part of the spectrum must be exclusively due to the Q- spin. The positions of these E and A features are better consistent with the following principal values: g,, = 2.0061; gVv= 2.00485; g,, = 2.0027 (14) which are close to the values given in (1 3) for the BRC quinone. The reduction in g anisotropy or the corresponding change of the isotropic g value falls within the range known for quinone radical~.~~ The effect of different orientations of the Q- 2 tensor principal axes, Le., the Q- molecular axes, with respect to the dipolar axis (29) Okamura, M . Y . ; Feher, G. Biochim. Biophys. Acra 1972, 267, 222. (30) Hales, B. F. J . Am. Chem. Sac. 1975, 97, 5993. (31) Cast, P.; De Groot, A.; Hoff, A. J. Biochim. Biophys. Acra 1984, 723, 52. ( 3 2 ) CRC Handbook of EPR-Spectra of Quinones and Quinols; Ed. Petersen, J . A,, Ed.; CRC Press: Boca Raton, FL, 1985.
Stehlik et al.
A
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1
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A
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861.7
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863.7
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865.7
B,/mT
Figure 2. (a) Experimental EPR spectrum extracted from the full data set of Figure 1. The curve shows the average within the digital time window from 200 to 250 ns after the laser flash. (b) Simulationsof the spectrum with the dipolar interaction vector z d parallel to the three principal axes of the tensor gQ. aQand @Q are the polar coordinate angles . QQ = 90°, @Q = 0’. Le., of zd in the principal axis system of g ~ Top: zD parallel to &?Qui center: CIQ = 90°, @Q = 90°,Le., z D parallel to gQyy; bottom: aQ = Oo, PQ = Oo, i.e., z d parallel to gQ,z.The g factors used are indicated at the top of the simulated spectra. gQ, = 2.0061, gQ = 2.00485; gQs = 2.0027, gp = 2.0026, J = 0 mT, D = -0.12 mT: !:ne width AB(Pi) = 0.27 mT, AB(Q-) = 0.20 mT. I
I 861 7
I
I
I
I
I
I
863 7
I
I 865 7
Bo/mT Figure 3. Separation of the contributions of two groups of transitions in Scheme I1 and Table 11: 24, 13 (assigned to Q-) and 34, 12 (assigned to P+) for the spectrum of Figure 2b (top) ( z d parallel tog&). Solid line: P+, AB(P+) = 0.27 mT; dotted line: Q-, AB(Q-) = 0.20 mT.
zd is demonstrated in Figure 2b. Obviously, only an orientation close to zdllg, is compatible with the experimental spectrum. Note that according to the BRC structure (see Table I) zd(P+Q-) has the smallest angle (30’) with the x axis of the Q molecule. The sign of the ESP pattern is essentially determined by the indisputable fact of a singlet precursor state and the negative sign of the P+Q- dipolar coupling parameter according to Table I. Figure 3 shows the separate contributions of the transition pairs (Scheme I1 and Table 11) 24, 13 (mainly Q-) and 34, 12 (mainly P+) for the spectrum of Figure 2b (zdIlgx,). The structure in the P+ part results from the dipolar coupling. It shows up more clearly if the Gaussian line width of P+ is reduced. With the experimental line width AB(P+) = 0.27 mT this structure is wiped out to a large extent, because the line width becomes larger than the dipolar coupling D. From further simulations we have to conclude that with the dipolar coupling and line-width parameters fixed as taken from independent experimental information, the high-field part of the
The Journal of Physical Chemistry, Vol. 93, No. 4, 1989 1617
ESP of Photosynthetic Reaction Centers I
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863.1
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865.7
861.7
BolmT
863.7
865.7
BohT
Figure 4. (a) Introduction of anisotropy in the tensor & gp,, i =
=
g, = 2.00285;gp, = gll = 2.0022.Dotted line: ap = 00, i.e. zd p 2 h e I to gil;dashed line: a p = 90°,i.e., zd perpendicular to gll; solid line: a p = 45'. a pis the polar angle of the vector zd in the gpcoordinate system.
Since k p is axially symmetric, variation of the azimuthal angle flp is not needed. The ordinate of this figure is compressed by a factor of 2. All other parameters as in Figure 2b (top) (zd parallel to g e ) . (b) Solid line: a p = 45'; dotted line: a p = 35'; dashed line: a p = 5'.
Figure 5. (a) Variation of the exchange coupling J. Solid line J = 0; dotted line J = 0.005 mT; dashed line J = -0.005 mT. All other parameters are as in Figure 4 solid line. Note the pattern in the high-field part is very sensitive to even small values of J. Arrows concern remarks in text. (b)Variation of the dipolar coupling D. Solid line D = -0.12 mT; dotted line D = -0.18 mT; dashed line D = -0.08 mT. All other parameters are as in Figure 4 solid line.
experimental spectrum (Figure 2a) cannot be simulated by variation of the g,,(Q-) component or of the orientation of the tensor of Q- with respect to zd while keeping an isotropic g(P+) = 2.0026. 4 . Possible Role of g Anisotropy of P+. Experimental data on the g anisotropy of P+are rather scarce. A small but significant anisotropy has been d e m o n ~ t r a t e din~ ~single crystals of BRCs ( R . sphaeroides) by using Q-band EPR (35 GHz). It was not possible to distinguish the different RCs in the unit cell. Critical model assumptions had to be used to infer the following parameters for the axially symmetric tensor 3 P ) : g, = 2.00285
gll = 2.00220
(15)
g,,denotes the magnetic field orientation parallel to the out-of-plane axis of the chlorophyl constituents of the donor P. On general grounds gl,will be the smaller component close to the free-electron g value. Anisotropic contributions due to spin-orbit interaction are restricted to g,. Figure 4 presents simulations with the g(P) anisotropy given in ( 1 5 ) . With respect to the b(Q) tensor the orientation of the dipolar axis zd was kept fixed at zd/lgxx. The orientations of the g(P) tensor axes with respect to zd were varied as given with the polar angle a p = f(gil,Zd). Note that the relative orientation of the tensors g(P) and g(Q) varied accordingly. As'seen in Figure 4a the relatively small g(P) anisotropy gives rise to much larger contributions in the P+ range. For the orientation a p = Oo (dotted line) the upfield part becomes already dominant compared with the downfield part in contrast to the experimental spectrum (Figure 2a). For intermediate orientations (Figure 4b) reasonable simulations are obtained. As expected, a sign change occurs in the upfield part when a ppasses through the region of the magic angle (-54'). The correct sign of the polarization pattern requires orientations 0 < a p C 54O. At this point it is interesting to compare this again with the BRC structure.'"I6 gll(P+)is expected to be oriented parallel to the membrane plane or perpendicular to the overall C, symmetry axis of the reaction center. In contrast the dipolar axis zd is predicted to be closer along the c2axis with L(Zd,CZ) 30' (see Table I). zd is also closest to the g,,(Q) component direction. Hence, from this analogy we expect that within *30° we have gJQ) 11 zd Ig,,(P). However, for zd Igll(P)or cyp = 90' Figure 4a gives the wrong sign of the ESP pattern in the P+ range. On the other hand, a p 45O is in surprisingly good agreement with the experimental spectrum (Figure 2a) and still compatible with
-
-
(33)
Allen, J . P.; Feher, G . Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 4795.
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B,lmT
865.7
Figure 6. (a) Variation of the polar angle aQaway from i d parallel to dotted line LYQ = 70'; dashed line CYQ = 50'. All other parameters and as in Figure 4 solid line. (b)Variation of the azimuthal angle fiQ away from z d parallel to gQxx.Solid line flQ = 0'; dotted line PQ = 20'; dashed line f i =~ 40'. All other parameters are as in Figure 4 solid line.
gQxx.Solid line aQ = 90';
a RC structure analogous to that of BRC. It is important to note once more that the good agreement between experimental and simulated spectra is obtained with parameters inferred from independent experimental information rather than with fitting a number of open variables. For completion we present simulations in Figures 5 and 6 where other interaction parameters (see Table 111) are varied around those values taken for the solid curves in Figure 4. When small values of the exchange coupling constant J are permitted (Figure 5a) only the spectral contributions in the P+ range are affected, yet very sensitively. Note that the relative amplitudes of the two upfield absorptive signals (see arrows in Figure 5a) are influenced by the sign of J. Variation of the dipolar coupling constant D (Figure 5b) seems to affect the amplitude and not the overall shape of the ESP spectrum. Rather large variations of D are compatible with the experimental result. On the other hand, the value of D obtained from the analogy to the BRC structure seems particularly reliable. Generally, one can expect that both the dipolar axis zd and the glldirection are rather fixed axes within the R C structure. In
1618 The Journal of Physical Chemistry, Vol. 93, No. 4, 1989 I
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335.2
337.2 8, ImT Figure 7. (a) Experimental spectrum taken at the X band. Microwave frequency 9.400 GHz, microwave power 20 mW, time window 450-500 ns after laser excitation, scan range 4 mT. (b) Simulation with the same parameters as used in Figure 4 solid line except for the appropriate frequency and magnetic field range. (c) Separation of P+ and Q- con-
tributions. contrast, the quinone acceptor may reorient with the transition to the charge-separated state P+Q- as compared to the ground state. The same angles aQ and PQ introduced before may be used to describe the various orientations of the g(Q) tensor with respect to the fixed dipolar axis zd. Figure 6 presents simulations where the orientation of zd and the g(P) tensor axes are kept constant but the g(Q) principal axes are varied with respect to zd. Significant changes of the ESP pattern are seen only if the orientation departs by more than f20° from the chosen central value zd 11 gxx(Q). 5. Comparison with Other Experimental ESP Patterns. Figure 7a presents the X-band spectrum (9 GHz) corresponding to the data shown in Figure 2a for the K band (24 GHz). Also shown in Figure 7 are the simulations using the parameter sets of Figure 4 (solid lines) except for the different magnetic field range. Clearly, the reduced g anisotropy effect in the X-band is responsible for the simpler E/A/E overall pattern. Note the additional absorptive (A) feature on the upfield part of the spectrum, which is well resolved at the K-band (Figure 2a) but often hard to observe in the X-band with an insufficient signal-to-noise ratio. We want to emphasize that this X-band pattern has been observed in virtually all early-transient EPR spectra investigated so far and found to be common to a variety of PS I reaction centers as well as BRCs with the non-heme iron removed or decoupled (for a review see, e.g., ref 1). Due to increased hyperfine coupling, ESP patterns of protonated whole algae and PS I particles exhibit a broader but similar E/A/E pattern, which remains unresolved even at the K-band (24 GHz) (see Figure 3 of ref sa). The spectrum of iron depleted BRC of R26 has been found to be virtually undistinguishable from those of PS I particles. To this admittedly fairly low level of detail we take this observation as a justification for our use of structural information, so far available only for BRC, to guide the simulation of the ESP pattern in PS I. Hopefully, ESP patterns of deuteriated BRCs with improved spectral resolution in the K or Q band will soon be avaiable and allow more direct tests. A particular property of the presented interpretation is the anisotropic character of the ESP pattern, implying a strong orientation dependence if the magnetic field orientation is changed
Stehlik et al. with respect to a fixed axis system in the RC structure. Indeed, strong orientation dependence has been reported by McCracken and S a ~ e for r ~chloroplast ~ preparations dried on Mylar film. Although the spectra are taken with time resolution 2 2 ks and are expected to contain not only the pure P+A,- state the characteristic E/A/E pattern is observed for random orientation. A clearcut result in their strong orientation dependence is a pronounced E feature at the downfield end of the spectrum when Bo is parallel to the membrane normal and this feature is absent for Bo perpendicular. With respect to the BRC structure (Table I) the orientation Bo parallel means Bo aligned close to zd(P+Q-) as well as g,,(Q). As we have seen, only for this orientation the ESP pattern starts with an E feature on the downfield side (see Figure 2b) but with A for all other orientations. Therefore, this orientation dependence34is compatible with our interpretation. Orientation-dependent studies with better spectral resolution are in progress to confirm these preliminary conclusions. The simulations presented in this paper rely heavily on the better spectral resolution obtained in deuteriated material. So far only freeze-dried whole algae S. liuidus have been studied at higher than X-band frequencies. At lower spectral resolution several transient EPR studies are published for a variety of species and PS I preparations (in part reviewed in ref 1). Additional ESP signals have been seen at longer times which gain importance in view of the new interpretation scheme. Specifically we refer to data obtained on whole cells of different photosynthetic algae.35 While the typical ESP pattern discussed here and assigned to transitions in the coupled P+A,- pair (E/A/E pattern; see Figure 7) is observed at early times, another E/A pattern confined to the region of the P+ signal dominates at later times, but it was unobservable with our perdeuteriated algae of S. liuidus. We have also seen this later signal in chloroplast sample^.^' So far this signal has usually been interpreted as a “multiplet” effect seen on P+ resulting from the singlet-triplet mixing in the short-lived primary radical pair P+Ao-.’ We would like to emphasize once more that this is excluded if EPR detection occurs during the lifetime of a coupled spin pair as expected from the kinetics of the charge separation, (1). As a consequence a radically different approach to the interpretation of this additional ESP pattern appears necessary. We suggest the following possibility based again on the notion that EPR detection occurs within a coupled spin pair. If P+ is still coupled to a later acceptor subsequent to A , , presumably an iron-sulfur center with a large g anisotropy (for a review see ref 17), then EPR transitions within this pair are likely to be observed only within the narrow range of the P+ signal as all other transitions are spread over a wide spectral range. Furthermore, the large range of principal values of FeS centers between 2.07 and 1.86 (ref 17) restricts efficient singlet-triplet admixtures to specific orientations within the random powder pattern. Only the P+ powder pattern will contribute significantly to the observable spectrum. Simulations of the observed E/A pattern with this concept are presented el~ewhere.~’ Similar E/A patterns limited to the P+ spectral region have been observed in bacterial RCs after replacement of the quinone acceptor associated with a lengthening of the P’I- lifetime.36 Again the kinetics permits only detection in the P’( FeQ)- pair, which constitutes a coupled pair according to the BRC structure. The ESP concept suggested above provides an alternative interpretation of this signal consistent with the independent kinetic and structural information available. For completion we note that this additional E/A type ESP spectrum can also be due to the conventional CIDEP related mechanism (section 2.6). However, the condition for this would be that during the observation of this E/A pattern in the P+ range (34) McCracken, J. L.; Sauer, K. Biochem. Biophys. Aria 1983, 724, 83. McCracken, J. L. Thesis, University of California, Berkeley, 1983. (35) (a) Manikowski, H.; McIntosh, A. R.; Bolton, J. R. Biochim. Biophys. Acta 1984, 765, 68; (b) Proc. Congr. Amp. Roma 1986, 542; (c) Proc. 3rd Inf. School, Ustron, Poland 1987. (36) Gunner, M. R.; Robertson, D. E.; LoBrutto, R. L.; McLaughlin, A , C.; Dutton. P.L. Prog. Photosynrh. Res. 1987, 1. 2, 217.
ESP of Photosynthetic Reaction Centers P+ is not coupled anymore to the acceptor carrying the transferred electron.
4. Discussion and Conclusions We briefly summarize the main line of reasoning. The first stable charge-separated state of photosystem I in untreated active centers under physiological conditions (room temperature) renders a polarized EPR spectrum. It is observable by transient EPR in the time range from a few 10 ns up to several microseconds. Within this time window as a consequence of the well-established kinetics, the transferred electron resides initially at the first stable acceptor A,, assumed to be a quinone type molecule. This corresponds to the established quinone nature of the respective acceptor in bacterial and photosystem I1 RCs. On the basis of the known BRC structurel4-I6 and thus the known spin dipolar coupling (Table I) the charge-separated state P+860QA-constitutes a coupled spin pair with correlated spins due to their origin from the precursor singlet state P*860. From the independent kinetic and structural information it is mandatory that only the correlated spin pair mechanism (section 3.2) is appropriate to explain the transient EPR signals observed in BRCs. This conclusion with respect to the ESP mechanism remains valid, although EPR observation occurs after removal of the non-heme iron because neither the kinetics15 nor the relevant structural details are expected to change significantly. While there is no significant difference in the electron-transfer kinetics of PS I and BRC, the analogy to the known structure of BRCs is a pure working hypothesis so far. However, on the basis of this analogy the charge-separated state P'700AI- in PS I has to be considered as a correlated coupled spin pair as well. In fact, the appropriate ESP mechanism (section 2.5) renders reasonable simulations of the observed ESP patterns. Furthermore, it is satisfying that the resulting orientation of the AI quinone acceptor with respect to the reaction center structure turns out to be close to that of the QA site in the BRC structure. In this context we like to point out that the basic structural feature considered here to be analogous between PS I and BRC is hardly critical. It reduces essentially to the following cross feature which is not expected to vary considerably between different reaction centers: The donor Pa60 (BRC) or PTo0(PS I) is located on the periplasmic (inner) side of the membrane while the acceptor QA or A I is on the cytoplasmic (outer) side. As a consequence the vector connecting the two is approximately perpendicular to the membrane plane and represents the dipolar zd of the predominant interaction in the coupled spin pair. It seems quite reasonable that this specific feature is common to PS I and BRC even if the structures of the respective reaction centers turn out to be significantly different. In conclusion the correlated spin pair ESP mechanism is mainly responsible for the observed polarized transient EPR spectra because alternative ESP schemes employed earlier are not consistent with the independent kinetic and structural information. In addition, the correlated pair mechanism renders considerably better simulations of the more resolved transient EPR spectra of deuteriated material at higher microwave frequencies.
The Journal of Physical Chemistry, Vol. 93, No. 4, 1989 1619 Within this ESP mechanism the experimental EPR spectra are most sensitive to the orientation of the A I quinone acceptor. Its long molecular axis (in-plane, perpendicular to the C O bond) must be aligned within less than 20° along the dipolar axis zd(P+,dI-). It should be noted that such an orientation is close to that obtained for QA in BRC for the ground state. Future EPR experiments on BRCs with the resolution available for PS I in Figure 1 or better, taking into account the refined ground-state structure, will allow to determine the QA reorientation in the charge-separated state to rather high accuracy and to improve preliminary conclusions drawn from optical experiment^.^^ Finally, we want to remark that the transient EPR spectra of photosynthetic reaction centers represent the first case of correlated spin pairs studied in a rigid structure, i.e., retaining the effects due to the anisotropic spin interactions, which in turn permit the evaluation of relevant structural information already demonstrated in ~ o l u t i o n Therefore, . ~ ~ ~ ~ the ~ ~experimental ~ and interpretative methods applied here to photosynthetic reaction centers are expected to be useful to study transient spin pairs generated in frozen solution, polymers, or even crystals as long as their lifetime is sufficiently long, i.e., longer than IO-* s.
Note Added in Proof. As one referee pointed out, the D values may be estimated starting from eq 8 of ref 38: D / g p = 3/gp(r' - 3 z 2 / r 5 )
-
-1.Sg/3/r3
-
which simplifies for distant point charges due to z r. With r = 2.84 nm this yields D / g p = -0.12 mT, in agreement with the corrected value in Table 1. Note that all D values given in ref 22 have to be scaled by the same factor of about 2, as done in ref 39. As demonstrated in Figure 5b the ESP pattern is not altered significantly by the absolute value of D; mainly its overall amplitude is affected, at least within the range of D values relevant here. As a consequence the ESP pattern is not very sensitive to the distance between P+ and Q-, instead far more sensitive to the orientation between the respective 2 tensor axes and the dipolar axis zd.
Acknowledgment. We thank Marion C. Thurnauer and her colleagues at Argonne National Laboratory for fruitful collaboration. Encouragement, valuable discussions and continuous advice contributed substantially to this work. The deuteriated whole algae Synechococcus liuidus were prepared and supplied by H. Crespi and H. DaBoll (Argonne). We are grateful to A. Ogrodnik (Munich) for the data listed in Table I based on recent atomic coordinates. M. Plato helped with programs and computational support. A. J. van der Est contributed to the final simulations. The work was supported by the Deutsche Forschungsgemeinschaft (Sfb 312). (37) Kleinfeld, D.; Okamura, M. Y.; Feher, G. Biochemistry 1984, 23, 5780. ( 3 8 ) Carrington, A.; McLachlan, A. D. Introduction t o Magnetic Resonance; Harper & Row: London, New York, 1967; pp 117f. (39) Lersch, W.; Lendzian, F.; Lang, E.; Feick, R.; Mobius, K.; MichelBeyerle, M. E. J . Magn. Reson., in press.
