J. Phys. Chem. 1995,99, 7195-7196
Spectral Analysis Is Suitable To Decompose Overcrowded Resonance Raman Spectra of Metalloporphyrins and Yields Reliable Depolarization Ratios
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Reinhard Schweitzer-Stenner," Esko Unger, Gerasimos Karvounis, and Wolfgang Dreybrodt
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FBI -Institute of Experimental Physics, University of Bremen, P.O. Box 330440, 28334 Bremen, Germany
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Received: January 3, 1995
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Spiro and co-workers' have measured and compared the complex Raman spectra of the heme group embedded in yeast iso-1-cytochrome c with some of its isotopomers. Their findings lead them to the conclusion that most of the particular strong DPR dispersions earlier observed in our laboratory for the Raman lines v4, v19, and v21 of horse heart ferrocytochrome c2 result from spectral overlap with adjacent lines rather than from symmetry lowering perturbations. To check the reliability of the reported DPR values, we have reinvestigated the polarization properties of the above Raman lines by measuring polarized Raman spectra of ferrocytochrome c at various excitation wavelengths with comparatively high spectral resolution (i.e., 1.9-2.4 ~ m - ' ) . ~Thus, we reaffiied the earlier reported DPR dispersion for v21 (at 1312 cm-l) and v4 (at 1360 cm-l), whereas the corresponding DPR dispersion of the very intense ~ 1 band 9 (at 1583 cm-l) was found to result in part from an overlap with the adjacent v2 mode. Moverover, it was shown that other Raman lines (Le., v2, v20) also show significant dispersion of their DPR. Hu and Spiro have now published a reply" in which they claim that the spectral crowding in the Raman spectra of ferrocytochrome is too intense to allow a reliable decomposition into single Raman lines. To support their argument for the case of the Raman line 191, they have measured the polarized spectra of yeast iso-1-ferrocytochrome c and its 2,4-di(a-dl)-isotopomer (Le., deuteration of the CH groups attached at the thioether bonds) between 1200 and 1350 cm-l at 11 K with three different excitation wavelengths. Indeed, the spectra of the natural abundance (n.a.) sample taken at 413 nm (B excitation) show that the v21 band overlaps with at least two lines at 1302 and 1317 cm-l. They are absent in the corresponding spectra of the isotopomer and are therefore assigned to the C,H bending modes of the thioether bridges. The DPR value of the isolated 1 9 1 of the above isotopomer is close to 3 (not 5 , as mentioned in ref 4) at 413, 521, and 531 nm. From this observation the authors conclude that the DPR of v21 does not show any significant dispersion. Spiro and c o - w ~ r k e r shave ~ , ~ performed their measurements at 11 K to obtain a slightly better separation of overlapping bands. It is normally very difficult to obtain correct DPR values from heme proteins in an aqueous solution under this condition, because the scattered light is partially depolarized by multiple scattering in the ice even if one uses a backscattering geometry. This reduces the apparent DPR dispersion signficantly and makes comparison with our data difficult. In order to check the reliability of our previous analysis of the v21 and other relevant Raman bands, we have subjected our spectra to a new line shape analysis using the program MULTIFIT recently developed in our laboratory, which allows better and faster fitting of experimental spectra than the previously used LAB CALC. The natural Lorentzians of the Raman lines have now been convoluted with Gaussian profiles to account for the spectrometer function. Furthermore, we 0022-3654/95/2099-7 195$09.00/0
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Figure 1. Polarized (parallel and perpendicular to the polarization of
the incident laser beam) Raman spectra of horse heart ferrocytochrome between 1280 and 1450 cm-' measured with 514 nm excitation. The spectra are analyzed by a novel program called MULTIFIT. The experimental data are plotted as squares. The solid lines result from the fit to the experimental data. Relevant spectral lines derived from the analysis are plotted at the bottom of the figures. strictly assigned each Raman band to the very same half-width for all excitation wavelengths investigated. Finally, we directly used a linear base line in the fit to the data. As an example, Figure 1 shows the polarized Raman spectra measured with 5 14 nm excitation (QV) excitation together with single Raman bands resulting from the spectral decomposition. It turns out that in contrast to 520 nm spectrum shown by Hu and Spiro' the perpendicular component of the v21 band exhibits only a small shoulder which can be attributed to a Raman line at 1322 cm-'. Owing to its low intensity, the parallel component of this line is barely detectable. This band is absent in the spectra taken with higher excitation wavelengths (data not shown). The parallel component of v21 is overlapped by a band at 1317 cm-' and may indeed result from C,H bending. Its DPR cannot determined with sufficient accuracy, but the band seems to be polarized. Table 1 lists the DPR values of v21 as derived from the present analysis. The values obtained from a previous study are given in parentheses. A major discrepancy is only obtained for 514 nm excitation where due to the consideration of a Raman line at 1317 cm-' we have now obtained a larger DPR value of 7.9. 0 1995 American Chemical Society
7196 J. Phys. Chem., Vol. 99, No. 18, 1995
Comments
TABLE 1: DPR Values of the Raman Lines VZI (1312 cm-l), v4 (1360 cm-I), and M (1367 cm-l) Obtained at Different Excitation Wavelengths" wavelength (nm) YZI (1312 cm-') v4 (1360 cm-I) M (1367 cm-l) 457 472 488 496 501 5 14
1.2 (2.2) 1.5 (1.7) 3.0 (2.1) 2.2 (1.9) 2.9 (1.6) 7.9 (4.6)
0.16 (0.15) 0.19 (0.18) 0.22 (0.21) 0.16 (0.19) 0.14 (0.16) 0.43 (0.5)
0.70 (0.51) 0.53 (0.47) 1.0 (0.5) 1.8 (0.5)
"The corresponding DPR values reported in ref 7 are given in parentheses for comparison.
However, this makes the DPR dispersion of this Raman band even more pronounced. We like to comment on another issue addressed in the papers of Hu et al.' and Hu and Spiro! They claim that the v4 mode which they observed at 1364 cm-I is overlapped by an inverse polarized Raman line at 1365 cm-l in the n.a. spectrum of yeast cytochrome c. This is inferred from Raman spectra of 15Nisotopomers, in which these bands appear at 1357 (v4) and 1363 cm-'. The latter, which was attributed to a symmetric CH3 bending mode of the methyl substituents (M band), is not detectable with 413 nm excitation but appears significantly more intense than v4 in the Q-band region. Spiro and co-workers argue that v4 band of the 15N-isotopomercannot be observed at all with Qv excitation, but that is not evident from the spectra of the 15Nisotopomer shown in Figure 9 of ref 1, which clearly exhibit a at least depolarized shoulder at 1357 cm-l. Moreover it may well occur that 15N-deuterationchanges the eigenvector of the v4 mode in a way that alters the Raman cross section in the Qv-band region. Finally, it must again be taken into account that the position of the v4 band may be different at 300 and 11 KS5 In fact, we found from the 457 nm spectra that v4 is positioned at 1360 cm-l rather than at 1364 cm-l. Cartling6 has reported Raman spectra of ferrocytochrome c measured at 77 K with 413 nm excitation which exhibit the v4 line at 1363 cm-l. Therefore, it is likely that the overlap between v4 and M band is more pronounced in the spectra of Spiro and cow o r k e r ~than ~~~ in ours. Our present spectral analysis yields additional Raman bands at 1367 and 1375 cm-l. The former.
which increases its relative intensity toward Qv-band excitation, has been previously attributed3 to the M band observed by Hu et al.' The yet unassigned inverse polarized 1375 cm-' band was already recognized in the previous analysis (cf. the 488 nm spectra in Figure 1 of ref 3) but not treated consistently in all fits. The new DPR values of the v4 band and the 1367 cm-l band are listed in Table 1. Comparison with our previous results (given again in parantheses) reveals good agreement for all excitation wavelengths besides 514 nm, at which the DPR value of v4 is now slightly lower (i.e., 0.42 instead of 0.5). Two DPR values of the 1367 cm-l band are now significantly higher (1.0 and 1.8 instead of 0.5 at 501 and 514 nm, respectively). This finding makes it even more apparent that this band is indeed identical with the M band. Finally, we like to comment on the argument of Hu and Spiro4 saying that the DPR dispersion recently observed for the v4 band of Ni(II)-octaethyltetraphenylporphyrin may be due to spectral overlap between this band and two inverse polarized bands at 1352 and 1372 cm-l. This argument is incorrect. As we have outlined in detail under "Material and Method" in ref 7, a rather sophisticated analysis was employed to decompose the extremely complex spectra of this substance. By that means we could determine the DPR dispersion of the two bands at 1352 and 1372 cm-l, thus eliminating their influence on v4. A more detailed presentation of these data is under way. References and Notes (1) Hu, S.; Moms, I. K.; Singh, S. P.;Smith,K. M.; Spiro, T. G. J . Am. Chem. SOC. 1993, 115, 12466. (2) (a) Bobinger, U.; Schweitzer-Stenner, R.; Dreybrodt, W. J . Raman Spectrosc. 1989, 20, 191. (b) Schweitzer-Stenner, R.; Bobinger, U.; Dreybrodt, W. J. Raman Spectrosc. 1991, 22, 65. (3) Schweitzer-Stenner, R. J. Phys. Chem. 1994, 98, 9374. (4) Hu, C.; Spiro, T. G. J. Phys. Chem. 1995, 99, xxxx. (5) Schweitzer-Stenner,R.; Jentzen, W.; Dreybrodt, W. In Proceedings of the Vth International Conference on Spectroscopy on Biological Molecules; Theophanides, T., Anastassopoulou, J., Fotopoulos, N., Eds.; Kluwer Academic Publishers: Dordrecht, 1993; p 31. (6) Cartling, B. Biophys. J. 1983, 43, 191. (7) 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. J. J. Phys. Chem. 1993, 97, 3701. JP9501270
