8266
J. Phys. Chem. B 1998, 102, 8266-8277
•The Radical Pair State P•+ 700A1 in Photosystem I Single Crystals: Orientation Dependence of the Transient Spin-Polarized EPR Spectra
Andreas Kamlowski,†,‡ Stephan G. Zech,§ Petra Fromme,§ Robert Bittl,*,§ Wolfgang Lubitz,§ Horst T. Witt,§ and Dietmar Stehlik*,† Institut fu¨ r Experimentalphysik, Freie UniVersita¨ t Berlin, Arnimallee 14, 14195 Berlin, Germany, and Max-Volmer-Institut fu¨ r Biophysikalische Chemie und Biochemie, Technische UniVersita¨ t Berlin, Strasse des 17 Juni 135, 10623 Berlin, Germany ReceiVed: March 31, 1998; In Final Form: July 10, 1998
•The light-induced, charge-separated state P•+ 700A1 in single crystals of Photosystem I (PSI) from the cyanobacterium Synechococcus elongatus is investigated with transient, direct-detection EPR spectroscopy. The orientation of the phylloquinone head group of A1 within the PSI reaction center is determined from the •orientation dependence of the spin-polarized X-band EPR spectrum of the radical pair P•+ 700A1 made up of the primary donor, P700, and the acceptor, A1. From the angular dependence of the overall spin-polarization pattern an upper limit ∠(c, zd) e 30° is evaluated for the angle between the crystallographic c-axis, collinear with the membrane normal, and the dipolar axis, zd, connecting the electron spin density centers of P•+ 700 and . A partially resolved hyperfine coupling (hfc) is assigned to the hfc tensor of the 2-methyl group of A1. A•1 Its A| principal axis encloses an angle of β ) 35°-55° with c. Simulations of the rotation patterns support a lower limit for the angle ∠(c, zd) g 25° with a larger error than for the upper limit. zd is confirmed to be parallel to both the gxx principal axis of g(A•1 ) and to the CdO carbonyl bonds within (5°. With respect to rotation around the gxx axis, the angle between the (c, zd) plane and the quinone plane of the A•1 head group can only be specified within an upper limit of 60°. Together with independent knowledge about the location of A1 within the PSI reaction center, a nearly complete structural model for the head group of the functional A1 cofactor is achieved.
1. Introduction In oxygenic photosynthesis, Photosystem I (PSI)1a serves as a light-induced plastocyanin:ferredoxin oxidoreductase, reducing NADP+ (see e.g. refs 1b and 2 for recent reviews on PSI). The electron acceptor side of this membrane-integral pigmentprotein complex contains three terminal iron-sulfur (FeS) centers, FX, FA, and FB, which classify PSI as an FeS-type reaction center (RC) (alternatively Type I RC).3 Preceding the FeS centers, a quinone functions as an electron acceptor called A1.4b It was shown that A1 is equivalent to a protein-bound phylloquinone (vitamin K1 (VK1); see refs 1b, 7, and 8 for reviews) of which two per PSI RC were identified biochemically.9,10 A1 exhibits unusual redox properties as compared with quinones in Vitro or with structurally similar ones in the Type II RC of purple bacteria (bRC). Moreover, the EPR spectroscopic properties, e.g. g-tensor anisotropy and hyperfine couplings are significantly different for A•1 in PSI as compared with both singly reduced VK1 and other semiquinones in Vitro 11-13 All these properties directly reflect and to Q•A in bRC. the important influence of the protein environment as well as the differences of the quinone binding sites in the two types of reaction centers. * Authors to whom correspondence should be addressed. E-mail:
[email protected], fax: +49 30-838-6081. Bittl@ chem.TU-Berlin.DE, fax: +49 30-314-21122. † Freie Universita ¨ t Berlin. ‡ Present address: Institut fu ¨ r Organische Chemie, J. W. Goethe Universita¨t Frankfurt am Main. § Technische Universita ¨ t Berlin.
Considering the limitations of the X-ray data set of PSI at presently 4.0 Å resolution,14 the binding pocket of A1 (or A′1) as well as the position and, even more, the orientation of the A1 (or A′1) head group have not been assigned. More recently, Schubert et al.5 attributed a rather large spherical electron density to the naphthoquinone head group of A1. This assignment was largely based on recent pulsed EPR results for the radical pair •15-17 and single crystals.6 P•+ 700A1 of PS I in frozen solution Both the absolute value, i.e., the distance between the spin •- 15-17 density centers of the radical ions P•+ and 700 and A1 , orientational information of the associated dipolar axis zd with respect to the unit cell axis system of PSI single crystals6 were obtained. However, these data do not provide orientational information on A1 or relative orientations with respect to other cofactors. Transient EPR spectroscopy proved to be most useful for this purpose. In particular, a nearly collinear arrangement of the dipolar axis zd and the CdO axes of the quinone has been established (see ref 11 and earlier work cited therein). Singlecrystal studies provide higher accuracy for such orientational information, and in addition the orientation with respect to the crystal axes. Independent information on the orientation of the CdO axes of the quinone and of the quinone plane with respect to the membrane plane was obtained recently from a CW-EPR study of photoaccumulated A•1 in two-dimensionally ordered PSI preparations.12 The single-crystal data will confirm these orientational data very well. In addition, the agreement provides a useful check of the nontrivial assumption that the information
S1089-5647(98)01702-7 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/26/1998
•Radical Pair State P•+ 700A1
J. Phys. Chem. B, Vol. 102, No. 42, 1998 8267 A•1
obtained on the photoaccumulated state12 is identical to that on the functionally active transient radical pair state •P•+ 700A1 investigated here. Transient EPR results on PSI single crystals are presented here which have been obtained in conjunction with the pulsed EPR experiments reported in ref 6. The broader concept behind such complementary experiments is the determination of the orientation dependence of magnetic interaction properties (e.g. g tensor, hyperfine and interradical ion coupling) of all photoexcitable paramagnetic cofactor states with respect to the crystal axes and with respect to each other. From this the location and orientation of the cofactors and their relative orientations can be derived provided the relation between the magnetic principal and the molecular axis system is known. In the case of the PSI single crystals from Synechococcus elongatus, studies on stationary paramagnetic species published so far include ESEEM and ENDOR spectroscopy of the oxidized 18,19 and CW-EPR on the photoreducible primary donor, P•+ 700, - 20 In the latter case, the iron-sulfur centers FA and FB . determination of the relative orientations of the principal axes of g(FA ) and g(FB ) permitted to obtain the orientation of the protein subunit PsaC, carrying both 4Fe-4S clusters, FA and FB.21 An obvious extension would be the correlation of even more relative tensor orientations. The goal would be to provide a sufficiently complete set of relative tensor orientations to determine the full orientational arrangement of the cofactors including information on the active branch. As a step in this direction, this paper presents relative magnetic tensor orientations of the transient radical ion pair •P•+ 700A1 in relation to the tensor orientation of the iron-sulfur cluster FA , all measured in the same single crystal. From the orientation dependence of both the spin-polarization pattern and a partially resolved hyperfine structure (hfs), the orientation of the A•1 head group is obtained. Combination with independent structural information permits a nearly complete model of A•1 within the PSI reaction center. This model relies primarily on orientational information from intramolecular properties of the A•1 acceptor. In addition, the data contain an appreciable amount of information on the interaction of A•1 with the protein environment, i.e., on the specifics of the A1 binding site. This will be the subject of the second paper22 which combines all available results to conclude on a model for the A1 binding site within the PsaA/PsaB heterodimer protein of the PSI reaction center. 2. Materials and Methods The EPR experiments were carried out on a Bruker ESP380E X-band FT-EPR spectrometer equipped with a Bruker ER4118XMD-5W1-EN dielectric ring resonator placed in a helium flow cryostat (CF 935, Oxford) with temperature control system (ITC4, Oxford). The time-resolved EPR signals were collected in direct-detection mode, by feeding the dc signal from the microwave-detection diode directly into a digital storage oszilloscope (LeCroy 9450) (see e.g. ref 23) controlled by a homewritten acquisition routine. The light source in these experiments was a frequency-doubled Nd:YAG laser (GCR 130, Spectra Physics) working at λ ) 532 nm with a fwhm of about 8 ns. The light intensity incident at the resonator was approximately 3 mJ per pulse. •The two-dimensional EPR data sets for P•+ 700A1 , S(B0, t), were taken at T ) 80 K with a microwave power of 215 µW (-30 dB). For each single-crystal orientation 128 time traces were averaged at 128 field points. Transient EPR spectra were
extracted from the complete data sets by a digital boxcar procedure as described in ref 23; a boxcar window of 1.5 µs width was used with a delay of t ) 0.5 µs after laser pulse excitation at t ) 0 µs. The CW-EPR spectra of the FeS center FA were recorded at T ) 18 K in the dark after laser irradiation during slow cooling from 110 to 18 K. The applied microwave power and amplitude of the magnetic field modulation are given in the respective figure captions (see also ref 20). A resonator background spectrum measured without sample was subtracted in each case. Using laser excitation, only the iron-sulfur center FA is reduced. Unfortunately, FB could not be reduced in detectable amounts by this procedure even at higher irradiation temperatures up to 110 K. Sample rotation was carried out in a home-built goniometer which allows for rotation around an axis, r, perpendicular to the static magnetic field, B0. The actual rotation angle is referred to as R. CW-EPR spectra of the iron-sulfur center FA were recorded every 5° for a total of 200°, whereas in case of the •transient EPR spectra of P•+ 700A1 steps of ∆R ) 10° were used. Trimers of PSI from Synechococcus elongatus were prepared following in principle the method described in ref 24 and 25 and single crystals grown.25 Both procedures were modified as outlined in ref 26 and 27. As in the case of our previous studies,6,20,28 the rod-shaped needle-like single crystal form was used. The crystallographic c-axis is oriented parallel to the long morphological needle axis. The unit cell belongs to the space group P63. It is made up of two trimers, i.e. six PSI monomers, each of which constitutes one asymmetric unit. The crystallographic axes, 3h and 21, as well as the threefold axis, which relates the PSI monomers within each trimer, are all parallel to the crystallographic c-axis. In an EPR experiment, the invariance of the EPR signal to 180° rotations around an axis perpendicular to B0 leads to the effective reduction of the space group P63 to D6 point symmetry. Furthermore, the crystallographic c-axis is collinear with the membrane normal, n.29 Prior to mounting, the single crystals were incubated and soaked with buffer (5 mM MES (pH 6.4), 0.02% β, Ddodecylmaltoside, 2 M sucrose and 10 mM sodium ascorbate) essentially as described in refs 20 and 28. The incubated crystals were mounted either in a notch perpendicular to the long axis of a suprasil quartz rod or in a bore of 1 mm diameter parallel to this rod. Thus, the crystal c-axis is oriented either perpendicular or parallel to the rotation axis r, respectively. After mounting and removal of virtually all buffer, the crystal was shock frozen in liquid N2. The frozen crystal was finally transferred into the precooled He-flow cryostat. 3. Results In this study, laser pulse irradiation permitted the generation of two different paramagnetic states. First, the transient radical ••ion pair P•+ 700A1 . At T ) 80 K, electron transfer beyond A1 is inhibited in some PSI centers and charge recombination from •P•+ 700A1 to the ground state occurs. This excitation cycle takes place in one- to two-thirds of the PSI centers30,31 (see ref 1 for a review). Second, in the remaining PSI centers electron transfer leads to stable charge separation between P•+ 700 and the iron, which can be characterized via their CWsulfur center FA EPR spectra. With this, the orientation of the crystals can be 20,28 First, the calibrated with the known orientation of g(FA ). calibration results with FA will be reported, followed by the orientation dependence of the transient spin-polarized EPR •spectra of P•+ 700A1 .
8268 J. Phys. Chem. B, Vol. 102, No. 42, 1998
Kamlowski et al.
Figure 1. Angular dependence of the CW-EPR spectra of the reduced iron-sulfur center FA in a PSI single crystal with nearly the c-axis as the rotation axis (c || r) (SC-p; see Table 1) from Synechococcus elongatus. Experimental parameters: T ) 18 K; incident microwave power: 6.8 mW (-15 dB); 0.5 mT modulation amplitude at 100 kHz. The field scale of the spectra was accommodated such that all refer to a microwave frequency of 9.76 MHz. Subtraction of a resonator background spectrum created an artifact in the g ) 2 region. Solid lines represent simulation of the orientation dependence for the FA resonance positions as function of the rotation angle, R, with a misalignment between the c-axis and the rotation axis, r, of 5°. For the orientation of the principal g-tensor axis, see Table 1.
3.1. Determination of the Crystal Orientation: Angular Dependence of the EPR Signals of FA . The orientation of each single crystal could be determined accurately from the known angular dependence of the CW-EPR spectra of ironsulfur center FA , since the g-tensor anisotropy of g(FA ) is large compared to the unresolved EPR line width. Already at X-band microwave frequencies the orientation dependence of the EPR lines of FA is well resolved. In Figure 1 the angular dependence of the EPR signals of FA is presented for a PSI single crystal (SC-p) with its crystallographic c-axis nearly parallel to the axis of rotation. If c is collinear with r, only up to three signals should occur for FA. Due to the 21 screw axis, the respective PSI monomers become pairwise magnetically equivalent. Obviously, in case of this single crystal (Figure 1) the desired orientation has been achieved only approximately: in some angular regions more than three (orientation dependent) EPR signals are resolved, indicative of a small misalignment. This has been quantified by simulating the angular dependence based on the published 20,28 In Figure 1 the orientations of the principal axes of g(FA ). solid curves represent a satisfactory simulation. Variation of the angles of misalignment allowed the determination of the angle between r and the c-axis: ∠(c, r) ) 5° ( 3°. Several single crystals were oriented with c perpendicular to the rotation axis, c ⊥ r. CW-EPR spectra of FA are shown in Figure 2 for two examples (crystals: SC-s1 and SC-s2). When c is parallel to the magnetic field, B0, all signals coincide. This
Figure 2. Orientation dependences of the CW-EPR spectra of FA for two single crystals in which the crystallographic c-axis was adjusted in the plane of rotation. (a, top) Single crystal SC-s1 (see Table 1). Microwave power: 22 mW (-10 dB), 0.48 mT modulation amplitude. (b, bottom) Single crystal SC-s2. Microwave power: 86 mW (-4 dB); 0.54 mT modulation amplitude. See caption to Figure 1 for the other experimental details. The solid lines are simulations of the orientation dependence (cf. Figure 1 and Table 1), both with an angle between c and the rotation axis of |∠(c, r) - 90°| ) 0°, but different Euler angles, φ (see Table 1).
particular orientation is chosen to set the experimental rotation angle to R ) 0° (or c || B0). In addition, the rotation patterns exhibit a characteristic mirror symmetry with respect to R ) 90° (and R ) 0°), indicating how well c is oriented within the plane of rotation (see refs 20 and 28 for comparison). Simulations of the FA angular dependencies show that in both cases
•Radical Pair State P•+ 700A1
J. Phys. Chem. B, Vol. 102, No. 42, 1998 8269
TABLE 1: Orientations of the Cartesian Axis System of the Single Crystals Used with Respect to the Laboratory Axis Systema Euler anglesb
single crystal
φ
θ
SC-p SC-s1 SC-s2 SC-s3 SC-s4
0 27 0 15 15
5 90 90 95 90
ψ 30 + R R R R R
remark Figures 1 and 3 Figures 2a and 4a Figures 2b and 4b ref 20 ref 28
Principal Axis System of g(FA ) to Crystal Axis System 47 64 0 for all crystals a The crystallographic c-axis and two axes perpendicular to c span the Cartesian crystal axis system (CAS). The laboratory axis system (LAS) is defined by the magnetic field, B0, the rotation axis, r, and the third axis perpendicular to both B0 and r. b The rotation matrix, REuler, defined by the Euler angles is as follows (see ref 11 for comparison): REuler ) Rz(ψ)Ry(θ)Rz(φ). The angle, R, corresponds to the chosen angle of rotation around the axis, r.
the alignment is quite good, as quantified by an angle between c and r of ∠(c, r) ) 90° ( 3°. Note that the spectra at R ) 90° (c ⊥ B0) are quite different for the two crystals (cf. Figure 2, a and b). These two spectra must occur in the rotation pattern for the crystal orientation c || r (Figure 1). Indeed, they are equivalent to those at R ) -20° + n60° and 10° + n60° (n ) 0, 1, 2, 3). Simulations of the rotation patterns indicate that a rotation around the c-axis by ∆R ) 27° ( 3° + n60° relates the two crystal orientations in Figure 2 with respect to one another. In fact, this is close to the maximum difference possible (30°) in accordance with the symmetry pattern shown in Figure 1. The relative orientations of all PSI single crystals investigated are collected in Table 1. The respective crystal orientations are characterized by Euler angles for the relative orientation of laboratory and crystal axis system (Table 1). [The laboratory axis system (LAS) is defined by the magnetic field, B0, the rotation axis, r, and the third axis perpendicular to both B0 and r. The crystallographic c-axis and two axes perpendicular to c span the Cartesian crystal axis system (CAS).] All orientation information is referred to one common (Cartesian) crystal axis system. With respect to the unit cell crystal axis system, c, a, and b, a calibration angle is left open. This angle corresponds to a rotation around the c-axis common to both axis systems. For the determination of an appropriate calibration angle, see discussion in section 4.2. 3.2. Orientation Dependence of the Spin-Polarized EPR •Spectra of the Radical Pair P•+ 700 A1 . In this section, the •orientation dependence of the transient EPR spectra of P•+ 700A1 in PSI single crystals will be presented. For each of the crystals the CW-EPR spectra of FA have been measured as well. 3.2.1. Rotation with c Parallel to the Rotation Axis. In •Figure 3 the transient spin-polarized EPR spectra of P•+ 700A1 are presented as a function of the rotation angle, R, with the crystallographic c-axis nearly parallel to the rotation axis r; the misalignment has been determined to be ∠(c, r) ) 5° ( 3° (see above and Figure 1). Thus, for all rotation angles, c is virtually perpendicular to the external magnetic field, B0. In Figure 3, the overall signal amplitude is almost constant and a predominant A/E-polarization pattern is observed. Note, that the spectra show no net polarization. This is expected because •the radical pair P•+ 700A1 is generated in a pure electronic singlet •state. For comparison, the spectrum of P•+ 700A1 in frozen solution is included at the top of Figure 3. Obviously, the overall spectral width is different, with the single-crystal spectra
Figure 3. Angular dependence of the transient X-band EPR spectra •of the radical pair P•+ 700A1 in single crystal SC-p. See Figure 1 for the corresponding angular dependence of the CW-EPR specta of FA obtained on the same crystal. The rotation axis, r, is within 5° parallel •to the crystallographic c-axis. At the top, the spectrum of P•+ 700A1 in •frozen solution (powder spectrum of P•+ A ) is included for com700 1 parison. Experimental parameters: T ) 80 K; excitation wavelength: 532 nm; microwave frequency and power: 9.76 GHz and 215 µW (-30 dB); average of 128 events at each of the 128 field points; time window for digital boxcar integration: (0.5-2.0) µs.
being narrower. In particular, for this crystal orientation, the spectra do not extend as far downfield as the powder spectrum. Qualitatively, this implies that the principal gxx-axis of g(A•1 ) (lowest field spectral component) must be closer to parallel than perpendicular to the crystallographic c-axis. The effect of orientation change is significantly different for the emissive (high field) than for the absorptive low-field part of the spectra (see Figure 3): The emissive part changes much less than the absorptive part. The angular dependence of the absorptive part, however, is indicative for a partially resolved hyperfine structure (hfs) (see below). 3.2.2. Rotation with c Perpendicular to the Rotation Axis. In Figure 4, a and b, the orientation dependence of the transient •EPR spectra of P•+ 700A1 is depicted for the same two single crystals (SC-s1 and SC-s2), both oriented with c ⊥ r. The corresponding CW-EPR spectra of FA are given in Figure 2. With the crystallographic c-axis parallel to the magnetic field, c || B0 (R ) 0° and 180°), all six radical pairs per unit cell become equivalent: a maximum E/A-polarization pattern is detected in this case for both crystals. In addition, a hfs in the low-field emissive part is clearly resolved and corresponds to a quartet splitting. Moreover, the spectra at this orientation extend •as far downfield as the powder spectrum of P•+ 700A1 , indicating that the angle between the field and the gxx principal axis of g( A•1 ) is quite small. From Figure 4 (c ⊥ r) it is obvious that an increase of the angle between c and B0 away from 0° diminishes the overall E/A-amplitude and smears out the hfs considerably. At rotation angles between 50° and 60° a change in the overall polarization
8270 J. Phys. Chem. B, Vol. 102, No. 42, 1998
Figure 4. Angular dependence of the transient X-band EPR spectra •of the radical pair P•+ 700A1 in PS I single crystal SC-s1 (a, top) and SC-s2 (b, bottom). See Figure 2 for the corresponding angular dependence of the CW-EPR specta of FA obtained on the same crystals. The c-axis is perpendicular to the rotation axis, r. The rotation •angle was set to R ) 0° for c || B0. At the top, the spectrum of P•+ 700A1 in frozen solution is included for reference. See caption to Figure 3 for the experimental parameters.
pattern from E/A to A/E is observed, together with some reappearence of a characteristic hfs. Note that this angular range includes the Magic angle of θMA ≈ 54.74° (tan θMA ) x2) at which the dipolar coupling is zero. The sign of the coupling is opposite at angles above or below θMA. However, it is not obvious why the change in the polarization pattern occurs in •this range. The dipolar vector, zd, between P•+ 700 and A1 is
Kamlowski et al. expected to enclose a specific angle with c. According to the unit cell symmetry, the six dipolar vectors are arranged equally spaced on a cone with this angle. Note that for the field orientation with ∠(c, B0) ) θMA, only the projections of zd onto c are at the Magic angle, not the dipolar vectors themselves. In principle, the angles between B0 and each of the six dipolar vectors are different from the Magic angle. Thus, a cancellation of oppositely polarized contributions is responsible for the observed change in the overall polarization pattern. This cancellation can account for the reappearence of some apparent hfs. For different orientations of the radical pairs with respect to B0, different effective hyperfine couplings are expected. Hence, cancellation will not be complete for each of the hyperfine components. For angles above 60°, a local maximum of the overall A/E pattern occurs at R ) 90° (c ⊥ B0). Note, however, that the two spectra at R ) 90° for the two single crystals are different (cf. Figure 4, a and b). Comparison with the spectra obtained at the crystal orientation c || r (Figure 3) shows that they are nearly equivalent to those at R ) -20° + n60° and R ) 10° + n60°, respectively. This is consistent with the calibration obtained with the CW-EPR signals of FA which showed that the two crystals are oriented with respect to each other by a rotation of 27° around the c-axis (see previous section). 3.3. Qualitative Analysis and Simulation of the Orientation Dependence of the Spin-Polarized EPR Spectra. Two features characterize the orientation dependence of the transient •EPR spectra of P•+ 700A1 in PSI single crystals: the change in sign and amplitude of the spin-polarization pattern, and the resolved hyperfine structure. From this the orientation of the quinone acceptor A1 with respect to the PSI complex can be evaluated based on (i) the CCRP1a model (see refs 32, 33, and 34 for application to radical pairs in photosynthesis; cf. e.g. refs 35-37 for reviews) and (ii) the known magnetic tensor •parameters of the radical pair P•+ 700A1 (cf. Table 2 and ref 11). An additional information is the angle between the crystallographic c-axis and the dipolar vector, zd, which can be confined to a narrow angular range. First, the required basics of the CCRP model will be described. Then, a qualitative analysis of the A1 orientation will be followed by quantitative simulations of the orientation dependence. Basic Concept of the CCRP Model. According to the CCRP •concept the spectral properties of a radical pair like P•+ 700A1 are governed by tensorial quantities: the g tensors of both radical •ions, i.e. g(P•+ 700) and g(A1 ), the interradical electron spin coupling tensor (predominantly dipolar with principal axis zd), and the hyperfine coupling tensors of all nuclei to both electron spins. Due to the tensorial character, the relative orientations of the respective principal axes as well as the orientations of these axes to the magnetic field, B0, are important. Without hfc the system of two coupled spins S ) 1/2 in a magnetic field has four energy levels. In general, all four transitions (∆mS ) (1) are EPR active. For a given set of parameters, a four line stick spectrum is expected as displayed in Scheme 1. Since the electron transfer from 1P/700 to A1 is fast compared to the spin dynamics, the spin system is populated in a singlet state. The well-known antiphase pattern occurs without net spin polarization. For spin-spin coupling small compared to the g factor difference, the two pairs of antiphase lines are centered approximately at the respective effective g values of the individual spins. The splitting between the two antiphase lines and their relative sign is proportional to the spinspin coupling. Two important properties of the spin system •P•+ 700A1 should be noted. For virtually all orientations of the
•Radical Pair State P•+ 700A1
J. Phys. Chem. B, Vol. 102, No. 42, 1998 8271
TABLE 2: Internal Magnetic Parameters of the Radical •Pair P•+ 700A1 Spin-Spin Coupling parameter
ref -170 ( 4 1 ( 0.5 g-Tensors
D (µT) J (µT)
15, 17 15, 17
parameter
gxx
gyy
gzz
ref
g(P•+ 700) g(A•1 )
2.0030 2.0062
2.0026 2.0051
2.0023 2.0022
43a 11
Hyperfine Coupling (hfc) parameter A•1 :
A(2-CH3)
A| (MHz)
A⊥ (MHz)
ref
12.8
9.0
39, 40
EPR Line Widthsb parameter
without hfc
with hfc
ref
∆B(P•+ 700) (mT) ∆B(A•1 ) (mT)
0.7 0.7
0.7 0.335
11 this work
Orientational Parameters Euler angles (deg) parameter g(P•+ 700)
to
g(A•1 )
R 83
β
γ
ref
128
10
11
polar angles (deg) parameter
θ
φ
ref
zd in g(A•1 ) A(2-CH3) in g(A•1 )
90 90
0 60
11
Recently, two new sets of principal values for g(P•+ 700) have been published.50 However, at X-band microwave frequencies this does not affect the calculated spectra, due to the small g-tensor anisotropy. Moreover, the simulation is restricted to account for the low-field part b only, which is exclusively dominated by g(A•1 ). The EPR line widths are used to convolute the stick spectrum. “Without hfc” means that the specific hfc tensor A(2-CH3) is not used in the simulation, whereas if it is included a reduced residual line width has to be applied to convolute the resonances due to A•1 . a
SCHEME 1: Stick Spectrum of the Correlated Coupled •Radical Pair P•+ 700A1 for a Given Set of Parameters (See Text for Details)
field B0 with respect to the principal axis systems of g(P•+ 700) ), the difference in the effective g-values is positive: and g(A•1 •+ ∆g ) g(A•1 ) - g(P700) > 0. Hence, in good approximation the low-field pair of lines can be assigned to transitions of the •+ •spin on A•1 . The spin-spin coupling of P700A1 is almost exclusively dipolar. The exchange coupling is at least 2 orders of magnitude smaller than the D value of the dipolar coupling (see e.g. ref 17). Therefore, the splitting of the two lines assigned to A•1 is determined by D and the angle between the dipole vector and the magnetic field, ∠(zd, B0). For an angle smaller than the Magic angle (∠(zd, B0) < θMA), a polarization
pattern E/A/E/A is expected as shown in Scheme 1, i.e. the lowest field line appears in emission. Correspondingly, if ∠(zd, B0) > θMA, a polarization pattern A/E/A/E is predicted. In the case of hfs, which in first approximation can be accounted for by Gaussian broadening of each of the four lines, progressive cancellation will occur. This shows up at first for the two inner lines, if the EPR line width, ∆B, of the radical •ions P•+ 700 and A1 (cf. Table 2) becomes comparable with or larger than the splitting due to ∆g. In case of ∠(zd, B0) < θMA, the spectrum is expected to collapse into an overall E/A polarization pattern. Correspondingly, an overall A/E pattern should occur for ∠(zd, B0) > θMA. Spin-Polarization Pattern. In the case of the spin-polarized EPR spectra in frozen solution, the angle between zd and the gxx principal axis of the quinone g tensor can qualitatively be determined from the spectral line shape at low field (cf. e.g. refs 11, 34, and 38). Similarly, inspection of the single-crystal spectra immediately gives information on the angle ∠(c, zd). First, at the orientation c || B0 an overall E/A polarization pattern requires this angle to be smaller than the magic angle θMA. Second, this upper limit by θMA can be reduced further with the observation of an exclusive A/E polarization pattern for all orientations with c || r. For angles ∠(c, zd) larger than the complement to the magic angle, the opposite E/A pattern should have been observed. Thus, ∠(c, zd) has to be also smaller than the complement to the magic angle
∠(c, zd) < 90° - θMA ) 35.26°
(1)
An additional indication that this upper limit can be reduced further is related to the experimental observation that the overall amplitude of the spectra at c || r hardly varies with changing field orientation (cf. Figure 3). However, a reliable assessment of how much this upper limit can be reduced must be deferred to quantitative simulations including the hyperfine coupling (see section 3.3.1). The upper limit of ∠(c, zd) directly translates into structural restrictions for the orientation of the A1 head group, since the dipolar axis zd was shown to be collinear to both the gxx principal axis of g(A•1 ) and the CdO carbonyl bonds (see Table 2, ref 11). Hence, the angle between the CdO bond of A1 (the gxx axis) and the c-axis must also be smaller than 35°. ResolVed Hyperfine Structure. In addition to the change in the sign of the polarization pattern, a partially resolved hfs characterizes the orientation dependence of the transient EPR •spectra of P•+ 700A1 . This hfs can be described by the angular dependence of both the splitting of the four equally spaced lines and the modulation depth which has a maximum at c || B0 (cf. Figure 4). Furthermore, the splitting occurs in the low-field part of the spectrum. These properties are indicative of the hfs due to the 2-methyl group of A•1 for the following reasons: (i) •+ Comparing CW-EPR spectra of stable A•1 and P700 at X-band, •only that of A1 shows distortion from Gaussian line shape, indicating a partially resolved hfs (see e.g. ref 12 and references therein). (ii) The low-field part in the transient EPR spectrum (see above). (iii) In is dominated by resonances of A•1 ENDOR, TRIPLE, and ESEEM studies on P•+ 700 in the PSI single crystal18,19 no large hfc tensors could be detected. (iv) ENDOR and TRIPLE on A•1 clearly indicate a dominant hfc attributed to the 2-CH3 group.39,40 Thus, the partially resolved •quartet hfs in the transient EPR spectra of P•+ 700A1 can be •assigned to the methyl group of A1 at position 2. The principal values of this axial hfc tensor are known39-41 and are given in Table 2. The effective hfs obtained at c || B0 falls in
8272 J. Phys. Chem. B, Vol. 102, No. 42, 1998
Kamlowski et al.
the range Aeff ) 11.6-10.4 MHz. With the orientation dependence of an axially symmetrical hfc tensor:
Aeff ) [A⊥2‚sin2 β + A|2‚cos2 β]1/2
TABLE 3: Orientation of the Quinone Acceptor to the Crystal Axis System (CAS) and Angles between the c-Axis and the Principal g-Tensor Axes R(g(A•1 ), CAS)
(2)
Euler angles (deg)
the orientation of the principal axes with respect to the c-axis can be evaluated. The angle between c and the A| component, therefore, is
β ) ∠(c, A|(2-CH3)) ) 35°-55°
(3)
In a first approximation, the A| principal axis can be assumed to be parallel to the C(2)-CH3 bond connecting the naphthoquinone ring and the CH3 substitutent. This approximation is the better, the more the hyperfine coupling tensor is determined by only the spin density at C(2), FπC(2) (for discussion see section 4.3). Hence, the angle between the C(2)-CH3 bond of the 2-methyl group and crystallographic c-axis is also expected to be 35°-55°. For c || r, i.e. c ⊥ B0, this angle β results in an Aeff value between A⊥ ) 9.0 MHz and about 11.5 MHz, depending on the orientation of this hfc tensor with respect to B0. The partially resolved hfs in the transient EPR spectra for c || r (cf. Figure 3), depending on the rotation angle, corresponds to splittings between 9 and 10 MHz, in agreement with the expected values. However, due to the very low modulation depth, the error is quite large. Furthermore, due to the crystal symmetry, different orientations of the radical pairs with respect to B0 occur at this crystal orientation, and the observed hfs is a superposition of up to three different Aeff values. Finally, according to the molecular structure of the VK1 head group the angle between the CdO carbonyl group and the C(2)CH3 axis is 60°. Hence, the angle of this CdO group with respect to the c-axis is equal to or larger than 60° - (35°55°). Because zd is collinear with the gxx principal axis of 11 g(A•1 ), this represents a lower limit for the angle between zd and c. The limiting case is consistent with the simulation results
∠(c, zd) g 25°
R
(4)
This lower limit complements the upper limit evaluated qualitatively from the orientation dependence of the polarization pattern (see above). The resulting range of angles ∠(c, zd) corresponds well to that obtained by independent methods. The angle between the quinone gxx-axis and the membrane plane was determined to 63° ( 10°.12 Using the independent result of collinearity between the zd and the gxx-axis of A•1 , the angle ∠(c, zd) is 27° ( 10°. The same result, however, directly and with higher accuracy was obtained when this angle was calculated from the orientation dependence of the dipolar coupling together with the principal D value known from studies •15-17,42 ∠(c, z ) ) 27° ( 5°.6 of P•+ d 700A1 in frozen solution: This angle will be used in the simulations of the orientation dependence of the transient EPR spectra (see below). Finally, we note, that for the limiting case β ) 35° the sum of the determined angles (∠(c, zd) and ∠(c, C(2)-CH3)) adds up to 60°, i.e. the axes, CdO (gxx), zd, C(2)-CH3, and the c-axis lie in the same plane. The angle between the plane (c,zd) and that of the naphthoquinone head-group of A•1 at present can only be specified within the upper limit of 60°. Again, this is in agreement with the angle of 76° ( 10° determined between the quinone and membrane plane in ref 12. 3.3.1. Simulation of the Orientation Dependence. The qualitative results obtained in the previous section can be refined by quantitative spectral simulations based on the CCRP concept. However, a large set of parameters is needed to calculate the
β
-27
c
γ
90 ∠(c,gii) (deg)
0
gxx
gyy
gzz
27
63
90
spectra. The quality of the conclusions depends on how many parameters are known from independent studies. Fortunately, •most of the “internal” parameters of the radical pair P•+ 700A1 , e.g. principal g values,11,12,43 relative orientation of principal axes with respect to the dipolar vector zd,11 and coupling constants, D and J,15-17,42 (see Table 2) are known to a high degree of confidence (see Discussion). The qualitative analysis gave strong indications that the partially resolved, orientation dependent hfs has to be assigned to the hfc of the 2-methyl group of A•1 . This assignment is •confirmed by simulation of (i) the powder spectrum of P•+ 700A1 and (ii) the full orientation dependence of the spectra. In Figure 5a two simulations are compared with the measured X-band •powder spectrum of P•+ 700A1 . In one simulation (Figure 5a, dotted line) no specific hfc is included, only an isotropic line broadening is taken into account. In the other (Figure 5a, dashed line), the specific hfc due to the 2-CH3 group is included. In the latter case agreement with the measured spectrum is remarkably improved. Note that only a single specific hfc is required for this result. Corresponding simulations of experimental spectra in K and W band have been achieved (not shown). The main discrepancy concerns the up-field part of the X-band spectrum (Figure 5a) (the same applies to the simulations at higher frequencies). This field region includes •+ one of the g(A•1 ) and all g(P700) contributions, with a large extent of spectral overlap. Therefore, in a first step our simulations are directed to only the low-field part of the transient EPR spectra which is dominated by g(A•1 ). In Figure 5b, the experimental transient EPR spectrum (bottom) for the orientation c || B0 is again compared with the two simulations without and with the hfc tensor A (2-CH3) (dotted and dashed line, respectively). The overall E/A polarization pattern is reproduced in both simulations. However, the partially resolved structure for the low-field side is simulated well only if the anisotropic hfc is included. This provides further support for the assignment of the resolved hfs to the 2-methyl group of A•1 . For the simulations, the angle between the c-axis and the dipolar connecting axis, zd, has been specified to ∠(c, zd) ) 27° 6 (see above). Finally, the complete orientation dependence was simulated as shown in Figure 6 for the two crystal orientations, c || r and c ⊥ r (Figure 6, a and b), respectively, with the same set of parameters (see Tables 1-3). In the case of the orientation c || r (Figure 6a), the angular dependence of the spectral line shape at low field, i.e., the partially resolved hfs, is reproduced quite well. In addition, the overall amplitude of the spectra hardly varies with orientation as experimentally observed. For the orientation c ⊥ r (Figure 6b), several comments should be added. The general properties of the angular dependence, e.g., the sign change of the polarization pattern, and the (re)appearance of the hfs, are simulated satisfactorily. As expected, the up-field line shapes are not simulated as well (see above).
•Radical Pair State P•+ 700A1
Figure 5. Comparison of experimental and simulated spin-polarized •EPR spectra of P•+ 700A1 . (a, top) Spectra in frozen solution (powder spectra). Solid line: experimental spectrum (cf. e.g. Figure 3, top). Dotted line: simulation based on the CCRP concept without explicit consideration of anisotropic hfc. Dashed line: simulation including the 39,40 The parameters anisotropic hfc tensor of the 2-CH3 group of A•1 . are given in Table 2. (b, bottom) Spectra at c || B0. Bottom (solid line): experimental spectrum (cf. Figure 4b, R ) 0°). Dotted line: simulation without the specific hfc to the methyl group. Dashed line: simulation with this hfc. Simulation parameters: Tables 2 and 3. The vertical dashed lines are a guide for the eye and should emphasize the quartet hfc.
J. Phys. Chem. B, Vol. 102, No. 42, 1998 8273
Figure 6. Comparison of the experimental orientation dependence (left panel) and the simulation (right panel) of the transient spin-polarized •spectra of P•+ 700A1 . (a, top) Orientation with c || r (SC-p; cf. Figure 3). (b, bottom) Orientation with c ⊥ r (for SC-s2; cf. Figure 4b). Simulation parameters: Tables 1-3.
The largest modulation depth due to the hfs occurs at c || B0 in agreement with experiment. Furthermore, for rotation angles R ) ∠(c, B0) > 0°, the reduction of the hfs depth is reproduced, although details of the partial loss of resolution with increasing angle between c and B0 are not. This may be due to additional
8274 J. Phys. Chem. B, Vol. 102, No. 42, 1998
Figure 7. Structural model of the orientation of the electron acceptor A1 in PSI with respect to the crystallographic c-axis. On the right, the equivalent alternative orientation of A1 is shown. The two orientations cannot be distinguished due to the inherent C2 symmetry. An angle of ∠(c, zd) ) 27° 6 has been used for the average angle. For the limiting case of β ) 35° and orientation of gyy, see text.
anisotropic hyperfine coupling which leads to stronger “cancellation” of resolved hfs. On the other hand, the reappearance of resolved hfs is simulated well. Indeed, the chosen orientation of the magnetic tensors, i.e., the orientation of the radical pair (cf. Tables 2 and 3), can account for the nearly complete cancellation of the oppositely polarized spectra around the magic angle. So far, all simulations have been calculated with an angle ∠(c, zd) ) 27°. Additional simulations with different angles indicate that the qualitative upper limit of 35° can be lowered to ∠(c, zd) e 30°. For angles greater 30°, the overall amplitude of the simulated spectra begins to show a significant angular dependence, which is incompatible with the experimental results of Figure 3. •In conclusion, the X-band spectrum of the P•+ 700A1 in frozen solution as well as the angular dependence of this spin-polarized spectrum in PSI single crystals can be simulated on basis of the CCRP concept. Only the anisotropic hfc of the 2-CH3 group of A•1 needs to be included specifically. Additional hyperfine couplings contribute within the Gaussian line broadening. 4. Structural Model of the Quinone Acceptor A1 4.1. A1 Orientation with Respect to the Membrane Normal or Crystallographic c-Axis. Orientations have been determined for the dipolar axis, zd, with respect to the c-axis and also for selected A1 quinone molecular axes, specifically the gxx principal tensor axis (short molecular in-plane axis x, along the CdO double bonds) and the hyperfine tensor axis along the C(2)-CH3 bond (see Tables 2 and 3). They can be combined to a structural model for the naphthoquinone part of the native phylloquinone acceptor A1 as shown in Figure 7. The essential features are the quinone x-axis along the CdO double bonds (parallel to zd) encloses an angle of 27° ( 5° to the c-axis (membrane normal, n) consistent with the angular interval obtained in this study. In addition, the quinone plane is assumed to orient perpendicular to the membrane plane in Figure 7. This model is the consequence of the following individual results: (i) The gxx-axis (along the CdO double bonds) is parallel to zd. Compared to earlier results11 the error for this angle could by now be reduced to (5°. (ii) For the angle ∠(c, zd) the upper
Kamlowski et al. limit has been determined to e30°. This limit has been concluded from the angular dependence of the overall polarization pattern (see Figures 3 and 6a). The lower limit, ∠(c, zd) > 25°, is consistent with overall simulation results and hyperfine data but carries a large error. (iii) The angle between the planes of (c,zd) and of the quinone headgroup is only specified by an upper limit of 60°. In view of the larger error and lack of information on the direction of rotation, the average angle zero is chosen for Figure 7, corresponding to the limiting case β ) 35°. 4.2. A1 Location within the PSI Reaction Center. The structural model in Figure 7 does not include yet the information about the distance between the radical ion centers P•+ 700 and A•1 , which is available from the pulsed EPR studies on the same single crystals. The ESEEM modulation of the so-called out-of-phase echo directly reflects D and J. A value of D ) -(170 ( 4) µT has been obtained for PSI of Synechococcus elongatus15,17 and Synechocystis PCC 6803.42 With the known distance dependence of D, a center-to-center distance between •the respective spin densities of P•+ 700 and A1 of r ) |zd| ) 2.54 ( 0.03 nm could be evaluated. Within experimental error this distance is identical to that determined for PSI from spinach.16 Besides the precise distance value, the ESEEM data for the single crystal yield the angle ∠(c, zd) ) 27° ( 5°.6 The agreement with the angular interval obtained in this work gives strong support for the respective evaluation procedures. The angle of 27° has been used to position the center of A1 (taken as the spin density center in the middle of the CdO bonds). With this, the A1 position and orientation is determined within the PSI reaction center, except for a final reference to the crystallographic a- or b-axis, which cannot be obtained from the EPR results. The A1 position is specified only to a torus around an axis parallel to the c-axis which passes through the spin density of P•+ 700 and is close to the quasi C2-symmetry axis of the general cofactor arrangement and protein framework of the PSI reaction center; see ref 6 for more detail in connection with the recent X-ray structure data.5,14 Note that the angular dependence of the radical pair spectra in Figures 3 and 4 are correlated with that of the FA center (Figures 1 and 2) but neither of them with respect to the crystal axes a and b. For various technical difficulties an independent axis determination by X-ray scattering under maintained crystal orientation did not work out in our case. Alternatively, the crystal axis orientation can be obtained from crystals with clearcut morphological shapes which reflect the crystal axes directly. A corresponding orientation assignment used in ref 6 has been subjected to an additional calibration experiment, which has been performed on a large crystal with obvious parallel surface planes. While these planes are certain to contain the growth direction (c-axis) it has been assumed (not proven yet) that they contain also the a- (or b-) axis. Rotation of the crystal around an axis parallel to the macroscopic plane leads to a magnetic field orientation parallel to the plane normal, i.e., perpendicular to both the c- and a-axes. The corresponding EPR spectrum of FA is readily identified in the angular dependence of Figure 1 (modulo 60°). Within (5° the spectra with a fourfold lowfield degeneracy at R ) 65° + n60° correspond to the orientation B0 perpendicular to the crystalline a- (or b-) axis. The remaining sixfold degeneracy can be reduced to twofold by the reasonable assumption that the A1 position must be close to the average acceptor plane. With this additional information, Figure 8 presents the EPR determined location and orientation of A1, now fixed within the established cofactor arrangement as known from the latest
•Radical Pair State P•+ 700A1
Figure 8. Orientation of the electron acceptor A1 with respect to the cofactor arrangement of PSI. The spin density center of A1 is positioned with the angle ∠(c, zd) ) 27° obtained from ESEEM results on P•+ 700 6 A•1 in PSI single crystals. This corresponds to the electron density center obtained from the X-ray data set at 4.0 Å resolution.5 Top: Cofactor arrangement viewed along the axis connecting the Mg atoms of the two Chla entities constituting P700 (called eC1 and eC1′ in ref 5). The crystallographic c-axis and the membrane normal, n, are in the plane of the paper. The axis zd connecting the (spin density) centers of •P•+ 700 and A1 is shown together with the direction of the gxx principal axis of g(A•1 ). Bottom: Projection onto the crystallographic a,b plane. The view direction is along the pseudo-C2-symmetry axis from the stromal side, i.e. from the FeS clusters to P700. As a reference to the overall crystal structure the axis connecting the C2 axis and the crystallographic threefold axis (triangle) is chosen and makes an angle with a of 42° (Krauss, N., Klukas, O., personal communication). In ref 5 it is introduced as axis H. The projection of the axis connecting the terminal two FeS centers onto the a,b plane and this axis encloses an angle of 6°.5
electron density map at presently 4.0 Å resolution.5,14 In Figure 8 (top), a view perpendicular to the c-axis is shown, along the line connecting the Mg atoms of the Chla entities constituting P700. This projection plane is close to the plane of the Chla electron acceptors with the trimer C3 axis to the left. The bottom part of Figure 8 represents the projection along the quasi C2 symmetry axis from the stromal side. Of course, it is not known which branch is the active one for electron transfer. On the other hand, EPR observes the active A•1 state within the •A . With the increasfunctional charge separated state, P•+ 700 1 ing number of distinct molecular properties (resolved g, and hyperfine tensors etc.) determined from the EPR spectra, A1 is
J. Phys. Chem. B, Vol. 102, No. 42, 1998 8275 more and more likely to represent only one of the two quinones confirmed to be present per PSI reaction center; i.e., only one branch is active. Note that the choice of a specific A1 site in Figure 8 is arbitrary. It has been made for easier comparison and places A1 in the same position as the one (of two) quinone center positions, assigned in the X-ray structure and termed QK.5 In Figure 8 (bottom), some additional axes have been included for easier comparison to the overall PSI reaction center structure: The axis connecting the C3 trimer axis (full triangle) and the quasi C2 symmetry axis. This axis has been termed H in ref 5. The angle between this axis and the crystallographic a-axis is ∠(H, a) ) 42° + n60° (Krauss, N.; Klukas, O., FU Berlin, personal communication). Furthermore, the axis H encloses an angle of 6° with the projection of the axis connecting the centers of the two terminal FeS centers.5 In addition, the axes connecting the respective Mg atoms of the Chla entities of P700 and A0 are shown (compare to ref 5). The suggested QK site falls just slightly outside the range of (3° established for the maximum deviations of the acceptor Chl centers from the average acceptor plane. 4.3. Comparison with Other Independent Data. The •orientation dependence of the P•+ 700A1 spectrum has already been studied in partially oriented broken chloroplasts.44,45 The information content of these data is remarkable. However, at the time of these early studies, the appropriate spin-polarization mechanism, the CCRP model, had not been established yet. It should be noted that reinterpretation of the 1983 data would render similar structural parameters for A•1 as obtained now. Hence, the data of refs 44 and 45 have been confirmed by the single-crystal data presented in Figure 4 with better hyperfine resolution. Along the lines of refs 44 and 45, MacMillan et al.12 evaluated the orientation of A1 with PSI particles partially oriented on Mylar sheets.12 Within the respective experimental accuracies, our results (cf. Table 3) are consistent with the angle of the gxx principal axis of g(A•1 ) with respect to the membrane plane (or membrane normal) obtained in ref 12. This agreement confirms the nontrivial expectation that the results for the photoaccumulated A•1 state are the same as those from •+ •A•1 in the functional P700A1 state. Recently, an independent 46 study with data relevant to the position of A•1 appeared, which is based on the relaxation enhancement induced by the nearby reduced iron sulfur center FX . The model in Figure 8 is also consistent with these data. Part of the orientational information on A1 in this paper is derived from hyperfine data, in particular the limiting case, that the long molecular in-plane axis, y, of the naphthoquinone head group of A1 is oriented approximately within the (c,zd) plane which is perpendicular to the membrane (see Figures 7 and 8). This result relies on the further independent experimental data and assumptions: (i) For A•1 in PSI the (relatively large) principal hfc tensor elements, A| and A⊥, of the methyl group in position 2 (cf. Figure 7 for the numbering) are known accurately from ENDOR and TRIPLE experiments.39,40 (ii) The principal axis of the large A|-component is aligned close to the C(2)-CH3 bond direction. (iii) The hfc tensor is axially symmetric. Statement (i) has been confirmed by further cwand pulsed-ENDOR studies on A•1 (ref 41 and Lendzian, TU Berlin, and MacMillan, Univ. Frankfurt, personal communication). Statements (ii) and (iii) are well established if the only relevant spin density resides on the carbon atom C(2), FπC(2), and deviations from axial symmetry are small. In case of large spin densities located asymmetrically on adjacent ring carbon atoms this could change. Note, however, that non-axial
8276 J. Phys. Chem. B, Vol. 102, No. 42, 1998 symmetry of A(2-CH3) matters here only to the extent that it influences the evaluated angle, β, and the lower limit for ∠(c, zd). Since the deviation from axial symmetry is small, the influence on the orientational information is negligible. From the broadening of the corresponding signal in the pulsedENDOR spectrum of A•1 in frozen solution at 10 K, this anisotropy is at most ∼0.4 MHz as compared to an isotropic hfc of Aiso ) 10.3 MHz (ref 41 and Lendzian and MacMillan, personal communication). For comparison, in case of Q•A in bRC all principal components of the methyl hfc tensor have been obtained, and the deviation from axial symmetry is 0.4 MHz as compared to Aiso ) 4.5 MHz (Lendzian, Isaacson, Feher, personal communication).] Generally, for semiquinones the spin density at the carbonyl oxygen atoms is large (see e.g. ref 47). Due to the dipolar coupling between the electron and the proton spin, this may introduce additional deviations of the A| axis orientation (statement (ii)). However, in the case of Q•A in bRC of Rb. sphaeroides the A| principal axis is within a few degrees collinear to the bond direction of the methyl group (refs 48 and 49, and Lendzian, Isaacson, Feher, personal communication). A first set of W-band ENDOR studies of semiquinones in ViVo as well as in Vitro48,49 indicates that the angle between the gxx principal axis and the A| principal axis of the methyl hfc tensor is at least 60° within the error margin. Hence, in the case of A•1 the angle, ∠(gxx, A|), is also expected to be at least 60°. Improved accuracy with respect to the orientation of the gyy axis can soon be expected from high-frequency transient EPR studies (Q- and W-band) on PSI single crystals, which are in progress in our laboratories. 5. Final Remark This paper aims at the location and orientation of the A1 acceptor in the PSI reaction center. From the experimental data a virtually complete structural model of the quinone head group of A1 has been derived. In addition, the experimental data contain also information (mostly less direct) on the interaction with the protein environment. Such additional information will be combined in the accompanying paper22 with independent structural and spectroscopic data from the literature as well as with structural modeling studies in order to determine characteristic properties of the A1 quinone binding site. These properties are related to those of the QA binding site in bacterial reaction centers, the structural and functional properties of which are known to much greater detail. Note Added in Proof. The PSI single-crystal data, presented in this paper, have recently been supplemented by transient EPR spectra at Q-band and pulsed ENDOR spectra at X-band on the same crystal samples (S.Z. and R.B.). Preliminary evaluation of both, the better-resolved g-anisotropy and the more accurate Aeff value at B0 || c, yields an angle between the planes (c,zd) and of the A•1 head group which is close to the upper limit of the range determined in this paper (
