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J. Phys. Chem. 1994, 98, 9314-9319
Revisited Depolarization Ratio Dispersion of Raman Fundamentals from Heme c in Ferrocytochrome c Confirms That Asymmetric Perturbations Affect the Electronic and Vibrational Structure of the Chromophore's Macrocycle Reinhard Schweitzer-Stennert Biophysics Research Division, University of Michigan, 930 North University, Ann Arbor, Michigan 481 09 Received: June 3, 1994; In Final Form: July 14, I994@ We have remeasured the depolarization ratio of some high-frequency Raman fundamentals from heme c in horse heart ferrocytochrome c at different excitation wavelengths provided by an argon ion laser. All investigated Raman lines exhibit a significant dispersion of their depolarization ratios. These results contradict recent suggestions by Hu et al. (J.Am. Chem. SOC.1993,115,12466) made on the basis of Raman experiments on various isotopomers of the heme c group in yeast ferrocytochrome c and confirm earlier studies by Schweitzer-Stenner et al. (J.Raman Specrrosc. 1991, 22, 65). The depolarization ratio reflects asymmetric distortions of the heme symmetry imposed by the central Met 80 ligand, the peripheral thioether bonds, and the protein environment. The latter gives rise to nonplanar distortions which are reflected by antisymmetric and symmetric contributions to the Raman tensor to A*, and AI, modes of the heme c macrocycle, respectively.
Introduction It is widely appreciated that resonance Raman spectroscopy provides an excellent tool to study structure and dynamics in heme proteins' owing to the selective enhancement of heme vibrations by excitation in the visible region between 380 and 600 nm. While many researchers have used the ground state frequencies of Raman-active modes to elucidate even subtle structural changes2 of the porphyrin macrocycle induced for instance by axial ligation of the central metal,3 changes of its spin c~nfiguration,~,~ displacement of its central metal atom? and relaxation processes within the protein envir~nment,~.' our laboratory investigated the mechanism governing Raman line intensities under different excitation conditions.8 To this end we measured the depolarization ratio (DPR) of some structuresensitive Raman lines as a function of the excitation wavelength. The DPRs of all macrocycle lines exhibited by model porphyrins with a pure D4h symmetry (for instance, Ni(I1)-porphin) are nearly independent of the excitation energyg and identical to their excitation values, i.e., 0.125 for symmetric AI, modes, 0.75 for asymmetric B1, and B2, modes, and infinite for antisymmetric Azg modes. Asymmetric distortions imposed by peripheral substituents and heme-protein interaction, however, give rise to a significant DPR dispersion, which has been found to be very sensitive to changes in the protein environment caused by proton binding to distinct amino acid residues involved in the regulation of oxygen binding.8s10 Rather strong dispersion effects have been found for Raman lines of horse heart ferrocytochrome c . ~ , ~The ' oxidation marker line v4 at 1362 cm-', for instance, which is normally polarized owing to the AI, character of its normal mode in an unperturbed D4h symmetry, has been shown to become inverse polarized between its QOand Qv resonance positions exhibiting a maximal DPR of 7," while it maintains its polarized state (DPR = 0.140.18) in the Soret band region."J2 Similar strong dispersion effects were obtained for the Raman lines of the antisymmetric modes v19 (1312 cm-') and ~21(1582cm-~) and the asymmetric modes Y ~ (1620 O cm-') and ~ 3 (1177 0 cm-I). These depolarization dispersion curves (DPD) and the corresponding reso-
'
Permanent address: Institute of Experimental Physics, University of Breman, 28359 Bremen, Germany. Abstract published in Advance ACS Abstracts, September 1, 1994. @
nance excitation profiles (REPS) were then subjected to a theoretical analysis based on a fifth-order, time-dependent perturbation approach which explicitly accounts for multimode contributions to the Raman cross section.11c The derived vibronic coupling parameters suggest that the porphyrin macrocycle in ferrocytochrome c is perturbed by antisymmetric AQ and asymmetric B1, distortions which were assigned to the axial ligands (in particular Met 80) and the two thioether bonds between the heme and the apoprotein. In particular, the A2,type distortions were found to be significantly stronger than in other heme proteins like myoglobin or hemoglobin.*J1 Our analysis and interpretation of the above ferrocytochrome c data were recently questioned by Spiro and co-~orkers.'~ These authors measured and compared the complex Raman spectra of reduced and oxidized yeast iso-1 cytochrome c with its meso-d4, pyrro1e-l5N, 2,4-di(a-d1), and 2,4-di(b-d2) isotopomers. Their data lead them to the conclusion that the oxidation marker line at 1364 cm-I overlaps with an inverse polarized line at 1365 cm-', which was assigned to the in-phase bending mode of the 1,3,5,8-methylsubstituents of the porphyrin macrocycle. For the sake of simplicity we refer to it as the M-band (M = methyl) throughout the paper. While the v4 mode dominates in the Soret band region owing to its strong FranckCondon coupling within the B state, the inverse polarized M-band was suggested to become significant in the Q-band region. Consequently, the authors concluded that the large DPR values of the v4 mode reported in our earlier studies (DPR = 7) result from the contribution of the nearby methyl band. Its resonance enhancement and inverse polarization were rationalized in terms of a model which assumes this mode to be vibrationally mixed with the antisymmetric v21 mode at 1314 cm-I. Resonance excitation within the B band (at 413 nm) revealed two small depolarized lines at 1302 and 1317 cm-', which were assigned to 2,4 C,H bending modes. Hu et al.I3 suggested that these lines may interfere with the normally inverse polarized line at 1314 cm-' (v21),thus giving rise to the strong decrease of its DPR (from 20 to approximately 1) in the preresonance region between Bo and Qv. The even stronger DPR dispersion reported for the antisymmetric v19 mode at 1587 cm-l-its DPR decreases to 0.25 in the B-band region1'-was tentatively explained by its overlap with the nearby polarized line of the
0022-3654t94t2098-9374$04.50tO 0 1994 American Chemical Society
J. Phys. Chem., Vol. 98, No. 38, 1994 9375
Letters symmetric v2 mode, which is comparatively strong in the Soret band region. The DPRs of some depolarized lines dealt with in ref l l c which are comparatively isolated, i.e., v10 (1620 cm-'>, vll(1548 cm-'), and ~ 1 (1213 3 cm-l), were considered as constant in spite of some serious deviations from their D4h expectation values. In view of the importance of polarization dispersion effects for the investigation of the heme symmetry and its dependence on the environment, the results and conclusions of Hu et al.13 prompted us to reinvestigate the polarization properties of some ferrocytochrome c Raman lines. To this end we measured polarized horse heart ferrocytochrome spectra between 1200 and 1700 cm-' at different excitation wavelengths between 514 (QV excitation) and 457 nm (preresonance B excitation) with comparatively high spectral resolution (1.8-2.4 cm-') at room temperature. All spectra were subjected to a thorough and consistent line shape analysis. This procedure provided reliable depolarization ratios for the Raman lines v2, v4, v10, v11, and v21 and to some extent also for v37, ~ 3 0 ,v20, and the Raman band Hu et al.13 assigned to the bending motion of the methyl substituents.
Material and Methods Preparation of Samples. Horse heart cytochrome c was obtained as a generous gift from Prof. Sandford A. Asher and was used without further purification. The lyophylized material was dissolved in 0.05 M potassium buffer adjusted to pH = 8.0. By adding a few grains of sodium dithionite, each sample attained the ferrous state. No oxidation was observed during the experiments. The final concentration of the samples was 1 mM.
Resonance Raman Spectroscopies. The exciting radiation was obtained from a Coherent Arzf ion laser. The laser beam, polarized perpendicular to the scattering plane, was focused onto a sample adjusted to a temperature of 10 "C. The average laser power at the sample was 50 mW. The scattering volume within the probe was imaged onto a spectrometer slid of 200 pn width to provide a spectral resolution between 2.4 (at 457 nm excitation) and 1.9 cm-' (at 514 nm excitation). A polarizer between sample and entrance slit of the Spex 1403 CzemyTurner double monochromator, which is equipped with 2400 groove/mm holographic gratings, enabled us to measure the intensity of the two components polarized perpendicular (Ipp) and parallel (Ipa)to the polarization of the incident laser beam. To eliminate the different transmission of the spectrometer, a polarization scrambler was placed between polarizer and entrance slit. The scattered light was dispersed by the above monochromator and detected by a photomultiplier. The data were digitized and stored on a Datamate computer from where they were transferred to a personel computer for further analysis. Spectral analysis was carried out by using the program Lab Calc from Galactic Industries Corp. Some spectra, in particular those measured with perpendicular polarization, were slightly smoothed by the Savitsky-Golay method to eliminate the highfrequency part of the noise. Care has been taken in order to avoid any distortion of the spectral bands by this procedure. The spectra were corrected for the linear background and finally analyzed by employing the Lab Calc curve-fit program. Several attempts with different guess values were made to fit a spectrum in order to estimate the statistical error of the fitting parameters. The depolarization ratios e of thus identified spectral lines were calculated as
e = IppJIpa
(1) The polarized components of a single line exhibit the same
TABLE 1: Depolarization Ratio Values Derived from the Band Shape Analysis As Described under "Materials and Methods'" (a) DPR of Raman Lines between 1300 and 1420 cm-'
457 472 476 488 496 501 514
2.2 (1.8) 1.7 (1.9) 2.0(2.0) 2.1 (2.3) 1.8 (1.9) 1.6 (2.0) 4.6 (4.5)
0.15 (0.15) 0.18 (0.18) 0.146(0.19,0.14') 0.21 (0.25) 0.19 (0.2) 0.165 (0.18) 0.5 (0.5)
1.2 1.1 2.3 0.51 0.47 0.49 0.50
1.04 4.0 1.3 2.51 2.26 2.2 3.4
0.55 0.5 0.57 0.37 0.27 0.25 0.66
DPR of Raman Lines between 1500 and 1650 cm-I
1 (nm) v11 v19 v2 v34 VI0 0.84 (0.79) 5.19 (0.25) 0.14 0.28 0.83 (0.81) 457 0.21 0.42 0.63 (0.84) 5.4(1.5) 488 0.57 (0.78) 2.0 1.99 0.75 (0.5) 514 0.81 (0.78) 36 (20) a Corresponding values reported in ref 1lb are given in parentheses for comparison. "he statistical errors are 20% for v21, 5-10% for v4, and 20-30% for VZO, ~ 2 9 ,and the M band. From ref 1la. The statistical errors are 5% for v11, 20-40% for v2 and vl9, 30-40% for v34, and 5% for VIO. spectral half-width. Therefore, their heights could be used as a measure of the corresponding intensities. The final plots of the spectra and the corresponding Raman bands were produced out by utilizing the GRAMS386 software package. The collection cone of the backscattering radiation has a halfangle of 15O. This reduces the error in DPR due to the finite collection angles and allows a rather exact determination of even large depolarization ratios. 14,15 To eliminate possible errors due to an insufficient depolarization by the polarization scrambler, the latter was adjusted so that the measured depolarization ratio of the 216 cm-' line in the CC4 Raman spectrum was identical with its expectation value of 0.75 at all excitation wavelengths.16
Result and Discussion Figure 1 shows the polarized spectra of ferrocytochrome c between 1280 and 1450 cm-' measured at 457, 488, and 514 nm excitation. Corresponding spectra of the region between 1500 and 1650 cm-' are depicted in Figure 2. The single lines displayed therein result from deconvoluting the spectra as described under "Material and Methods". The polarized components of a single line were fitted using the same halfwidth and frequency position. In addition, we have measured the polarized spectra between 1300 and 1450 cm-' at 472, 476,496, and 501 nm excitation. These data were also subjected to a thorough line shape analysis. The DPRs obtained from all fits are listed in Table 1. The results can be classified as follows: 1. The oxidation, marker line v4 (at 1361 cm-' in our spectra) is nearly isolated at 457 nm excitation and can be nicely fitted by a single Lorentzian. The corresponding depolarization value of 0.15 agrees well with earlier data." A perfect fit to the data, however, requires consideration of a small line at approximately 1369 cm-', which exhibits a DPR of about 1.2. At 488 nm excitation the perpendicular component of this line clearly shows up as a shoulder of the v4 line, which becomes even more apparent at wavelengths closer to the corresponding Qv resonance position (Figure 1). This line is inverse polarized in the preresonance region of the B band but becomes depolarized with increasing excitation wavelength and exhibits a minimum of its DPR at 496 nm. Higher DPR values can be expected for the region between the Qv and QO resonance positions, where
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Raman Shift [cm-11 Figure 1. Polarized Raman spectra of ferrocytochrome c between 1280 and 1450 cm-I measured with 457 nm (upper panel), 488 nm (middle panel), and 514 nm (lower panel) excitation. The spectral lines derived from the spectral analysis are plotted on the bottom of each panel. The digitized spectra are displayed by dotted points. The solid lines result from the fit to the data. antisymmetric contributions to the scattering tensor dominate because of constructive interference In spite of this band's overlap with v4 the DPR of the latter could be determined with an accuracy between f 0 . 0 2 (at 488 nm) and 0.05 (at 514 nm) and was found to exhibit a significant dispersion, which is depicted in Figure 3. All DPR values are close or even identical to that obtained in our earlier studies.*J1 This in particular holds for the DPR at 514 nm, which is found to be 0.5 in this and our earlier studies." A comparison of these results with the data reported by Hu et al.13 lead us to the conclusion that the Raman band at 1369 cm-' is identical with the M band these authors attributed to the symmetric bending motion of the 1,3,5,8-methyl substituents.
They reported a somewhat lower frequency (i.e., 1365 cm-l), but this value was derived from a Raman spectrum of the meso-d isotopomer based on the assumption that the v4 band is absent at 520.8 nm excitation. In view of the present results this is unlikely to provide a very accurate value. To explain the resonance enhancement and the inverse polarization of the M band, Hu et al.13 proposed vibrational mixing of the methyl bending modes with the antisymmetric v21 mode at 1312 cm-'. Strictly speaking, this argument is physically wrong, since a normal mode cannot mix with another vibration in the harmonic approximation. On the other hand, the sensitivity of the M band to 15N isotopic sub~titution'~ suggests that at least NC, s (s = stretch) vibrations of the
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J. Phys. Chem., Vol. 98, No. 38, 1994 9377
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6000
4000
n
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3Ooo 2060
loo0 0 1500
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Raman Shift [cm-11 Figure 2. Polarized Raman spectra of ferrocytochrome c between 1500 and 1650 cm-' measured with 457 nm (upper panel), 488 nm (middle panel), and 514 nm (lower panel) excitation. The spectral lines derived from the spectral analysis are plotted on the bottom of each panel. The digitized spectra are displayed by dotted points. The solid lines result from the fit to the data.
macrocycle contribute to its normal coordinate. While vibrational mixing between internal modes of peripheral substituents and local vibrations of the heme c macrocycle are indeed possible,17 it is difficult to imagine that this would significantly involve NC, s, from which the methyl group is separated by two bonds, i.e., ClCp and C,Cp. Hence, it remains presently unclear whether methyl vibrations are at all involved in the normal mode of the M band, in particular because unambiguous experimental evidence for this assignment has not yet been provided. Hu et al.13 observed another weak band overlapping with v4 at Qv excitation, which they assigned to a combination mode of ~ 1 and 5 ~ 2 4 . We have observed this band by our line shape
analysis, thus eliminating it as a possible source of error in determining the DPR of the v4 mode. 2. The DPR dispersion of the v21 band at 1312 cm-' (Figures 2 and 3) is very close to that found in our earlier studies," where we rationalized this observation as resulting from asymmetric A2g and B1, perturbations of the macrocycle. Hu et al.I3 have argued that this dispersion may be caused by two adjacent lines at 1302 and 1317 cm-' appearing at 413 nm excitation, which the authors assigned to bending modes of the two CH groups at the thioether bonds. Our data unambiguously show that v21 has a weak but clearly detectable parallel component at 1312 cm-'. We found the parallel component of another weak band at 1298 cm-', which could be separated
9378 J. Phys. Chem., Vol. 98, No. 38, 1994
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by our line shape analysis. It may indeed result from the above bending modes. A line at the higher energy side of 1 9 1 could not be identified in our spectra. It should be mentioned that Hu et al.13 observed a weak band at 1314 cm-l in the spectra of meso-d4 isotopomer, which they assigned to a propionate mode. It is comparatively weak and exhibits an inverse polarization. This rules out that its overlap with the v21 band produces the above DPR dispersion. 3. In our earlier studies we reported a particular strong DPR dispersion of the Y19 line at 1582 cm-'. While it was found to be inverse polarized between the Qo and Qv resonance positions (e = 20), a considerable small DPR of 0.3 was obtained in the preresonance region of the B band. In agreement with Hu et al.,I3 the present analysis reveals that this observation was in part caused by a spectral overlap with the comparatively strong v2 line at 1591 cm-', which is polarized in the B band region. The ~ 1 line 9 was now found to be inverse polarized even at 457 nm excitation, but its DPR is still dispersive and increases significantly upon approaching the Qv resonance position (Table 1). Interestingly, the DPR of the Y Z mode, which is mainly C K p stretch and of AI, type in an undistorted DG symmetry?J7 exhibits a drastic dispersion in that it increases from 0.14 at 457 nm to 2.0 at 514 nm. This observation indicates that this mode is subject to strong antisymmetric A2g distortions.ll 4. The DPR values obtained for the B1, type modes vll(1544 cm-') and Y I O(1619 cm-') are close to the values reported in ref 11 at 457 and 514 nm excitation, whereas those derived from 488 nm spectra are somewhat different (Table 1). The DPR values of other lines at 1398 cm-I (190,Azg in D4h), 1403 cm-' (V29, B2, in D4h) and 1603 cm-' (~34,E, in D4h), which were not analyzed in our earlier studies, are subject to larger statistical errors because of their comparatively low intensity and some spectral overlap with adjacent lines. There i s no doubt, however, that all these lines exhibit a strong DPR dispersion (cf. Table 1 and Figure 3). This holds in particular for ~34,whose very existence in the Raman spectrum proves that the porphyrin macrocycle is affected by symmetry lowering perturbations. Taken together, our results provide strong evidence that asymmetric perturbations of the porphyrin macrocycle give rise to a significant DPR dispersion of ferrocytochrome c Raman lines. With the exception of the ~ 1 line, 9 we could practically reproduce the DPR values reported in earlier studies from our laboratory." The analysis presented in ref l l c suggests that the DPR dispersion is caused by comparatively strong AQ and B1, distortions which together lower the heme symmetry to C,.
We rationalized these distortions as being caused by electrostatic interaction between Met 80 and the inner part of the porphyrin macrocycle.llcJ8 In the meantime crystallographic studies on yeast cytochrome c have revealed a nonplanar, &-like structure of the heme chrom~phore.'~As we have recently shown for Ni(II)-octaethyltetraphenylporphyrin, such nonplanar distortions admix antisymmetric Azg contributions to the Raman tensor of AI, modes and symmetric AI, elements to the tensor of antisymmetric Azg modes.20 This type of symmetry mixing is also operative for ferrocytochrome c and may indeed be caused by the nonplanarity of its heme c chromophore. That notion and the involvement of the thioether bridges are corroborated by the high DPR of the ~2 band at Qv excitation, which owing to the CpCp character of its normal mode should be highly sensitive to distortions of the pyrrole symmetry. Future experiments on modified cytochrome c are necessary to prove this interpretation.
Acknowledgment. I am grateful to Prof. Sanford A. Asher for providing horse heart cytochrome c and thank him, Prof. Wolfgang Dreybrodt, and Dr. Walter Jentzen for critically reading the manuscript. Moreover, I thank Prof. Samuel Krimm for an illuminating discussion on normal coordinate issues and Prof. John A. Shelnutt for sending me his paper on the structure of cytochrome c prior to publication. I am further indebted to Prof. Michael Morris for providing access to his MS-DOS computer facilities for the final analysis of the data and preparation of the figures. Finally, I acknowledge that this study has been carried out when I was a Max Kade fellow and Visiting Research Scientist in the laboratory of Prof. Samuel Krimm at the University of Michigan in Ann Arbor. References and Notes (1) (a) Friedman, J. M. Science 1985, 228, 1274. (b) Rousseau, D. L.; Friedman, J. M. In Biological Application of R a m n Spectroscopy; Spiro, T. G., Ed.; John Wiley & Sons: Chichester, 1988; p 33. (2) Li, X.-Y.; Spiro, T. G. In Biological Application of Raman Spectroscopy; Spiro, T. G., Ed.; John Wiley & Sons: Chichester, 1988; p 1. (3) (a) Shelnutt, J. A.; Rousseau, D. L.; Friedman, J. M.; Simon, S. R. Proc. Natl. Acad. Sci. USA. 1979, 76, 4409. (b) Asher, S. A. Methods Enzyml. 1981,76,371. (c) Debois, A.; Lutz, M.; Banerjee, R. Biochemistry 1978, 18, 1510. (4) Spuo, T. G.; Strekas, T. C. J. Am. Chem. SOC. 1974, 96, 338. (5) (a) Spaulding, L. D.; Chang, C. C.; Yu, N. T.; Felton, R. H. J . Am. Chem. SOC.1975, 97, 2517. (b) Spiro, T. G.; Stong, J. D.; Stein, P. J . Am. Chem. SOC.1979, 101, 2648. (6) Temer, J.; Spiro, T. G.; Nagumo, M.; Nicol, M. F.; El-Sayed, M. A. J . Am. Chem. SOC. 1980, 102, 3238. (7) (a) Friedman, J. M.; Lyons, K. M. Nature (London) 1980, 284, 570. (b) Friedman, J. M.; Stepnoski, R. A.; Stavola, M.; Ondrias, M. R.; Cone, R. L. Biochemistry 1982, 21, 2022. (c) Friedman, J. M.; Scott, T. W.; Stepnoski, R. A.; Ikedo-Saito, M.; Yonetani, T. J . Biol. Chem. 1983, 10, 10564. (8) Schweitzer-Stenner, R. Q.Rev. Biophys. 1989, 22, 381. (9) Unger, E.; Bobinger, U.; Dreybrodt, W.; Schweitzer-Stenner, R. J . Phys. Chem. 1993, 97, 9956. (10) (a) Schweitzer-Stenner, R.; Wedekind, D.; Dreybrodt, W. Biophys. J . 1989, 55, 703. (b) Schweitzer-Stenner, R.; Dreybrodt, W. J . Raman Spectrosc. 1992, 23, 539. (11) (a) Schweitzer-Stenner, R.; Dreybrodt, W. J . Raman Spectrosc. 1985, 16, 11. (b) Bobinger, U.; Schweitzer-Stenner, R.; Dreybrodt, W. J . R a m n Spectrosc. 1989, 20, 191. (c) Schweitzer-Stenner, R.; Bobinger, U.; Dreybrodt, W. J . Raman Spectrosc. 1991, 22, 65. (12) Remba, R. D.; Champion, P. M.; Fitchen, D. B.; Chiang, R.; Hager, L. P. Biochemistry 1979, 18, 2280. (13) Hu,S.; Moms, I. K.; Singh, S. P.; Smith, K. M.; Spiro, T. G . J . Am. Chem. SOC.1993, 115, 12466. (14) Deb, S. K.; Bonsol, M. L.; Roy, A. P. Appl. Spectrosc. 1984, 38, 500.
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Letters (15) Stichtemath, A. Doctoral Thesis, Bremen, 1993. (16) Fukushi, K.; Kimure, M. J. J . Ruman Spectrosc. 1979, 8, 125. (17) Li, X.-Y.; Czemuszewicz, R. S.; Kincaid, J. R.; Stein, P.; Spiro, T. G . J . Phys. Chem. 1990, 94, 41. (18) Kubitscheck, U.; Dreybrodt, W.; Schweitzer-Stenner, R. Spectrosc. Lett. 1987, 19, 681. (19) (a) Hobbs, J. D.; Shelnutt, J. A. Submitted for publication. (b) Hobbs, J. D.; Majumder, S. A.; Luo, L.; Sickelsmith, G. A.; Quirke, J. M.
E.; Medforth, C. J.; Smith, K. M.; Shelnutt, J. A. J. Am. Chem. SOC.1994, 116, 3261.
(20) (a) Stichtemath, A.; Schweitzer-Stenner, R.; Dreybrodt, W.; Mak, R. s. W.; Li, X.-Y.; Sparks, L. D.; Shelnutt, J. A.; Medforth, C. J.; Smith, K. M. J. Phys. Chem. 1993, 97, 3701. (b) Schweitzer-Stenner, R.; Stichtemath, A.; Dreybrodt, W.; Medforth, C. J.; Smith, K. A. Biophys. J . 1994, A137.
