The g-Factor Anisotropy of Plant Chlorophyll a•+

VOLUME 104, NUMBER 30, AUGUST 3, 2000

© Copyright 2000 by the American Chemical Society

LETTERS The g-Factor Anisotropy of Plant Chlorophyll a•+ Peter J. Bratt,† Oleg G. Poluektov,‡ Marion C. Thurnauer,‡ J. Krzystek,§ Louis-Claude Brunel,§ Joshua Schrier,|,⊥ Ya-Wen Hsiao,⊥ Michael Zerner,⊥,¶ and Alexander Angerhofer*,† Department of Chemistry, UniVersity of Florida, Box 117200, GainesVille, Florida 32611, Chemistry DiVision, Argonne National Laboratory, 9700 South Cass AVenue, Argonne, Illinois 60439, Center for Interdisciplinary Magnetic Resonance, National High Magnetic Field Laboratory, Florida State UniVersity, 1800 East Paul Dirac DriVe, Tallahassee, Florida 32310, Department of Chemistry, Saint Peter’s College, 2641 Kennedy BouleVard, Jersey City, New Jersey 07306, and Quantum Theory Project, UniVersity of Florida, Box 118440, GainesVille, Florida 32611 ReceiVed: March 24, 2000; In Final Form: June 8, 2000

High-field EPR experiments at 12 T/330 GHz were performed on chlorophyll a radical cations in methylene chloride at low temperatures. Using fully deuterated chlorophyll it was possible to obtain the principal components of the rhombic g-tensor as gR ) 2.00329 ( (5 × 10-5), gβ ) 2.00275 ( (8 × 10-5), and gγ ) 2.00220 ( (8 × 10-5). Protonated chlorophyll radicals did not give enough spectral resolution to yield the g-anisotropy from the EPR spectrum. This was true even at higher field/frequency combinations up to 24 T/670 GHz. Semiempirical calculations were performed using the INDO/S method (ZINDO) which yielded good agreement with the experimental data.

1. Introduction Chlorophyll a (Chl a) is the main pigment in the photosynthetic system of higher plants. It serves both as the main energy carrying component of the antenna complexes as well as the primary donor in the photosynthetic reaction center protein (RC). During the primary event an electron is transported through a chain of pigments in the RC yielding an electron-hole pair * To whom correspondence and requests for materials should be addressed at: Alexander Angerhofer, Chemistry Department, University of Florida, Gainesville, FL 32611. Phone: (352) 846 3281. Fax: (352) 392 0872. E-mail: [email protected]. † Department of Chemistry, University of Florida. ‡ Argonne National Laboratory. § Florida State University. | Saint Peter’s College. ⊥ Quantum Theory Project, University of Florida. ¶ Our friend and colleague, Mike Zerner, passed away on February 2, 2000.

which is used to drive subsequent dark reactions.1 The primary donor is a special pair of chlorophyll molecules which was recognized by EPR and ENDOR spectroscopy of its radical cation2-4 and later confirmed by X-ray crystallography of the RC protein of purple photosynthetic bacteria.5 The electronic structure of the primary donor radical cation of plant photosystem I (PS I), P•+ 700, has been studied extensively by EPR, electron epin echo envelope modulation (ESEEM), and electron nuclear double resonance (ENDOR) spectroscopy.6-14 These studies indicate that P•+ 700 is a weakly coupled dimer where the spin density is either asymmetrically distributed over the dimer halves or may be explained by an admixture of the first excitedstate MO to a localized doublet ground state. A complementary approach to characterizing the electronic structure of the primary donor radical cation is to determine the anisotropy of the g-tensor, which reflects the symmetry properties of the primary donor. Furthermore, these parameters

10.1021/jp001126l CCC: $19.00 © 2000 American Chemical Society Published on Web 07/06/2000

6974 J. Phys. Chem. B, Vol. 104, No. 30, 2000 play a crucial role in the interpretation and correct simulation of the spin-polarized EPR spectra of the light-induced radical pair.15-17 At X-band, (9-10 GHz), the frequency at which EPR measurements are traditionally performed, the spectra of both •+ are featureless Gaussians dominated by P•+ 700 as well as Chl a unresolved proton hyperfine splitting.2 To resolve the g-tensor, it is necessary to perform the EPR experiment under high magnetic field conditions provided that the g-strain (or gheterogeneity) is small enough to allow the full spectral resolution of the (generally) rhombic EPR powder spectrum. This approach also affords orientational selectivity for an isotropic sample since the low and high-field edges of the powder spectrum can be assigned to molecules being aligned parallel to the external magnetic field along two different principal axes. Thus, when high-field ENDOR at the necessary frequencies (g330 GHz) becomes routine it should be possible to clarify some of the remaining issues raised by the low-field ESEEM and ENDOR experiments regarding the spin distribution over the dimer radical cation.14 Here, we report on high-field studies of Chl a•+ cation radicals in vitro in methylene chloride frozen solution where the fields have been increased sufficiently to afford the necessary resolution. Interestingly, it was not possible to obtain full resolution of the g-tensor for protonated Chl a even by going to fields as high as 24 T. This was due to a concurrent increase in line width indicating severe g-strain in this matrix. The g-anisotropy therefore was obtained with perdeuterated Chl a which exhibited line widths narrow enough to observe the full rhombic g-tensor. 2. Materials and Methods Protonated and deuterated Chl a was extracted from cells of Scenedesmus obliquus.18 For extraction of the deuterated Chl a 99% deuterated cells were used. Procedures for deuteration of Scenedesmus obliquus cells were described by Crespi et al.19 The Chl a was treated with iodine in stoichiometric quantities to prepare the radical cation in methylene chloride. The samples were prepared under nitrogen gas atmosphere and frozen in liquid nitrogen before being transferred to the sample holder. The high field EPR spectrometer has been described before.20 Field calibration and g-factor determination were performed as previously described using Mn2+ (0.05%) in MgO as a standard.21 From sweeps taken over the entire Mn2+ spectral range, we can obtain a linearity of our field sweep (main coil in persistent mode) of better than 0.5 mT over 50 mT, corresponding to an accuracy of approximately 0.05 mT within the sweep range of the primary donor radical cation spectrum. Previously, the accuracy of the calibration against the Mn2+ was limited by the Mn2+ line widths at low temperature.21 With repeated heating/pumping cycles, however, it is possible to achieve line widths of ∼0.1 mT for the Mn-standard even at cryogenic temperatures, which increases the absolute accuracy of the calibration. The absolute error obtained using this calibration method is estimated as being of the order of δg ) (5 × 10-5. We state a somewhat larger error of (8 × 10-5 for gβ and gγ because of the effects of dispersion on the highfield side line shape (as discussed below). For the relative errors of the g-values (errors in the determination of ∆g) we estimate a value of δ∆g ) (2 × 10-5. To obtain the anisotropic g-tensor the spectrum was simulated with a powder average, using (a) the SIMFONIA package supplied by Bruker Instruments Inc., and (b) the “ddpowhe” software package kindly given to us by Dr. J. Telser. The fits obtained this way are reasonable but not perfect. The fits can be improved by weighting the contributions from the three

Letters

Figure 1. Solid Line: EPR spectrum of deuterated Chl a•+ radical cations in methylene chloride at 10 K. EPR frequency was 327.282 GHz. Far-IR power incident at the sample was about 100 µW. Field modulation was 2 G at 8 kHz. The spectrum is a result of the accumulation of 10 scans. Dashed Line: Example simulated spectrum with gR ) 2.00329 (low field line), gβ ) 2.00275, and gγ ) 2.00220 (high field line) with line widths of ∆VR ) 30 MHz, ∆Vβ ) 17 MHz, and ∆Vγ ) 35 MHz, and relative weights for the intensities of 1.25, 0.70, and 0.25, respectively. Solid Line through center with sharp peaks: EPR spectrum of the calibration standard (Mn2+ in MgO). Four low-field Mn2+ lines are visible as well as a single line at 11.6675 T of unknown origin.

principal orientations of the g-tensor differently (see Figure 1). As is often the case in high-field EPR spectroscopy, the observed line shape is a convolution of absorption and dispersion due to rapid passage effects. To correct for this the spectra were subjected to a Hilbert transform followed by a phase correction and back-transform. This resulted in improved (differentiated) line shapes as judged by inspection. Since this procedure may in principle change the apparent g-factors extracted from the spectrum, simulations were done on a series of corrected spectra in which the phase correction was varied over 30°. The largest shift was observed for the zero crossing field of the spectra thus affecting the error margin of mainly gβ. The simulation yielded values within (8 × 10-5 which is similar to the error estimated from the calibration procedure. Since the gγ peak appears as a shoulder on the high field side of gβ (see Figure 1) and thus is not as readily distinguishable as the gR peak, a similarly large error of (8 × 10-5 was assumed for gγ. Theoretical calculations were performed by the semiempirical INDO/S method using the ZINDO code as described by Hsiao and Zerner.22 To calculate the g-tensors, geometry-optimized structures were placed in a simulated methylene chloride solvent to approximate the experimental conditions (static dielectric constant of 7.77, refractive index of 1.4242, density of 1.3266 g/cm3). Three structures were used in the calculations: (a) a purely computational structure based on a molecular mechanics simulation (MM+ force field in vacuo) using the commercial software package Hyperchem, and (b) and (c) the two Chl a molecules in the protein subunit of the dinoflagellate peridininchlorophyll-protein antenna complex from Amphidinium carterae.23 Unmodified structures were taken as given by (a) the output of the Hyperchem calculation and (b,c) the coordinates contained in the Brookhaven Protein Data Bank entry 1PPR. Optimized structures were obtained by a geometry optimization using the ROHF model and INDO/1 parametrization within ZINDO using methylene chloride as the solvent. For the theoretical structure (a) all atom positions were optimized while

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J. Phys. Chem. B, Vol. 104, No. 30, 2000 6975

21,35 Chl a•+) and Theoretical TABLE 1: g-factor Anisotropy of the Primary Donor Cation Radical from Experiment (P•+ 700, Calculations (a - c)a

P•+ 700 Chl a•+ (a)* (a)† (b) (c)

temp (K)

105(gR - 2)

105(gβ - 2)

105(gγ - 2)

105∆gxx-yy

105∆gyy-zz

105(giso - 2)

ref

40 200 290 10

317(7) 307(7) 304 329(5) 291 301 290 289

264(7) 260(7) 262 275(8) 258 287 278 277

226(7) 226(7) 232 220(8) 197 223 219 221

53 47 42 54 33 14 12 12

38 34 30 55 61 64 59 56

269 264 266 275 249 270 262 262

21 21 35 this work this work this work this work this work

a The structure (a)* for the nonoptimized Chl a was obtained from a model built with the commercial software package Hyperchem. It was optimized in vacuo using molecular mechanics with the MM+ force field. A further optimized structure (a)† was obtained after geometry optimization using an ROHF calculation with the ZINDO/1 parametrization in a dielectric environment representative of methylene chloride. The structures (b) and (c) represent the two Chl a molecules from the PCP antenna protein of Amphidinium carterae (heteroatom groups 601 and 602 in the PDB file 1PPR from the Brookhaven Protein Data Bank, respectively).23 The hydrogens were added to the structure using the model building facility of Hyperchem. Geometry optimization was done only on the hydrogen atoms using the same methods as for (a)†.

for structures (b) and (c) only the hydrogen atoms (not given in the X-ray structure) were optimized. The wave function was calculated using the ROHF model within INDO/S followed by setting up the CI matrix for full single excitations for type 1 (doubly occupied to singly occupied molecular orbital) and type 2 (singly occupied to virtual molecular orbital) excitations. The diagonal elements of the CI matrix provided the state energies which, together with the orbital coefficients were then used as input for the g-factor calculation program g_rhf, kindly provided to us by Dr. J. To¨rring and modified according to Hsiao and Zerner.22 Two- and three-center integrals were retained for the determination of the orbital angular momentum matrix elements as suggested by To¨rring et al.24 3. Results and Discussion Figure 1 shows the EPR spectrum of deuterated Chl a•+ radical cations in methylene chloride at 10 K taken at an EPR frequency of 327.282 GHz together with a simulation (dashed line) and the Mn2+ calibration spectrum. The spectrum was acquired as the derivative of the absorption mode due to field modulation and clearly shows all three main peaks of the powder average. The simulated spectrum is just one of many used to probe the error margins of the spectral parameters (principal components of the g-tensor and anisotropic line widths). The main results from the simulations are the components of the g-tensor which are conservatively given as gR ) 2.00329 ( (5 × 10-5), gβ ) 2.00275 ( (8 × 10-5), and gγ ) 2.00220 ( (8 × 10-5). The anisotropic line widths used were in the following ranges: ∆VR ) 20 - 30 MHz, ∆Vβ ) 20 - 30 MHz, and ∆Vγ ) 30 - 50 MHz. These values are in excellent agreement with a very recent D-band EPR study of electrontransfer pathways in PS II, where the g-anisotropy for the deuterated ChlZ radical cation was measured.25 While the absolute g-values have not yet been fully analyzed in that study, the observed differences between gR, gβ, and gγ are the same as ours within experimental error. This is a clear indication that possible artifacts during sample preparation such as oxidation byproducts or clustering, as well as different solvent environments did not play a big role in determining the g-tensor. Protonated Chl a•+ was also investigated but only yielded an asymmetric and unresolved EPR line at 330 GHz and higher frequencies up to 670 GHz (data not shown). Contrary to plant and bacterial RC's in which the EPR line width remained more or less constant with field,21,26 the line width increased with the field for Chl a•+ in methylene chloride, indicating the presence of g-strain and making it impossible to obtain the g-anisotropy for the protonated Chl a•+ radical in the organic

solvent matrix. Assuming the same amount of g-strain for deuterated and protonated Chl a•+ the line widths in the spectrum of the protonated cation would be expected to be four times as large as those in the deuterated case. This would require a four times higher field (i.e., 46 Tesla) to achieve the same resolution for the protonated species, everything else being equal. Such fields were not available for the current study. Theoretical calculations were performed to predict the g-factor anisotropy of Chl a•+. We follow the procedure outlined by Hsiao and Zerner.22 The doublet radical wave function and energies were calculated using the ROHF model within the INDO/S approach. This semiempirical model is fast and has been used successfully for the calculation of the optical spectrum of systems as large as the bacterial RC in Rhodopseudomonas Viridis.27 It was therefore anticipated that it would also do well in determining the electronic g-factor and its anisotropy in large extended radicals such as Chl a•+. The calculations presented here (see Table 1) were performed using simulated as well as crystal structures of Chl a for the radical cation in a dielectric environment consistent with methylene chloride. The results of the calculation with the simulated structure (a) are remarkably good. gγ falls within the experimental error margin while gβ is slightly above and gR somewhat more below the experimental values. The prediction for the isotropic g-factor (giso ) 1/ ∑ 3 i)R,β,γgi), 2.00270, is remarkably close to the experimental value, 2.00275. When using the X-ray structure of the peridinin-chlorophyll-protein (PCP) complex from Amphidinium carterae23 the calculated values of gβ and gγ were also remarkably close to the experimental data (see Table 1). In this case only gR was off, i.e., too low and in fact much too close to gβ rendering the g-tensor almost axial. Generally, the theoretical model seems to underestimate the actual anisotropy in the x,yplane, and the largest error appears in the determination of gR. Despite this shortcoming, the degree of agreement with experiment is unusually high for such highly precise measurements. Subtracting 2 from the g-values, the relative error for the calculation of the optimized structure (a) from experiment is about 4% (for gβ) and the largest deviation for the predicted gR is 9%. This is encouraging and further refinements as suggested by Hsiao and Zerner22 may make this approach of general utility to predict and/or correct structure based on ESR data. Another piece of information coming out of the theoretical calculations is the g-tensor orientation with respect to the molecular frame. This is given in Table 2 and Figure 2. The orientation of the g-tensor is largely determined by the shape of the MO’s that are used in the perturbation derivation of g. In particular, the frontier orbitals are important as their energy

6976 J. Phys. Chem. B, Vol. 104, No. 30, 2000

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TABLE 2: g-tensor Orientation of the Primary Donor Cation Radical in Chl a•+ Obtained from the Theoretical Calculationsa structure

(a)* (a)† (b) (c)

angles between the principal axes of the g-tensor and the molecular frame ∠(gR,x0) ∠(gβ,x0) ∠(gγ,x0) 90.9 172.4 82.4 72.2 161.7 94.2 152.8 -115.8 80.2 -21.4 68.7 87.9

∠(gR,y0) ∠(gβ,y0) ∠(gγ,y0) 162.2 -91.4 107.7 162.2 -107.8 90.3 -115.5 -25.8 87.4 68.7 158.7 90.2

∠(gR,z0) ∠(gβ,z0) ∠(gγ,z0) 107.8 82.5 19.4 89.0 94.1 4.2 81.1 89.6 10.2 91.9 91.0 2.1

a The notation used in this table is as follows: x , y , and z are the 0 0 0 unit vectors of the molecular coordinate system. Its x-axis is defined by the vector combining the two nitrogens on pyrrole rings II and IV. The z-axis is defined by the cross product of x0 with the vector combining the nitrogens on pyrrole rings I and III, and the y-axis is defined by the cross product between x0 and -z0. This leads to a slight deviation of y0 from the line through N1 and N3 of usually not more than a couple of degrees due to the in-plane skew of the structures. The angles between each principal g-tensor axis and the three unit vectors of the molecular coordinate system given in the table are in degrees. (a)* is the unoptimized structure (a); (a)†, the optimized as explained in the text.

Figure 2. Structure of Chl a with molecular x- and y-axes (x0, y0) and the principal axes of the g-tensors for the calculations on structure (a)† (denoted by R and β for the gR and gβ orientations), and (b) (denoted by R′ and β′, respectively). Note that the g-tensor orientation of (c) is very similar to that of (b).

denominator is the smallest and will thus have a larger weight in the sum over all the participating MO’s.28 As expected, gγ points along the normal to the chlorin molecular plane (at least for the optimized structures). For structure (a)†, the R- and β-components of the g-tensor are rotated from the molecular axis system in the x,y-plane by approximately 17° (see Figure

2). This may very well be caused by the particular orientation of the ethyl group on carbon 2 and/or the carboxymethyl group on carbon 10. Similar effects on the predicted g-tensor orientation for the bacteriochlorophyll (BChl) a cation by different orientations of the acetyl group have been noted by Plato and Mo¨bius.28 The approximate alignment of the principal axis for the largest g-factor (gR) along the molecular y-axis in the calculation for structure (a)† is also in agreement with these earlier studies. For the two PCP Chl’s gR is approximately aligned along the x-axis (with an offset of about +26°). One should note that in all of these calculations gR is underestimated and much too close to gβ which creates an approximate degeneracy between these two principal axes and thus renders the tensor orientation in the x,y-plane somewhat unreliable. After having determined the principal components of the anisotropic g-factor it is interesting to compare them with data obtained earlier on the radical cation of the primary donor in plant photosystem I (see Table 1).21 Correspondence of all three g-values within the combined error margins of the previous experiments on PS I and those reported here is found for low temperatures. It is expected and has actually been shown theoretically that the g-factor is very sensitive to the local geometry of the radical in question.22 The agreement between the values found for Chl a•+ in vitro and those of PS I (P+• 700) at low temperatures thus seems to indicate that the spin distribution of the radical electron in the primary donor is more monomerlike than dimerlike. Such a model had been proposed early on in the literature by O’Malley and Babcock based on proton ENDOR data.29 Later ESEEM and ENDOR data were interpreted in terms of a dimer model similar to that in bacterial RC's with an asymmetry of the spin distribution of about 3:17 or g6:1.8-12 More recently, multifrequency ESEEM combined with ENDOR and isotopic substitution and model calculations by Mac et al. explain the primary donor cation radical with a monomeric spin density distribution, but with a 25% admixture of the first excited state to the ground-state orbital.14 Our data at first glance support the monomeric model since the ganisotropy of both Chl a•+ is within the error margins of that observed for P+• 700 at low temperatures. The largest deviation between the two data sets is seen for gR where the in vitro radical shows a somewhat higher value. This could be due to either an admixture of the first excited doublet state as postulated by Babcock and co-workers14,29 or to some matrix-induced rearrangement of spin density as suggested by Lubitz and coworkers.11,12 The observed temperature dependent decrease in the g-factor anisotropy in PS I1 suggests either a redistribution of electron spin density on the dimer or a rotation of the two dimer halves with respect to each other. If we assume that the observed temperature dependence is due to increased delocalization of the spin density over the dimer, a more symmetric dimeric spin distribution would exhibit a more isotropic g-tensor. This would be consistent with the primary donor radical cation of Helibacillus mobilis which only shows an axially resolved g-tensor (gR ≈ gβ) even up to 24 T.30 The latter species contains a dimeric 1:1 spin distribution.31,32 However, it should be noted that the ENDOR spectra of P+• 700 do not change significantly between frozen solution at 77 K and room temperature, making a temperature-dependent redistribution of the electron spin rather unlikely.10,33 In the case of RC's from the purple bacterium Rhodobacter sphaeroides R26 the observed g-anisotropy of P+• 870 is temperature-insensitive despite an expected temperature dependent change in electronic structure.26 Comparison with the g-factor

Letters anisotropy of monomeric BChl a also revealed little difference.34 This indicates that g-factor information alone is not sufficient to predict electronic structure. The real potential of high-field EPR to resolve this problem may therefore lie in the application of orientation-selective ENDOR which is now possible since the full g-anisotropy of many protonated chlorophyll radicals can be resolved by EPR above 12 T.21 Acknowledgment. This work was supported by the National High Magnetic Field Laboratory. The high-field Bitter magnet (up to 25 T with high homogeneity) was funded by the Keck foundation. L.C.B. acknowledges the support from the Human Frontiers in Science program (HFSPO grant RGO349). J.A.S acknowledges the support of the NSF-REU program while at UF. Theoretical calculations at QTP were supported by the Office of Naval Research and by a SUR grant from IBM. Work at ANL (M.C.T. and O.G.P.) was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, under contract W-31-109-Eng38. We thank A. M. Wagner for assistance in obtaining deuterated chlorophyll a. The EPR work in Gainesville was supported by NSF (grant DMR-9601864). We thank Joshua Telser for providing his ddpowhe package for spectral simulations and Jens To¨rring for his program g_rhf that was used for the g-factor calculations after minor modifications. References and Notes (1) Feher, G.; Okamura, M. Y. In The Photosynthetic Bacteria; Clayton, R. K.; Sistrom, W. R. Eds.; Plenum Press: New York, 1978. (2) Norris, J. R.; Uphaus, R. A.; Crespi, H. L.; Katz, J. J. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 625-628. (3) Norris, J. R.; Scheer, H.; Druyan, M. E.; Katz, J. J. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 4897-4900. (4) Feher, G.; Hoff, A. J.; Isaacson, R. A.; Ackerson, L. C. Ann. N. Y. Acad. Sci. 1975, 244, 239. (5) Deisenhofer, J.; Epp, O.; Miki, K.; Huber, R.; Michel, H. J. Mol. Biol. 1984, 180, 385-398. (6) Lubitz, W. In Chlorophylls; Scheer, H., Ed.; CRC Press Inc.: Boca Raton, FL, 1991. (7) Davis, I. H.; Heathcote, P.; MacLachlan, D. J.; Evans, M. C. W. Biochim. Biophys. Acta 1993, 1143, 183-189. (8) Ka¨ss, H.; Fromme, P.; Witt, H. T.; Lubitz, W. Biophys. J. 1994, 66, A228. (9) Ka¨ss, H.; Bittersmann-Weidlich, E.; Andre´asson, L.-E.; Bo¨nigk, B.; Lubitz, W. Chem. Phys. 1995, 194, 419-432. (10) Ka¨ss, H. 1995 Die Struktur des prima¨ren Donators P700 in Photosystem I - Untersuchungen mit Methoden der stationa¨ren und gepulsten Elektronenspinresonanz, Ph.D. thesis, Technische Universita¨t Berlin.

J. Phys. Chem. B, Vol. 104, No. 30, 2000 6977 (11) Ka¨ss, H.; Lubitz, W. Chem. Phys. Lett. 1996, 251, 193-203. (12) Ka¨ss, H.; Fromme, P.; Lubitz, W. Chem. Phys. Lett. 1996, 257, 197-206. (13) Mac, M.; Tang, X.-S.; Diner, B. A.; McCracken, J.; Babcock, G. T. Biochemistry 1996, 35, 13288-13293. (14) Mac, M.; Bowlby, N. R.; Babcock, G. T.; McCracken, J. J. Am. Chem. Soc. 1998, 120, 13215-13223. (15) van der Est, A.; Prisner, T.; Bittl, R.; Fromme, P.; Lubitz, W.; Mo¨bius, K.; Stehlik, D. J. Phys. Chem. B 1997, 101, 1437-1443. (16) Utschig, L. M.; Greenfield, S. R.; Tang, J.; Laible, P. D.; Thurnauer, M. C. Biochemistry 1997, 36, 8548-8558. (17) Berthold, T.; Bechtold, M.; Heinen, U.; Link, G.; Poluektov, O. G.; Utschig, L.; Tang, J.; Thurnauer, M. C.; Kothe, G. J. Phys. Chem. B 1999, 103, 10733-10736. (18) Svec, W. A. In The Porphyrins; Dolphin, D. Ed.; Academic Press: New York, 1978; Vol. V, pp 341-399. (19) Crespi, H. L.; Conrad, S. M.; Uphaus, R. A.; Katz, J. J. Ann. N. Y. Acad. Sci. 1960, 84, 648-666. (20) Hassan, A. K.; Pardi, L. A.; Krzystek, J.; Sienkiewicz, A.; Goy, P.; Rohrer, M.; Brunel, L.-C. J. Magn. Reson. 2000, 142, 300-312. (21) Bratt, P. J.; Rohrer, M.; Krzystek, J.; Evans, M. C. W.; Brunel, L.-C.; Angerhofer, A. J. Phys. Chem. B 1997, 101, 9686-9689. (22) Hsiao, Y.-W.; Zerner, M. C. Int. J. Quantum Chem. 1999, 75, 577584. (23) Hofmann, E.; Wrench, P. M.; Sharples, F. P.; Hiller, R. G.; Welte, W.; Diederichs, K. Science 1996, 272, 1788-1791. (24) To¨rring, J. T.; Un, S.; Knu¨pling, M.; Plato, M.; Mo¨bius, K. J. J. Chem. Phys. 1997, 107, 3905-3913. (25) Thurnauer, M. C.; Poluektov, O. G.; Lakshmi, K. V.; Reifler, M. J.; Brudvig, G. W. Paper to be presented at the “Thirteenth International Conference on Photochemical Conversion and Storage of Solar Energy”, Snowman, Colorado, 2000. (26) Bratt, P. J.; Ringus, E.; Hassan, A.; van Tol, H.; Maniero, A.-L.; Brunel, L.-C.; Rohrer, M.; Bubenzer-Hange, C.; Scheer, H.; Angerhofer, A. J. Phys. Chem. B 1999, 103, 10973-10977. (27) Thompson, M. A.; Zerner, M. C. J. Am. Chem. Soc. 1991, 113, 8210-8215. (28) Plato, M.; Mo¨bius, K. Chem. Phys. 1995, 197, 289-295. (29) O’Malley, P. J.; Babcock, G. T. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 1098-1101. (30) Bratt, P. J.; Heathcote, P.; Angerhofer, A.; Hassan, A.; Maniero, A.-L.; Brunel, L.-C. High Field EPR Spectroscopy of the Cation Radical Primary Electron Donors of Chlorobium limicola and Heliobacillus mobilis. Annual report; National High Magnetic Field Laboratory; 1998. (31) Rigby, S. E. J.; Thapar, R.; Evans, M. C. W.; Heathcote, P. FEBS Lett. 1994, 350, 24-28. (32) Bratt, P. J.; Muhiuddin, I. P.; Evans, M. C. W.; Heathcote, P. Photochem. Photobiol. 1996, 64, 20-25. (33) Mo¨bius, K.; Lubitz, W. In Biological Magnetic Resonance; Berliner, L. J., Reuben, J. Eds.; Plenum Press: New York, 1987;. (34) Burghaus, O.; Plato, M.; Rohrer, M.; Mo¨bius, K.; MacMillan, F.; Lubitz, W. J. Phys. Chem. 1993, 97, 7639-7647. (35) Prisner, T. F.; McDermott, A. E.; Un, S.; Norris, J. R.; Thurnauer, M. C.; Griffin, R. G. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 9485-9488.