J. Phys. Chem. 1985,89, 1856-1861
1856
ARTICLES Resonance Enhancement in the Ultraviolet Raman Spectra of Aromatic Amino Acids Richard P. Rava and Thomas G. Spiro* Department of Chemistry, Princeton University, Princeton, New Jersey 08544 (Received: August 9, 1984)
Raman spectra obtained with ultraviolet laser excitation at 200, 218, and 240 nm via a H2Raman-shifted Nd:YAG laser are reported for phenylalanine, tyrosine (and tyrosinate), and tryptophan in dilute (1 mM)aqueous solution; for tryptophan, the Raman spectrum with 266-nm excitation is also reported. Large changes in enhancement pattern are seen at the different excitation wavelengths, which can be understood in terms of dominant vibronic ( B term) scattering in resonance with the quasi-forbiddenLa and Lb states and Franck-Condon ( A term) scattering via the allowed B%bstates. Different enhancements for the different amino acids are associated with the red shifts of the electronic transitions and increasing allowedness of the quasi-forbidden states in the order phenylalanine, tyrosine, tyrosinate, and tryptophan. The tryptophan scattering efficiency diminishes sharply at 200 nm, above the allowed transition (218 nm),although the absorptivity remains high at this wavelength, and the scattering is distributed among many Raman modes. Both effects are suggested to arise from mixing of closely spaced electronic transitions via the nuclear motions (Born-Oppenheimer breakdown). The 850-cm-' doublet of tyrosine, which is due to a Fermi resonance between the ring breathing mode and the overtone of an out-of-plane mode, shows different intensity ratios with ultraviolet and visible excitation, attributable to selective enhancement of the polarizability associated with the ring-breathing mode. N
Introduction The availability of reliable lasers operating in the ultraviolet (UV) region makes it possible to study the resonance Raman (RR) spectroscopy of simple aromatic molecules. Ziegler and Hudson have demonstrated good quality R R spectra for benzene' and alkylbenzenes2 excited at 213 nm via the fifth harmonic of the Nd:YAG laser. Interesting enhancement patterns were observed, associated with the vibronic character of the forbidden La state, with which the laser was resonant. These spectra are of fundamental interest in their own right and are also important because of the potential of UVRR spectroscopy in probing the local environments of the aromatic amino acid constituents of proteins, phenylalanine, tyrosine, and tryptophan. Various approaches to UV excitation are available. Several studies with 257-nm excitation, obtained by frequency doubling the 514.5-nm line of the C W argon laser, have been p u b l i ~ h e d . ~ ? ~ In the near-UV region, C W Ar+ and Kr' lasers have useful lines in the neighborhood of 350 nm, while spectra excited between 266 and 335 nm have been reported via excitation with frequencydoubled flash-lamp-pumped dye laser^.^ For deep UV work, however, YAG-based laser systems currently give best results. Asher et al.5ahave described a system, based on mixing the YAG fundamental with the frequency-doubled output of a dye laser pumped by the same YAG laser, which is tunable from 217 nm to longer wavelengths; Raman excitation data for the benzene ring-breathing mode were reported. This group has also recently reported spectra of tryptophan, tyrosine, and phenylalanine (pH 12) excited with 225-nm, and myoglobin with 230-nm excitation.sa We have been attracted to the H2 Raman shift cell6 as a simple device for upconverting the YAG laser wavelengths. By focusing (1) L. D. Ziegler and B. Hudson, J. Chem. Phys., 74, 982 (1981). (2) L. D. Ziegler and B. S.Hudson, J . Chem. Phys., 79, 1134 (1983). (3) For example, see Y.Nishimura, A. Y. Hirakawa, and M. Tsuboi, Adu. Infrared Raman Spectrosc., 5 , 2 17 (1 979). (4) (a) L. Chinsky, A. Laigle, W. L. Peticolas, and P. Y. Turpin, J. Chem. Phys., 76, 1 (1982); (b) L. Chinsky and P. Y . Turpin, Biopolymers, 21, 277 (1982), and references therein; (c) D. C. Blazej and W. L. Peticolas, J . Chem. Pkys., 72, 3134 (1980). (5) (a) S.A. Asher, C. R. Johnson, and J. Murtaugh, Rev. Sci. Instrum., 54, 1657 (1983); (b) C. R. Johnson, M. Ludwig, S.O'Donnell, and S. A. Asher, J . Am. Chem. SOC.,106, 5008 (1984). (6) V. Wilke and W. Schmidt, App. Phys., 18, 177 (1979).
the 266-nm fourth harmonic output of the YAG laser into this device, one can produce usable output at 240, 218, and 200 nm, corresponding to the first, second, and third anti-Stokes quanta of the H2 stretching vibration. We have recently gathered R R spectra of the nucleotides at these wavelengths and analyzed their enhancement pattern^.^ In an earlier report, we demonstrated selective enhancement of tryptophan and tyrosine Raman bands at 218 and 200 nm, respectively,*and subsequently examined the R R spectra of the proteins insulin and a-lactalbumin at these
wavelength^.^ In this study we report R R spectra of phenylalanine, tyrosine, and tryptophan (see Figure 1 for the molecular structures) excited at 200, 218, 240, and 266 nm. Large changes in overall enhancement and in the relative enhancement patterns are observed at the different wavelengths, which can be understood satisfactorily in terms of varying contributions from Franck-Condon and vibronic (A and B terms)'O,l' scattering. For tryptophan, loss in overall enhancement with 200-nm excitation and redistribution of the intensity into many Raman modes are interpreted in terms of breakdown in the Born-Oppenheimer approximation, which may have general significance for R R spectra of molecules obtained at frequencies above their lowest transitions. Another point of interest concerns the -850-cm-' doublet of tyrosine, associated with a Fermi resonance interaction. The intensity ratio of this doublet changes markedly between spectra excited with visible or UV radiation; this behavior is shown to be consistent with selective UV enhancement of the unperturbed polarizability associated with the breathing mode fundamental.
Experimental Details The experimental apparatus for obtaining UVRR has been described previously.' The aromatic amino acids (Sigma) were dissolved in water at the concentrations (usually 1 mM) indicated in the figure captions. Typically, 10-30 laser pulses were accu(7) S.P. A. Fodor, R. P. Rava, T. R. Hays, and T. G. Spiro, J. Am. Chem. SOC..107. 1520 (1985). (8) R.'P. Rata andT. G. Spiro, J . Am. Chem. SOC.,106, 4062 (1984). (9) R. P. Rava and T. G. Spiro, Biochemistry, in press. (IO) J. Tang and A. C. Albrecht in 'Raman Spectroscopy", Vol 2, H. A. Szymanski, Ed., Plenum, New York, 1970, pp 33-67. (11) T. G.Spiro and P. Stein, Annu. Rev. Phys. Chem., 28, 501 (1977).
0022-3654/85/2089-1856$01 .50/0 0 1985 American Chemical Society
The Journal of Physical Chemistry, Vol. 89, No. 10, 1985 1857
RRUV Spectra of Aromatic Amino Acids PHENYLALANINE
TYROSINE
PHENYLALANINE
I
I
200nrn x2
--x
on
TRYPTOPHAN
218nrn
h Figure 1. Structure of the aromatic amino acid side chains.
TRYPTOPHAN TYROS I N E PHENYLALANINE
-
..-.-.-
1000
800
1200
1400
1600
Acrn-I
-**.****.
Rgure 3. Resonance Raman spectra of aqueous phenylalanine (2-6 mM)
with 200-, 218-, and 240-nm excitation. Intensities have been scaled relative to the intensity of the 3400-cm-' 0-H water stretching band as indicated by the multiplicative factors. Numbers in parentheses correlate with benzene mode assignments. For other assignments see Table 111.
30000
TYROSINE
pH 7
2 0 0 nrn I
;h 0
N -
20000 '
m
0
-
218nm
AT..';:-. 290
..... .... --._... . ... .. . e . .
275
260
. - a
245 2& WAVELENGTH (nm)
I
I
215
200
I
I
240nrn
U
x7
Figure 2. Absorption spectra of the aromatic amino acids between 185
and 300 nm, obtained with a Cary 14 spectrometer with 30 p M samples in a 1-cm cell, yielding OD = 1 for the strongest bands. mulated per point (1-2 cm-') for each spectrum, except for weaker signals (e.g., tryptophan at 200 nm) where up to 90 pulses were added. No time-dependent changes in the Raman spectra were observed, and the UV absorptions were unaltered after aquisition of the Raman spectra, indicating negligible sample degradation. All intensities were measured relative to the 34Wcm-l water band, which is not expected to show a strong wavelength dependence in the >200-nm region, as described in ref 7. Results Figure 2 displays UV absorption spectra for the amino acids under study; arrows mark the positions of the laser lines, at 200, 218,240, and 266 nm. The RR spectra are shown in Figures 3-6. Only the tryptophan spectrum was obtained at 266 nm; for phenylalanine and tyrosine, low signal strength and the onset of fluorescence obscured the spectra. The Raman intensities were measured relative to that of the -3400-cm-' 0-H stretching band of the solvent water molecules and were corrected for self-absorption.' Enhancement factors determined in this way are listed in Table I, In Figures 3-5, the R R spectra for a given amino
1
800
I
1000
I
1200
1
1400
I
1600
I
Acrn-' Figure 4. Resonance Raman spectra of aqueous tyrosine (pH 7, 1 mM)
with 200-, 218-, and 240-nm excitation. TABLE I: Molar Enhancement Factors (X10-2) Relative to the 3400-cm-' H20Band for Selected Raman Bands of the Aromatic Amino Acids;
amino acid band. cm-I 200 nm 218 nm 240 nm 266 nm 202 126 30 tyrosine, pH 7 1617 tyrosine, pH 11 1602 437 152 53 36 96 8 phenylalanine 1000 tryptophan 506 53 20S 1016 29b 'See ref 7 for the method of calculation. bEstimated from relative intensities of tryptophyltyrosine.* CValuesomewhat uncertain due to interference from tryptophan fluorescence at the water band.
~~
acid are scaled to show the relative enhancement at the different excitation wavelengths.
1858 The Journal of Physical Chemistry, Vol. 89, No. 10, 1985
Rava and Spiro
TABLE II: Aromatic Amino Acid Raman Band Assignments and Relative Intensities
obsd freq,cm-'
200 nm
1606 1586 1207 1182 1028 1000
10 8 5 5 2 10
1617 1601 1519 1443 1263 1210
10 7 0 1 6 9
1180 853 831
3 4 2
TYROSINE
200nm
re1 int at X, 218 240 266 nm nm nm assignmt',* A. Phenylalanine 10 6 8a 5 0 8b 3 5 C6HJ-C stretching 3 0 9a 0 2 12 5 10 1 (ring breathing)
B. Tyrosine, pH 7 10 10 8a 8 4 8b 0 2 19a 0 0 19b 0 0 7a 1 3 para-substituted benzene, totally symmetric stretch 4 7 9a 0 3 1 (ring breathing) 0 2 2 X 16a (out-of-plane)
C. Tyrosine, pH 11 1603 1575 1558 1517 1409 1264 1209
10 0 3 0 0 3 4
10 0 5 0 0 2 2
10 2 1 6 3 0 2
1174 852 832
2 2 1
4 1 1
7 2 3
1622 1578 1555
7 10 9
4 1 10
10 1 5
1496 1462 1434 1361 1342 1305 1256 1238 1148 1127 1016
4 4 4 4 7 1 2 7 3 5 5
2 2 1 4 4 2 0 3 1 1 10
0 3 0 3 5 1 0 1 1 1 8
880
2
3
2
762
4
9
5
8a
p H I1
I
x3
I
800
I
1000
1200
I
1400
I I1
I
1
J
1600
A cm-' Figure 5. Resonance Raman spectra of aqueous tyrosine (pH 11,l mM) with 200-, 218-, and 240-nm excitation.
8b 19a 19b 7a para-substituted benzene, totally symmetric stretching 9a 1 (ring breathing) 2 X 16a (out-of-plane)
D. Tryptophan 9
8a 8b 5 totally symmetric naphthalene-t ype stretchingC 1 19a 2 19b 2 6 n 3 14n 4 1 4 1 5 n 0 3 3 1 15or6NH 2 9 b 3 benzene and pyrrole ring- breathing out-of-phasec 2 skeletal vibration with appreciable N H pyrrole bendC 3 benzene and pyrrole ring-breathing in-phasec 10
"Benzene mode numbering from ref 16. *Pyrrole modes are indicated by n using the numbering scheme of ref 17. 'See ref 15.
Table I1 lists the observed frequencies and the relative intensities of the Raman bands at each wavelength. Band assignments are also given. For phenylalanine and tyrosine, these correspond to the analogue molecules toluene12and pcresol," which differ from the respective amino acids only in the replacement of a methyl proton by the a-carbon linkage (see Figure 1). In the case of (12) N. Fuson, C. Garrigou-Lagrange, and M.L. Josieu, Specrrochim. Acro, 16, 106 (1960). (13) R. J. Jacobsen, Specfrochim. Acra, 21, 433 (1965).
F i i 6. Resonance Raman spectra of aqueous tryptophan (1 mM) with 200-, 218-, 240-nm and 266-nm excitation. Numbers in parentheses correlate with benzene or pyrrole mode ( r )assignments.
TABLE III: Absorption Maxima (nm), Extinction Coefficient (M-I cm-I), and Transition Assignments for tbe Aromatic Amino Acids"
amino acid tyrosine, pH 7 tyrosine, pH 11 phenylalanine tryptophan
Ba.b
193 (36000) b 185 (36000) 218 (34000)
-
La 222 (7000) 240 ( 1 1000) 207 (7000) 273' (5500)
Lb
273 (1300) 293 (2400) 255 (300) 287' (4500)d
'States are labeled using the notation of Platt.'* *Difficult to determine because of OH- absorption. CAssignment from ref 15. dStrongly overlapped with L,.
tryptophan, recent assignment of the indole vibrational spectrum14 was used as a guide, together with the normal-mode calculations
The Journal of Physical Chemistry, Vol. 89, No. 10, 1985 1859
RRUV Spectra of Aromatic Amino Acids by Hirakawa et al.,I5 although the ahsence of a methylene substituent on the indole ring is associated with appreciable frequency differences, relative to tryptophan, for several of the modes. Where possible, the benzenet6 or pyrrole" (labeled T ) mode numbering systems were used to describe the vibrations.
Discussion Enhancement Mechanisms. The excited states of the aromatic chromophores under study are closely related to the parent benzene molecule (see Table 111). The first allowed (degenerate) T-T* transition of benzene (labeled Ba,bin Platt's notation") at 183 nm is preceded by two symmetry-forbidden transitions La and Lb whose absorption peaks are at 203 and 253 nm. Substituents on the benzene ring lower the symmetry and introduce some allowed character into the symmetry-forbidden transitions. In addition, the transition energies are shifted, and the degenerate Ba,btransition is split by the symmetry 10wering.'~ For phenylalanine, the substituent is a methylene group and the perturbation on the benzene electronic structure is slight. The La and Lb transitions are still weak, and the peak transition energies are close to those reof benzene, at 207, 255, and -185nm for La, Lb, and spectively. The perturbation is much larger for tyrosine, with a hydroxyl as well as a methylene substituent. La and Lb have appreciable intensity and are red-shifted relative to benzene (222 and 273 nm) as is the allowed transition, Ba,b,at 193 nm. The perturbation is largest for tryptophan, with a pyrrole ring fused onto the benzene ring. The Ba,bband is shifted down to 218 nm, and the La and Lb bands overlap at -280 nm. Assignments of Lb and La at 287 and 273 nm have been suggested for aqueous solution spectra,20while the Lb origin band of indole was located at 283.8 nm in a supersonic jet spectrum, although no evidence for the La state could be found within 2200 cm-' of the Lb In resonance with allowed electronic transitions, the dominant Raman scattering mechanism is the A (Franck-Condon) term in the scattering equationlo
-
((Mu)$(~p)$~(l[j)ciio)J (%r)A
=
J
((u:,
- upO) - uL + irejJ
+ cc
(1)
where Mu,pare electronic transition dipole moments, utj and uL are the energy of the j t h vibrational level in the electronic state e (similarly for up) and laser frequency respectively, ( k l l ) are Franck-Condon integrals, and C C is the complex conjugate of the first term. Totally symmetric modes are enhanced, along whose normal coordinates the exicted-state potential surface undergoes significant origin shifts. Because of the dependence on the square of the allowed electronic transition moment, this term is small for transitions that are only weakly allowed. Raman scattering may instead be enhanced via the B (vibronic scattering) term for weakly allowed transitions ((Mu)$(eO1halsO) (Mp):gC( 1I Q a b ) (40)I
where (eO1h,lso)/u:e is the vibronic coupling matrix element. This term is large if the weakly allowed resonant state is coupled vibronically to a nearby strongly allowed state. The vibronic determines the form of the vibronic coupling integral, ( eO1halsO) modes which are active in coupling the electronic states. These modes are enhanced in the Raman spectra and also lend strength to the absorption spectrum. In substituted benzenes, the La (14) A. Lautib, M. F. Lautib, A. Gruger, and S.A. Fakhri, Spectrochim. Acta, Part A , 36, 85 (1980). (15) A. Y. Hirakawa, Y. Nishimura, T. Matsumoto, M. Nakanishi, and M. Tsuboi, J . Raman Spectrosc., 7, 282 (1978). (16) E. B. Wilson, Jr., Phys. Rev., 45, 706 (1934). (17) R. C. Lord, Jr., and F. A. Miller, J . Chem. Phys., 10, 328 (1942). (18) J. R. Platt, J . Chem. Phys., 17, 484 (1949). (19) J. N . Murre11 in "The Theory of the Electronic Spectra of Organic Molecules", Wiley, New York, 1963, Chapters 6, 9, and 10. (20) Y. Yamatnoto and J. Tanaka, Bull. Chem. Soc. Jpn., 45, 1362 (1972). (21) R. Bersohn, U. Even, and J. Jortner, J . Chem. Phys., 80,1050 (1984).
transition couples to the a component of the Bq,btransition via the normal coordinate vgaand, to a lesser extent, ma vA. It couples to the b component of B, b via v8b. These three modes were shown by Ziegler and Hudsoni to be enhanced in the R R spectra of alkylbenzenes when excited at 213 nm, in resonance with La. The Lb transition is farther from the allowed B,, transition, and there are no vibrational modes that are as effective in mixing the two states. Consequently, the absorption strength of Lb is much less than that of La, in benzene and substituted benzenes, and it is not expected to be as effective at resonance enhancement of Raman bands. This is born out by the spectra to be discussed below. In benzene itself the La and Lb transitions are rigorously electric dipole forbidden, and the B term cannot contribute to resonance enhancement. Ziegler and Hudson' observed enhancement of the overtones of the benzene vibronic mixing modes v8 and 4,attributable to the C term in the scattering equation, which mixes the ground and excited states. They also observed intensity for the ring-breathing fundamental, vl, in excess of what was expected from preresonance enhancement via Ba,b. This extra intensity, also seen for alkylbenzenes? was likewise attributed to the C term. Phenylalanine. The R R spectra of phenylalanine (Figure 3) are very simple, only seven bands appearing above the background. The strongest enhancement is found at 218 nm, close to resonance with the La transition. This spectrum is quite similar to the ones reported by Ziegler and Hudson for alkylbenzenes at 213 nm. As expected, the dominant vibronic mixing mode, Yga, displays the greatest intensity, and the other mixing modes V8b and vga, at 1586 and 1182 crn-', are also prominent. Significant intensity is seen for the ring-breathing mode, u l , at 1000 cm-', and another totally symmetric mode, at 1207 cm-'. These two modes gain in relative intensity, as does a weaker symmetric ring mode ( u I z ) , at 1028 crn-', with excitation at 200 nm, as is expected from preresonance Franck-Condon scattering via the allowed Ba,btransition at 185 nm. The overall scattering, however, is a factor of 2 or 3 lower at 200 than at 218 nm, so that the absolute intensities of the symmetric modes are similar at these wavelengths. This observation supports the conclusion by Ziegler and Hudson's2 that the symmetric mode intensities seen in resonance with the La transition cannot be accounted for completely by preresonance enhancement from the allowed Ba,bstate, since one would then expect greater enhancement of the totally symmetric modes at 200 nm, and that an extra enhancement mechanism, perhaps via the C term, is required. With 240-nm excitation, which lies between the Lb and La transitions, the scattering is an order of magnitude weaker than at 218 nm. The observed features can probably be accounted for by preresonance enhancement from the La and higher-lying transitions. Tyrosine. The 218-nm laser line is also resonant with the La transition of tyrosine, whose R R spectrum at this wavelength (Figure 4) is even simpler than that of phenylalanine. Only the mixing modes V8a, V8b, and uga, at 1617, 1601, and 1180 cm-I, are seen with significant intensity. With 200-nm excitation, which is close to resonance with the tyrosine Ba,ballowed transition, the scattering is over twofold stronger than at 218 nm, and the totally symmetric modes at 853, 1210, and 1263 cm-I are prominent. These modes show negligible intensity at 218 nm, so that whatever extra mechanism is responsible for Franck-Condon scattering in phenylalanine at this wavelength is apparently not operative for tyrosine. The 240-nm line is between the La and Lb transitions of tyrosine. Overall Raman scattering is about threefold weaker than 21 8 nm and is dominated by the mixing modes vga, V'b, and uga In addition, a new band is seen in the 240-nm tyrosine spectrum, at 1519 cm-I, assigned to ring mode ~ 1 9 ~We . propose that this mode is effective in mixing Lb, but not La, with the allowed Ba,bstate. For tyrosine the Lb transition is much stronger than it is for phenylalanine or the alkylbenzenes (see Figure 2). The OH substituent induces appreciable allowed character into this transition, making the B term scattering more effective than it is for alkylbenzenes. Another feature of interest in the 240-nm spectrum is the reappearance of the 853-cm-I u I mode, and its prominent shoulder at 825 cm-'. This is the well-known Fermi doublet of t y r o s i r ~ e , ~ ~ . ~ ~
-
1860 The Journal of Physical Chemistry, Vol. 89, No. 10, 1985
which is ascribed to the interaction of vl with the overtone of the out-of-plane deformation mode, vIh. It disappears at 218 nm but reappears at 200 nm, albeit with greatly altered relative intensities of the two components. Moreover, there seems to be a distinct frequency shift and broadening of the low-energy shoulder in the 240-nm spectrum, suggesting the involvement of a third component in the band envelope, of unknown origin. The intensity at 240-nm implies an involvement of these modes in the vibronic character of the L b state, or else an additional Franck-Condon mechanism, perhaps via the C term, as discussed above for the ring-breathing mode of the alkylbenzenes. At pH 11 the tyrosine OH is deprotonated. The tyrosinate electronic transitions are red-shifted and intensified relative to tyrosine (Table 111). The R R bands (Figure 5) show characteristic frequency shifts, and there are also differences in the enhancement pattern. Thus the enhancement at 200 nm is even greater, relative to 218 nm, for tyrosinate than for tyrosine. Also, the tyrosinate totally symmetric modes at 852, 1209, and 1264 cm-I retain substantial relative intensity at 218 nm reflecting enhanced preresonance scattering and suggesting increased Franck-Condon character for the La scattering. In the 240-nm spectrum of tyrosinate, the vIga mode, a t 1517 cm-I, is quite prominent, and a new band is seen,at 1409 cm-I, assigned to vIgb,which apparently becomes effective in mixing with Ba,b An additional band is seen at 1575 cm-' whose assignment is uncertain. Tvptophan. The tryptophan R R spectra are far more complex, as expected from the larger number of atoms in the indole ring system. The strongest scattering is seen at 218 nm (Figure 6) in resonance with the Ba,btransition. Particularly prominent are the bands at 762, 1016, and 1555 cm-I, which have been assigned via a normal coordinate analysisI5to modes which can be described as in- and out-of-phase breathing motions of the benzene and pyrrole rings and a naphthalene-type stretch of the indole system, respectively. These modes can be expected to exhibit strong Franck-Condon activity in the allowed T-T* transition. Excitation at 266 nm also gives strong scattering, but with a different distribution of the intensity. The most prominent bands are those at 1622 and 1578 cm-I, assignable to the benzene modes vSa and vgb; these are the same modes that are effective in vibronic mixing for both phenylalanine and tyrosine. The 266-nm line is close to the -280-nm absorption band which is believed to contain both La and L b transitions of tryptophan. This is a strong ab= 5500 M-' cm-', see Figure 2), and it is clear that sorption the perturbation of the benzene symmetry is sufficient to render the La and/or L b transitions substantially allowed, thus accounting for the strong Raman enhancement. Neverthless, the prominence of the vibronic mixing modes vsa and v s b emphasizes the relationship to the benzene forbidden states and the residual importance of vibronic mixing in the long wavelength tryptophan transitions. With excitation at 240 nm, the Raman scattering is much weaker than at 218 or 266 nm, and the spectrum shows both the Franck-Condon and vibronic modes. It is of interest that vsa (1622 cm-I), but not v& (1578 cm-I), shows Franck-Condon as well as vibronic activity, since it retains much more intensity at 240 nm than does vgb, and it still remains moderately strong at 218 nm. Evidence for Born-Oppenheimer Breakdown in High-Lying Tryptophan States. We noted earliers a sharp drop in the tryptophan Raman scattering at 200 relative to 218 nm, even though the absorptivity is still high at 200 nm (see Figure 2). Figure 6 shows that the overall scattering is an order of magnitude weaker at 200 nm than at 218 nm. The 200-nm spectrum shows another interesting feature, namely, a redistribution of the intensity into a large number of modes. Whereas the 218- and 266-nm spectra are dominated by Franck-Condon and vibronic modes, respectively, both sets of modes are equally prominent at 200 nm, and numerous other bands have also gained significant relative intensity. (22) M. N. Siamwiza, R. C. Lord, M. C. Chen, T. Takamatsu, I. Harada, H. Matsuura, and T. Shimanouchi, Biochemistry, 14, 4870 (1975). (23) J. L. McHale, J. Raman Spectrosc., 13, 21 (1982).
Rava and Spiro We suggest that this intensity redistribution into many modes of the molecule is diagnostic for the excitation energy falling in a crowded region of electronic transitions, where the Born-Oppenheimer approximation is expected to break down. When electronic levels are close enough in energy (quasi-degenerate) small nuclear displacements can cause large changes in the electronic wave function. This phenomenon has been considered previously in the context of the Jahn-Teller effect, in which certain Jahn-Teller active modes are effective in breaking the degeneracy of an electronic level, and are strongly enhanced in the R R spectrum.24 Corrections for Born-Oppenheimer breakdown have also been applied, in the context of the Herzberg-Teller formalism, to the calculation of Raman excitation profiles.25 In these cases extra enhancements for specific modes are examined. Here we wish to emphasize the possible role of electron-nuclear coupling in inducing Raman activity into many modes and lowering the overall Raman scattering. If the wave function of a resonant molecular eigenstate 9,is written as a linear combination of Born-Oppenheimer wave functions (qSis the primary BornOppenheimer electronic wave function) \k, = a d s +
Ci bi"fii
(3)
then the resonant polarizability can be expanded in terms of these Born-Oppenheimer states and their mixing coefficients
It is evident that these states can provide intensity for a variety of Raman modes. The greater the number of states which contribute, the smaller are the coefficients, and since the coefficients enter the Raman polarizability expression as squares or cross products, the overall intensity can decrease by this mixing. In addition, it is possible that higher-lying excited states are subject to significant Duschinsky rotation, which might also distribute the intensity among many modes.26 This phenomenon may be of general significance for molecular Raman spectra excited with increasingly short wavelength lasers. The low-lying excited electronic states are frequently well separated and can give rise to strong Raman enhancements of a relatively few vibrational modes that are particularly well coupled to the state. At higher energies, the density of electronic states increases rapidly, and Born-Oppenheimer breakdown should be common. One can reasonably expect that excitation at energies above the lowest-lying allowed excited states of molecules will frequently be characterized by the appearance of many vibrational modes in the spectrum, and a loss in overall scattering efficiency, even though the absorptivity is high. Tyrosine Fermi Doublet and Resonance Enhancement. The dramatic change in the relative intensities of the components of the tyrosine Fermi double at -850 cm-I between 240 and 200 nm deserves comment. For both tyrosine and tyrosinate, the 853/831 cm-I ratio is greater than 2.0 at 200 nm, but about unity at 240 nm. With visible excitation, however, the ratio is 1.4 for tyrosine and 0.7 for tyrosinate.22 This variation in the ratio for different tyrosine entities is attributed to changes in the Fermi interaction associated with the state of the O H proton, and the ratio is diagnostic for different H-bonding modes of tyrosine in proteins.22 Unfortunately this ratio is much less discriminating in the UVRR ~ p e c t r a . ~ Since the doublet is due to mixing of the ring mode vl with the overtone of the out-of-plane deformation mode, q6a, we attribute the change in the intensity ratio to differential enhancement of the polarizabilities associated with the ring-breathing mode and the v16a overtone. The intensity ratio of the two bands involved (24) (25) (1973); (26)
M. Tsuboi and A. Y . Hirakawa, J . Mol. Spectrosc., 56, 146 (1975). (a) J. M. Friedman and R. M. Hochstrasser, Chem. Phys., I, 457 (b) M. Mingardi and W. Siebrand, J . Chem. Phys., 62, 1074 (1975). W. L. Peticolas, personal communications.
J . Phys. Chem. 1985,89, 1861-1865 in a Fermi resonance is given byz3
where I+ (I-) is the intensity of the higher (lower) frequency component, K = (Y&/ffb is the ratio of the unperturbed polarizability of the double quantum vibration, v,, to the single quantum vibration, vb, and 2( 2 12) u
Y=
'
(A'
+ 8V2)'12 - A
where A is the unperturbed frequency difference, 2v, - vb, and U is the perturbation energy, usually 3-10 cm-'. Of these parameters, only K depends on the laser wavelength, by virture of resonance effects on the polarizabilities. K is expected to have a small value, since the unperturbed polarizability of the overtone should be smaller than that of the breathing mode. It cannot be zero, however, since then I + / L = y-', independent of the laser wavelength. We expect K to be smaller with UV excitation, where the ring-breathing mode is in resonance, than with visible excitation, where off-resonance contributions may be relatively more important for the overtone of the out-of-plane mode. If K
