J. Phys. Chem. 1993,97,2015-2020
2015
Determination of the g Tensor of tbe Primary Donor Cation Radical in Single Crystals of Rhodobacter sphaeroides R-26 Reaction Centers by 3-mm High-Field EPR Robert Klette, Jens T. Thing, Martin Plato, and Klaus M6bius' Institute of Experimental Physics, Free University of Berlin, Arnimallee 14, D- IO00 Berlin 33,Germany
Birgit Bsnigk a d Wolfgang Lubitz Max- Volmer-Institute of Biophysical and Physical Chemistry, Technical University of Berlin, Strasse des 17. Juni 135,D-IO00Berlin 12,Germany Received: September 8, 1992;In Final Form: November 25, I992
Three millimeter (Wband, 95 GHz) EPR measurements were performed on single crystals of Rb. sphaeroides R-26 reaction centers at room temperature to determine the g tensor of the primary donor cation radical P865". Due to the applied high magnetic field (3.4 T), the magnetically inequivalent sites in the unit cell of the crystals could be resolved. Some of the results are corroborated by additional measurements on P865'+ in perdeuterated reaction centers from Rb. sphaeroides wild type 2.4.1, which could be crystallized for the first time. From this preparation the 3-mm EPR spectrum of P865" in frozen reaction center solution could be resolved into its three principal g tensor components. The principal directions of the g tensor in the single crystal reveal a breaking of the local Cz symmetry in the electronic structure of P865'' consistent with earlier ENDOR results for the hyperfine structure.
at frequencies as high as 95 GHz (W band) with correspondingly high magnetic Zeeman fields BO 3.3 T turned out to be sufficient to resolve individual g tensor components in the powder-type spectra of frozen RC solutions. For RC single crystals of Rb. sphaeroides R-26 first information on the g tonsor of P865'+ was obtained by Q band EPR." However, by using W band highfield EPR, in illuminated RC single crystals of Rb. sphaeroides R-26 even the P865*+lines of the two magnetically inequivalent sites in the xy symmetry plane of the crystal unit cell could be resolved, and their g factor rotation pattern in this plane could be measured.'2 The analysisof both the frozen-solution spectrum and the xy rotation pattern yielded the principal g tensor values and an estimate of the orientation of the g tensor principal axes system with respect to the C2 symmetry axis of the P865'+ dimer. Although in ref 12 W band EPR measurementsin only one crystal plane could be performed and, therefore, only limited conclusions about the direction of the g tensor principal axes in the crystal axes system could be given, an asymmetrical alignment of the g tensor with respect to the monomeric halves of the P865'+ dimer was deduced. In this communication we report on W band high-field EPR measurements of g tensor components of P865'+ obtained by the rotation patterns in all three symmetry planes of the RC single crystal. By this work the complete knowledge of the g tensor of P865'+ could be established. It will be shown that the analysis of the g tensor data fully corroborates the preliminary results presented in ref 12, Le., a breaking of C2 symmetry in the electronic structure of P865'+.
1. Inboductioa
The primary processes of photosynthesis involve light-induced electron-transfer steps to establish charge separation across the photosynthetic membrane. In photosynthetic reaction centers (RC's) these processes are controlled by both the threedimensional and electronicstructures of the donor and acceptor molecules which are embedded in the protein subunits. For RC's of two photosynthetic bacteria, Rp. uiridis' and Rb. sphaeroides R-26.2the three-dimensional structures have been determined to atomic resolution by X-ray crystallography. An appealing aspect of the X-ray structure is an approximate localC2 symmetry axis running through the primary donor 'special pair", P960 and P865, respectively. Detailed information on the electronic structure of the primary donor cation radical of Rp. viridis and Rb. sphaeroides, P960*+and P865'+, has been obtained by EPR and ENDOR (electronnuclear double resonance) spectroscopy. (For reviews, see refs 3 and 4.) Particularly the hyperfine tensor components, which can be determined by ENDOR and electronnuclear-nuclear triple resonance (TRIPLE) techniques, give detailed information on the unpaired electron distribution over the dimer halves of P.+ in RC single crystals.5v6 As the main result of these studies,a breakage of C2 symmetry in the electronic structure of the dimer cation radical states, P960'+ and P865*+ is indicated. The ratio of net spin densities was found to be approximately 2:l in favor of the L half of the dimer. This asymmetry could be rationalizedby small asymmetricdistortions of the dimer geometry and by the asymmetrical distribution of amino acid residues in the vicinity of the dimer. Since it was anticipated that the breakage of C2 symmetry in 2. Experimental Section the electronic structure of P.+ might represent an important functional factor in controlling the vectorial properties of The high-field EPR spectrometer, operating at 95 GHz (W photoinduced electron transfer in bacterial RC's, such as high band), wasdescribed earlier.13J4 Inorder toobtain the fullangular quantum yield and/or unidirectionality along the L protein information about the g tensor, a two-axis goniometer probehead branch238 it is desirable to test the results from hyperfine tensor (Figure 1) wasconstructedfor 3-mmEPR. Itconsistsofa Fabrymeasurementsby an independent probe for the electronicstructure Perot resonator (TEMw7 mode, near confocal mirror arrangein P.+:such a probe is the electronicg tensor, whose components ment) with the same microwave coupling mechanism as described and orientation reflect the symmetry properties of the electronic before.l4,l5 The capillary containing the sample protrudes into structure of the dimer. the resonator in the focal plane perpendicular to the resonator In contrast to earlier X band (9.5 GHz microwave frequ~ncy)~ axis. The capillary is fixed by an arm which can be rotated about the resonator symmetry axis by a gear drive with an angular and Q band (35 GHz)lo EPR measurements of the g factor, EPR OO22-3654/93/2097-2015$04.Oo/O
(6
1993 American Chemical Society
2016 The Journal of Physical Chemistry, Vol. 97, No. 9, 1993
e k
axis 1-3i I
n
Figure 1. Fabry-Perot resonator with goniometer for 3-mm EPR (Wband) with mounted RC single crystal (shown orientation B0)lcrystal c axis): a, mirrors (diameter 19 mm, distanceca. 10 mm); b, mirror supports (synchronously movable for frequency tuning); c, WR- 10 waveguide; d, microwave coupling unit;I5e, goniometer arm; f, gear for rotation of the arm about the resonator axis; g, arm support (movable for adjusting sample in center of resonator); h, rotatable sample holder; i, thread passing through arm e to sample holder h for sample axis rotation; k, quartz capillary with RC single crystal; 1, independently adjustable quartz capillary with Mn2+standard sample. Rotation axes: axis 1, gear driven rotation about the resonator axis; axis 2, thread driven rotation about the sample capillary axis. (For details see text.)
accuracy of 10.6% (ie., a reproducibility of 0.1' for a 180' rotation). The rotation covers a range of f140' relative to the direction of the magnetic field BO. A second rotation axis perpendicular to the first one is the samplecapillary axis. Rotation is carried out by a cotton thread passing through the arm to the sample holder. The relative precision of the angular setting is f0.4% (corresponding to f0.7' for a 180' rotation). Both rotation axes are independent of each other. A third rotation axis is provided by a 90' shift of the crystal around the second
Klette et al. axis prior to rotating it around the first axis. Thus, any orientation can be achieved without remounting the capillary containing the crystal. This is an important feature of the goniometer since it avoids additional alignment errors. To create the P865*+radicals, the crystals were illuminated by a filtered 50-W halogen lamp. A 5-cm water filter and subsequent edge filters restricted the excitation wavelengths to the range between 830 and 900 nm. Thus, the reaction centerswere excited predominantlyin the BChl dimer band at 865 nm. This prevented unnecessary heating of the sample by surplus light absorption. The light was guided into the Fabry-Perot via two quartz fiber bundles of 5-mm diameter,irradiatingthe samplefrom orthogonal directions. The light intensity was chosen to saturate the optical absorption. The sensitivity of the 3-mm spectrometer was optimized by using an InSb microwave detector (QFI/2, QMC Instruments Ltd., London). Working at 4.2 K, its noise equivalent power (NEP) is 9 X lO-l3 W/Hz*12reducingthe detector noise by almost 2 orders of magnitude compared to the conventional Schottky barrier beam lead diode (7 X 10-11 W/Hzl/2 NEP). Thereby, the signal-to-noise ratio of the EPR was increased by almost a factor of 20, and the modulation amplitude could be reduced to 0.05 mT (peak-to-peakvalue) to avoid modulation broadening. RCs of Rb. sphaeroides R-26 were isolated, purified, and crystallized as described before.16J7 The single crystals belong to the orthorhombicspace group E212121with four RCs per unit cell and one RC per asymmetric unit.16 For BOlying along a crystallographicaxis, all four sites are magnetically equivalent; in the crystal symmetry planes two mutually inequivalent sites remain. The typical crystal size was 0.3 X 0.6 X 3 mm, the needle axis being the crystallographicc axis. For the deuterated RC's, Rb. sphaeroides wild type bacteria were grown in D20. Their crystallizationwas successfulfor the first time. The crystal was mounted in a quartz capillary of 1-mm diameter and a wall thickness of 0.01 mm. It adhered to the wall of the capillary in a small drop of the mother solution from which it was grown. Thus, its long crystallographicc axis was self-aligned parallel to the capillary axis. To minimize dielectriclosses, excess aqueous solution was removed carefully. As a vapor reservoir, a small
b
a
-
A B
7
M
20G
d Figure 2. Three-millimeter EPR spectra of P~65*+from Rb. sphueroides R-26 RC single crystals at 12 OC for two different orientations in the uc plane: solid line, measured spectrum; large dots, COMPASS simulation;18 small dots, COMPASS deconvolution of the two magnetically inequivalent RC sites; X, the M I= */*line of the simultaneously recorded M2+standard (0.02% MnO in MgO powder). (a) Bollu: all four RC sites are magnetically equivalent. (b) L(B0.u) = 8 = 40° in the uc plane: two pairs of magnetically equivalent RC sites.
The Journal of Physical Chemistry, Vol. 97, No. 9, 1993 2017
EPR of Rhodobucter sphueroides R-26 Reaction Centers piece of tissue soaked with mother solution was placed inside the wax sealed capillary but outside the microwave field. Earlier X band ENDOR meas~rements~~ have shown irreversible structural changes upon freezing the crystals;consequently, care was taken to keep the crystals above 0 OC. Since temperatures above 25 OC would also damage thecrystals, all measurementswerecarried out at 12 OC. The RC single crystal was rotated by 180° about the three crystallographic axes u, 6, c. EPR spectra were taken every loo. At first the capillary containing the crystal was mounted perpendicular to BOin order to perform rotations in the crystal ub plane. Rotation of the crystal about one of the short axes u and b was executed by rotating the goniometer arm about the Fabry-Perot symmetryaxis. Toalign u or b parallel to this rotation axis, the crystal was rotated about the capillary axis c until the Ps65'+ resonance line did not show any splitting or broadening. The crystal alignment error in all three arrangements is estimated to be at most lo. Such an alignment error does not affect the principal g tensor values but is critical for the determination of principal axes directions in the crystal axis system. The g values were measured by simultaneously recording the hyperfine lines of a Mn2+ reference powder sample (0.02% MnO in MgO, g = 2.001 01 f 0.000 05, a = 8.710 & 0.003 mT)l2J5 serving as g factor and field calibration standard. The spectra were analyzed using the deconvolution program COMPASS.18 The resulting g values of the P865'+ lines of the two separated RC siteshaveanerrorofAg= f5 X 10-5. Theywereusedtocalculate the angular distribution function g(B) given by19
g'ie) = (g'),,cos2 e + (g'),, sin2e + 2(8),, sin e cos e
c axis
2.0020
io.
A0
1
b
l " I " 1 " l '
120.
1500
1eoe
a axis I
.rotation angle 8
+
3. Results md Discussion Figure 2a shows a 3-mm EPR spectrum of P865'+ with B& where all four RC sites are magnetically equivalent. In Figure 2b the lines from the two inequivalent sites show maximum separation in the (IC plane. The full line represents the experimental spectrum while the dotted line indicatesthe COMPASS simulation composed of three components with Gaussian line shapes: the Mn2+hyperfine line (MI = l/2, marked X), and the EPR lines of the two RC sites (small points). The corresponding rotation patterns (Figure 3a) reveal a phase shift of 2A8 = 64 f 1.5O between the two sites in the uc plane. The phase shift is defined by tan 2AB = 2gaC/(gaa-go), where AB is obtained from
+ gee) + '/2[(gaa- gcc12 +
which is equivalent to q 2.
900
(1)
g(e) = g,, cos2 e + g, sin2 e 2gii sin e cos e (2) so that the g tensor elements gii, gj,, and gijcan be determined directly from the rotation patterns g(0). The values of each gii calculated from two different crystal planes coincide within the experimental error. The errors of the rotation pattern fits are about Ag = f 2 X 10-5. Measurements on single crystals of deuterated RC's of Rb. sphueroides wild type 2.4.1 were restrictedto the bc plane because of rapid sample deterioration. However, well-resolved powder spectra (see next section) of these deuterated RC's could be obtained in a 1:1 mixture of glycerol and the same buffer solution in which the crystals were grown. These measurements were performed in the Fabry-Perot resonator under light illumination at 225 K. To extract the principal values of the g tensor, a simulation program by Burghaus14was used. The error is about & = 5 x 10-5.
4ga:]'/2
$0.
-i
b axis
+
*/2(gaa
6Lo
a axis
+
rotation angle I3
where the labels i and j denote pairs of crystal axes u, b, c. Since g = 2 bg with bg of the order of l e 3 , eq 1 can be linearized
de)
-t
+
cos 2(8 - At?) (3)
-I
C
2.0020
I
I
-900
I
I -600
I
I
,
I
-30°
I
c axis
baxis
I
J
,
Oo
I
I
1
30°
1
8 1 60'
I
I
,
I
90°
rotation angle 8 Figure 3. Rotation patternsofthegfactor of P865'+in the threesymmetry planes of a RC single crystal of Rb. sphaerofdes R-26. Theg values were obtained by a COMPASS deconvolution'8 of the partly resolved 3-mm EPR spectra (+). The two solid lines in each pattern are the leastsquares fits with eq 2 (satext). They belong to the pinvise magnetically inequivalent RC sites. The crossings of the curves wmspond to orientations, where BOlies parallel to one of crystallographic axes: (a) (IC plane, (b) ab plane, (c) bc plane.
Figure 3b shows the rotation patterns of the two sites in the ubplanewithasmallerphascshiftof2A~- 3 9 f 1.5O. Strikingly, any site splitting is missing in the bc plane (Figure 3c). The COMPASS deconvolution shows that the value of ga lies within the general experimental error of f5 X (see above). We therefore set gk = 0. We could affirm this choice by performing 3-mm EPR measurements on Rb. sphuerotdes wild type single crystals with perdeuteratcd BChl. The rmnance of the deuterated Pe6~'+does not show any site splitting in thebc plane. The EPR line width, which is reduced to 0.5 mT, does not show any systematic variation. The upper limit for kd is l e 5 .
Klette et al.
2018 The Journal of Physical Chemistry, Vol. 97, No. 9, 1993
( ) bewee0 tbe Priacipal Axe 2BELEgzfLy+j tensor 81 gar
0
gab
92
83
angle U
B Y U
B Y U
0
B
gab
&
U
gab gar
Figure 4. Three-millimeterEPR spectrum of Ps& in deuterated reaction centers of Rb. sphaeroides wild type 2.4.1 in frozen solution with 50% glycerol. X marks the M!= 1/2 line of the simultaneouslyrecorded Mn2+ standard (0.02% MnO in MgO powder).
Fitting the rotation pattern of both sites in all three planes with q 2 yields the components of the g tensor in the crystal axes system {a,bcJ: 2.002 98 iO.000 32 fO.OOO 40 g = &O.OOO 32 2.002 19 0 &0.00040 0 2.002 54
(
)
(4)
The values g,, gbb, and gab coincide within experimental error with former measurements in the ab plane.I2 The diagonalization of this tensor yields the principal values
g, = 2.003 29, ga = 2.002 39, gy = 2.002 03 with the average value
(5)
+
g, = '/3(ga go + gy) = 2.002 57 which is consistent with our W band measurements on frozen solutions but is distinguished by higher precision. Surprisingly, the smallest g value gr, is significantly below the free electron value,g, = 2.002 319. Thismaybeduetoarelativisticcorrection in the order of 2-3 X 10-4,as predicted theoreticallyby AngstLzo and/or to a considerable delocalizationof spin density into the u orbitals of the dimers2' The principal g values of P865'+ determined from the powder spectra of the deuterated RC's of Rb. sphaeroides wild type 2.4.1 (Figure 4) are
g, = 2.00 337, go 2.00 248, gr
2.00 208
(7)
with the average value of gi, = 2.002 64. The single-crystal Gaussian peak-to-peak line width used for simulating the powder spectra was 0.5 mT, in agreement with the experimental value from our single-crystal measurements (see above). The values show a slight systematical shift to higher gvalues, compared with nondeuterated Rb. sphaeroides R-26. This may be due to an orientational dependence of the line width, which is not modeled by the simulation program. Nevertheless, the deviation is still within the experimental errors. The additional important informationprovided by single-crystal measurements are the principal axes directions of the g tensor. Unfortunately, it is not possible to determine the sign of the offdiagonal elements solely from EPR results, but it should be provided by additional double resonanceexperiments. Since there is a 2-fold splitting in the rotation patterns, each sign belongs to one of the two inquivalent sites. Furthermore, by changing the symmetry plane, another combination of two sites forms the
Y
>0 0, gar> O), is given by the direction cosines 0.854
= (0.249).
0.303
B = (0.477
),
4.423
$ = (0.843 0.332
)
(8) 0.457 4.825 in the crystal axes system (a,b,c}. The eigenvectors of the other three g tensors differ from q 8 merely by changes in the signs of specific components. Although it is not possible to connect the BChl dimer of P865*+ unambiguously with the correct choiceof a principal axes system, a preselection can be tried by general considerations concerning the behavior of the g tensor of organic 7r radicals. In planar systems, such as the BChl monomer cation radical, the principal direction corresponding to the smallest eigenvalue, g,, lies near the normal to the molecular plane." A similar behavior is expected for &as'+ although theory is not yet able to translate the monomer gvalue data to the dimer case in a reliable way. To analyze our g tensors in this respect, a local coordinate system ( x y j }was constructed on the dimer ("dimer system"): the z axis is the average dimer normal (defining the average monomer planes as having the least sum of squares of distances to the four nitrogen atoms on the respective dimer half); the x axis is the projection of the Mg-Mg direction onto the plane normal to I . They axis then describes the approximate local C2 axis of the dimer. For positioningthe dimer system in the crystalaxes system, the recently determined X-ray structure2 was used. The angles between the dimer axes ( x y , z ) and the principal g tensor axes {a,@,y}are listed in Table I for all four g tensors. As follows from the argument mentioned above, the angles between the axes y and z should be small. Since the tensors g3 and 04 do not show the behavior typical for a 7r radical, they are most likely not the proper choice. The remaining choices, g1 and 02, both still show a significant deviation of the y axis from the z direction of 38O and 23O, respectively (Table I, Figures 5 and 6). The most importantresult, however, is the breakingof the local C2 symmetry; i.e., the @-axisdoes not coincide with the C2 axis in all cases. Rather, the deviation of the @ principal axis from the C2 axis is 32 & 4O for gl or 12 & 5.4O for 1 2 , respectively (Table I). These values lie clearly outside the experimental error of the angles. The breakage of C, symmetry is also outside possible errors of the X-ray structure analysis. Since the planes of the monomeric macrocycles are relatively well defined by the electron density around the dimer, the largest error in atomic positions is expected from a rotation around each of the normals to these planes. This rotational displacement was estimated to be at most 1 4 O for each macrocycle.6 The same error would have to be assigned to the
EPR of Rhodobacter sphaeroides R-26 Reaction Centers
The Journal of Physical Chemistry, Vol. 97, No. 9, 1993 2019
c2
c2
Y A
0
2
1
'd
I 4-
a
I
Figure 5. Tensor principal axes system of gl in P8&: (top) view from the averagedimernormal (zdirection) onto the monomerplanes; (bottom) view into the direction of the local Cz symmetry axis 0, axis).
Figure 6. Tensor principal axes system of g2 in P865": (top) view from the averagedimer normal (zdirection) onto the monomerplanes; (bottom) view into the direction of the local C2 symmetry axis 0, axis).
direction of the dimer C2 axis in the dimer xy plane. However, such a displacement of the C2 axis would only affect the angle between the C2 axis and the projection of the @ axis into the xy plane. In the gl case this angle is 30 f 3.7O (Figure 5, top). For g2it isonly 5.4f 3.4O (Figure6, top),whichcouldbecompensated by the rotational displacementerror. But the out-of-plane tilt of the @ axis of g2, being 10 f 4 O , remains unchanged (see Table I, where L(@,z) = 101O). The tilt angle of the @ axis of gl is 5 f 5' (Table I: L(@,z)= 96O). Thus, in both cases a significant deviation from the C2 direction remains, either in plane (gl) or as a tilt out of the BChl plane (g2).
in the Introduction, even strongerevidencefor symmetrybreaking in this state is supplied by recent ENDOR studieswhich revealed an asymmetricspin density distributionof p ~ / p M 2 in the dimer halves DL and DMof P865*+.6 Similar asymmetries in the spin density distribution have been observed by ENDOR in Pgm*+of Rp. uiridis RC's.23 Unfortunately,the result of the present highfield EPR work on g tensors cannot, at present, be backed up by quantum chemical calculations as was done for the hyperfine splittings. Calculations on g tensors are in progress; major difficulties are being encounteredin the calculation of the required numerous Q ?r excitationenergies in large dimers like P865*+.22 In conclusion, there remainsthe obviousquestion as to whether the asymmetry in the electronic structure is a general principle in the primary donors of the reaction centers of photosynthetic bacteria and whether this asymmetry is an important factor for the unidirectionality of electron transfer along the L protein subunit in the RC's. Analogous 3-mm EPR and ENDOR experiments at X and W bands on RC single crystals of other organisms with even more complex unit cell structure are in progress and will, hopefully, help to answer this question.
4. Conclusion
The g tensor of the primary donor cation radical P8& of the reaction center of Rb. sphaeroides R-26 could be completely determined. This was possible because of the enhanced g resolution of high-field EPR allowing to separate the two magneticallyinequivalent RC sitesin the symmetry planes of the single crystal. Principally, a 4-fold ambiguity remains, since the signs of the off-diagonal elements of the g tensor cannot be determined by EPR. In view of known properties of g values of organic r radicals, only two choices for the g tensor remain. A definite distinction could be provided by additional experiments, such as 3-mm ENDOR or TRIPLE-inducedX-band EPR. Such experiments are planned for the near future. Even on the basis of the present results, it is clearly shown that the local C2 symmetry of the three-dimensionalstructure of the dimer is broken for the electronic structure of the P865*+state. As has been mentioned
-
-
Acknowledgment. The authors thank Profes$ors G. Feher (University of California,San Diego), J. P. Allen (Arizona State University, Tempe), and D. C. Rees (California Institute of Technology, Pasadena) for providing the X-ray structural data of the RC of Rb. sphaeroides R-26 (refinement of Sept 1991). We are grateful to Mr. J. Claus from our machine shop, who skillfully built the goniometer. We thank Drs. F,Lendzian, 0.
2024 The Journal of Physical Chemistry, Vol. 97, No. 9, 1993 Burghaus, M. Huber,and Dipl. Phys. M. Rohrer for helpful discussionsand the refereesfor their constructivecriticism.This work was supported by the Deutsche Forschungsgemeinschaft (SFB's 312 and 337).
Refer"
md Notes
( I ) Michel, H.; Epp, 0.; Deisenhofer, J. EMBO J. 1986, 5, 2445 and references therein. (2) Feher, G.; Allen, J. P.; Okamura, M. Y.; Rees. D. C. Nature 1989, 339, 11 1 and references therein. (3) Lubitz, W. InChlo~~hylls;Schccr,H.,Ed.;CRCPress: BocaRaton, _ . FL, 1991; p 903. (4) Plato, M. In Chlorophylls; Schccr, H., Ed.;CRC Pres: Boca Raton, FL, 1991; p 1015. (5) LOUS,E. J.; Huber, M.; Isaacson, R. A.; Feher, G . In Springer Series in Biophysics; Michel-Beyerle, M.E., Ed.;Springer: Berlin, 1990; Vol. 6, p 45. Lendzian, F.;Endeward, B.;Plato, M.;Bumann, D.; Lubitz, W.; Mdbius, K.In Springer Series in Biophysics; Michel-Beyerle, M.E., Ed.; Springer: Berlin, 1990; Vol. 6, p 57. (6) Lendzian, F.; Endeward,B.; Plato, M.;Mdbius,K.;Wnigk, B.; Lubitz, W.; Huber. M;Isaacson, R. A,; Feher. G.Biochim. Biophys. Acra, to be. published. (7) Michel-Beyerle,M. E.; Plato, M.; Deisenhofer, J.; Michel, H.; Bixon, M.; Jortner, J. Biochim. Biophys. Acra 1988. 932, 52. (8) Plato, M.; Mdbius, K.; Michel-Beyerle, M. E.; Bixon, M.; Jortner, J. J. Am. Chem. Soc. 1988, 110, 7279.
Klette et al. (9) Norris. J. R.; Katz, J. J. In The Phorosynrheric Bacreria; Clayton, R. K., Sistro, W. R., Eds.; Plenum Press: New York, 1987; p 397. (IO) McElroy, J. D.; Fcher, G.;Msuzerall, D. C. Biochim. Biophys. Acra 1972, 262, 363. (1 1) Allen, J. P.; Feher,G. Proc. Narl. Acad. Sci. U S A . 1984,81,4795. (12) Burghaus, 0.;Plato. M.; Bumann, D.; Neumann, B.; Lubitz, W.; Mdbius, K. Chem. Phys. Lrrr. 1991. 185, 381. ( I 3) Burghaus, 0.;Tbth-Kischkat, A.; Klette, R.; Mdbius, K. J. Magn. Reson. 1988, 80, 129. (14) Burghaus, 0. Ph.D. Thesis, Free University Berlin, Berlin, 1991. (15) Burghaus.O.;Rohrer,M.;Gdtzingcr,T.;Plato,M.; MBbius,K.Meas. Sei. Technol. 1992, 3, 765. (16) Feher, G.; Allen, J. P. In Molecular Biology of the Phorosynrheric Apparatus; Cold Spring Harbor Laboratory: 1985; p 163. Allen, J. P.; Feher,
G.;Yeates, T. 0.;Komiya. H.;Rcts, D. C. Proc. Narl. Acad. Sci. U.S.A. 1987, 84, 5730. Allen, J. P.; Feher, G . In Crysrallizarion of Membrane
Proteinr; Michel, H., Ed.; CRC Press: Boca Raton, FL, 1991; p 137. (17) Bumann, D. Diploma Thesis, Free University Berlin, Berlin, 1989. (18) TrHnkle, E.;Lcndzian, F. J. Magn. Reson. 1989,84, 537. (19) Poole, C. P.; Farach, H. A. The Theory of Magnetic Resonance; Wiley-Interscience: New York, 1972; p 97. (20) Angstl, R. Chem. Phys. 1989, 132,435. (21) Prabhananda, B. S.; Felix, C. C.; Hyde, J. S.;Walvekar, A. J . Chem. Phys. 1985,83,6121. (22) Stone, A. J. Mol. Phys. 1964. 7 , 311. (23) Lendzian, F.; Lubitz, W.; Scheer, H.; Hoff, A. J.; Plato, M.; TrHnlde, E.; Mdbius, K. Chem. Phys. Lerr. 1988, 148, 377.
