7193
J. Phys. Chem. 1995, 99, 7193-7194
COMMENTS Apparent Depolarization Dispersion in Metalloporphyrin Resonance Raman Spectra Can Be an Artifact of Spectral Crowding
2,4-di(a-dl)-Cyt Bl2K
N.A.-CvC c
c
Songzhou Hu and Thomas G. Spiro* Department of Chemistry, Princeton Universiv, Princeton, New Jersey 08544 Received: November 18, 1994; In Final Form: January 25, 1995
Because of their unusual electronic properties, metalloporphyrins have provided fertile ground for the exploration of scattering phenomena in resonance Raman (RR) spectra.' The fust two singlet excited states, Q and B, are accessible to excitation with visible lasers, and their resonance enhancement patterns have been explored extensively.* The electronic transitions are subject to configuration interaction and vibronic m i ~ i n g .As ~ a result, resonance with the B transition primarily enhances totally symmetric modes via Franck-Condon scattering, while resonance with the Q transition primarily enhances non-totally symmerric modes #via vibronic scattering. In the idealized D4h symmetry of the metalloporphyrin chromophore, the depolarization ratios are fixed at the values Its, 3/4, and infinity for AI, modes, B1, and B2g modes, and Azg modes, respectively. Deviations from these values are evidence of symmetry lowering; in the lower symmetry, dispersion of the depolarization ratio with excitation wavelength is expected because of interferences from the separate contributions of the previously degenerate electronic transitionsFe Schweitzer-Stenner and co-workers have published extensive data on the variation of depdarization ratio with excitation wavelength for metallo~orphyrins~ and heme proteins5 and have analyzed the resulting dispersion curves in the framework of a fifth-order, time-dependent perturbation theory. However, spectral crowding makes it difficult to assure that the measurements are not affected by band overlaps. The porphyrin ring and its substituents have many atoms, and the density of vibrational states is high. Normal-mode analyses and studies of the effects of isotopic substitution have made it clear that mode coincidences are common.6 If two or more modes of different symmetry are accidentally degenerate, then dispersion is observed in the depolarization ratio of the composite band for the trivial reason that the relative intensities of the modes vary with excitation wavelength. We have recently demonstrated that mode overlaps do explain most of the depolarization dispersion reported for cytochrome c , by ~ ~identifying the overlapped modes in the spectra of isotopically labeled p r ~ t e i n .Schweitzer-Stenner ~ has published a rebuttal, in which the original claims for depolarization dispersion are reaffirmed, using curve-fitting procedures to resolve overlapped bands.8 However, no amount of curve fitting can reliably separate bands which are intrinsically broad and extensively overlapped. This point is illustrated by the depolarization characteristics of the Azg mode, vz1,shown in Figure 1. When the heme is labeled with deuterium at the C, positions of the two vinyl substituents, then the depolarization ratio stays essentially constant, at -5, whether the excitation is near resonance with the Q (530.9, 520.8 nm) or B (413.1 nm) transitions. This ratio is less than infinity, implying symmetry lowering, but there is little dispersion. Schweitzer-Stenner
520.8 nm
IO
530.9 nm
1250
I300
L J
m
1200
1250
1300
1350
Raman Shift (cm-')
Figure 1. Resonance Raman spectra of yeast isocytochrome c of natural abundance (left panel) and labeled with 2,4-di(a-rf,)-heme(right
panel). The sample preparation and experimental conditions are the same as reported in ref 7. reports a similar value, 4.6, with 514 nm excitation, but his values decrease markedly as the excitation approaches the B t r a n s i t i ~ n . ~This * ~ behavior is directly attributable to overlap with bands arising from C,H bending modes at 1302 and 1317 cm-' in the natural abundance spectra (Figure l), when the excitation is in resonance with the B transition. These bands have substantially lower depolarization ratios. With Q-band excitation, however, the CaH mode enhancement is diminished and contributes only weakly to the 2121 band envelopes, broadening it relative to the CaD isotopomer. (The small shoulder remaining in the isotopomer spectrum is attributed to incomplete isotopic substitution.) It is evident that curve fitting does not yield the true depolarization ratio, which is revealed in the isotopomer spectra. A similar overlap occurs for the totally symmetric mode v4, at 1364 cm-', which gives measured depolarization ratios near the expected l/g with B-band resonance, but higher values with Q-band resonance.5e We pointed out that this behavior results from accidental degeneracy with the umbrella modes of the four methyl s~bstituents.~ These modes give rise to antisymmetric scattering in Q-band resonance because an out-of-phase combination of these coordinates can mix with the skeletal coordinates that account for the nearby A2, mode, v~1.SchweitzerStenner claims to resolve the methyl band by curve fitting, leaving v4 with an elevated depolarization ratio, 0.5, using 514 nm excitation.8 But 214 is known7 not to contribute s i g " t l y to the 1365 cm-I band in the 520.8 nm excited spectrum because this band shifts only 2 cm-' upon 15N substitution in pyrrole rings, whereas the v4 shift, observed with 413.1 nm excitation, is 7 cm-l. Thus, the curve-fitting procedure is suspect in this case as well.
0022-3654/95/2099-7193$09.00/0 0 1995 American Chemical Society
7194 J. Phys. Chem., Vol. 99, No. 18, 1995
We also pointed out that the extremely large depolarization dispersion reportedSefor the Azg mode v19 (1587 cm-') was probably due to overlap with the A', mode v~ (1596 cm-'). Schweitzer-Stenner acknowledges this overlap but still claims a substantial residual dispersion after v2 and ~ 1 have 9 been deconvoluted by curve fitting.8 In view of the cited curve-fitting ambiguities, this claim needs support from isotopic data, in order to establish the degree of B-resonant enhancement of v19 and of Q-resonant enhancement of v2. The band overlap problem is not limited to cytochrome c. For example, Schweitzer-Stenner and co-workersk have invoked depolarization dispersion of v4 as additional evidence for symmetry lowering in NiOETPP (OETPP = octaethyltetraphenylporphyrin), in which the steric crowding of the ethyl and phenyl substituents imposes a saddling deformation on the p0rphyrin.l' This dispersion was noted to be absent for NiOEP? in which the steric crowding is absent. However, the v4 frequencies also differ in the two complexes$ 1383 cm-' for NiOEP but 1360 cm-' for NiOETF'P. The latter frequency is near coincidence with the methyl umbrella frequency, and it is possible that this overlap accounts for the depolarization dispersion, as it does i n cytochrome c. We do not, of course, deny the existence of depolarization dispersion and indeed rsported an example in cytochrome c, namely, a component of the\E, mode, v43 (1154 cm-'), which is polarized with 413.1 nm excitation but anomalously polarized with 520.8 nm e~citation.~ No overlapping band could be identified via isotopic substitution. However, assessment of the magnitude of the effect for a series of skeletal modes will require careful measurements on a series of isotopic species, in order to isolate the modes. In the absence of isotopomers, the overlap problem limits the utility of depolarization dispersion measurements. There are, however, other RR indicators of symmetry lowering that can be used to assess mole'cular distortion in metalloporphyrins and heme proteins. These include RR activation of out-of-plane modes7J2 and of in-plane modes of E,, ~ymmetry,~ as well as splittings of E, and E, modes.7 Acknowledgment. This work was supported by NIH Grant GM 33576.
Comments References and Notes (1) Spiro, T. G., Ed. Biological Applications of Raman Spectroscopy; Wiley: New York, 1988; Vol. III. (2) (a) Abe, M. Advances in Spectroscopy; Clark, R. J. H., Hester, R. E., Eds.; Wiley: New York, 1986; Vol. 13, 347-393. (b) Kitagawa, T.; Ozaki, Y. Stmct. Bonding 1987,64,71-114. (c) Spiro, T. G.; Czemuszewicz, R. S.; Li, X.-Y. Coord. Chem. Rev. 1990,100,541-571. (d) Spiro, T. G.; Smulevich, G.; Su, C. Biochemistry 1990, 29, 4497-4508. (e) Procyk, A. D.; Bocian, D. F. Annu. Rev. Phys. Chem. 1992,43,465-496. (3) Gouterman, M. In The Porphyrins; Dolphin, D., Ed.; Academic Press: New York, 1979; Vol. III, pp 1-166. (4) (a) Bobinger, U.; Schweitzer-Stenner, R.; Dreybrodt, W. J. Phys. Chem. 1991,95,7625-7635. (b) Unger, E.; Bobinger, U.; Dreybrodt, W.; Schweitzer-Stenner, R. J. Phys. Chem. 1993, 97, 9956-9968. (c) Stichtemath, A.; Schweitzer-Stenner, R.; Dreybrodt, W.; Mak, W. S. W.; Li, X.-Y.; Sparks, L. D.; Shelnutt, J. A.; Medforth, C. J.; Smith, K. M. J . Phys. Chem. 1993, 97, 3701-3708. ( 5 ) (a) Schweitzer-Stenner, R.;Dreybrodt, W. J . Raman Spectrosc. 1985,16, 111-123. (b) Schweitzer-Stenner. R.;Wedekind, W.; Dreybrodt, W. Biophys. J . 1986,49, 1077-1088. (c) Schweitzer-Stenner, R. Q. Rev. Biophys. 1989, 22, 381-479. (d) Bobinger, U.; Schweitzer-Stenner, R.; Dreybrodt, W. J . Raman Spectrosc. 1989, 20, 191-202. (e) SchweitzerStenner, R.; Bobinger, U.; Dreybrodt, W. J . Raman Spectrosc. 1991, 22, 65-78. (f) Schweitzer-Stenner. R.; Dreybrodt, W. J . Raman Spectrosc. 1992, 23, 539-550. (6) Li, X.-Y.; Czemuszewicz, R. S.; Kincaid, J. R.; Stein, P.; Spiro, T. G . J . Phys. Chem. 1990,94,47-61. (7) Hu, S.; Moms, I. K.; Singh, J. P.; Smith, K. M.; Spiro, T. G. J . Am. Chem. Soc. 1993, 115, 12446-12458. (8) Schweitzer-Stenner, R. J. Phys. Chem. 1994, 98, 9374-9379. (9) We note that our measurements are on samples frozen at 12 K. Although solid samples can scramble the polarization via multiple reflection, this problem is minimized for strongly absorbing samples.1° We used a relatively high concentration (-1 mM) of cytochrome c for this reason. The good agreement of our Q-resonant depolarization ratio with that of Schweitzer-Stenner shows the polarization scrambling to be minimal. (10) Strommen, D. P.; Nakamoto, K. Appl. Spectrosc. 1983, 37, 436. (11) (a) Shelnutt, J. A.; Medforth, C. J.; Berber, M. D.; Barkigia, K. M.; Smith, K. M. J . Am. Chem. Soc. 1991, 113,4077-4087. (b) Sparks, L. D.; Medforth, C. J.; Park, M.-S.; Chamberlain, J. R.; Ondrias, M. R.; Senge, M. 0.;Smith, K. M.; Shelnutt, J. A. J. Am. Chem. Soc. 1993,115, 581-592. (c) Barkigia, K. M.; Renner, M. W.; Furenlid, L. R.; Medforth, C. J.; Smith, K. M.; Fajer, J. J . Am. Chem. SOC.1993, 115, 3627-3635. (12) (a) Li, X.-Y.; Czemuszewicz, R. S.; Kincaid, J. R.; Spiro, T. G. J . Am. Chem. SOC.1989,111,7012-7023. (b) Piffat, C.; Melamed, D.; Spiro, T. G. J . Phys. Chem. 1993, 97, 7441-7450. JP943093D
