Analysis of Measured and Calculated Raman ... - ACS Publications

Sep 22, 2010 - Department of Chemistry and Biochemistry, Montclair State University, 1 Normal Avenue, Montclair, New Jersey 07043. J. Phys. Chem...
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J. Phys. Chem. A 2010, 114, 10897–10905

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Analysis of Measured and Calculated Raman Spectra of Indole, 3-Methylindole, and Tryptophan on the Basis of Observed and Predicted Isotope Shifts Senghane D. Dieng and Johannes P. M. Schelvis* Department of Chemistry and Biochemistry, Montclair State UniVersity, 1 Normal AVenue, Montclair, New Jersey 07043 ReceiVed: August 3, 2010

The aromatic amino acid tryptophan plays an important role in protein electron-transfer and in enzyme catalysis. Tryptophan is also used as a probe of its local protein environment and of dynamic changes in this environment. Raman spectroscopy of tryptophan has been an important tool to monitor tryptophan, its radicals, and its protein environment. The proper interpretation of the Raman spectra requires not only the correct assignment of Raman bands to vibrational normal modes but also the correct identification of the Raman bands in the spectrum. A significant amount of experimental and computational work has been devoted to this problem, but inconsistencies still persist. In this work, the Raman spectra of indole, 3-methylindole (3MI), tryptophan, and several of their isotopomers have been measured to determine the isotope shifts of the Raman bands. Density functional theory calculations with the B3LYP functional and the 6-311+G(d,p) basis set have been performed on indole, 3MI, 3-ethylindole (3EI), and several of their isotopomers to predict isotope shifts of the vibrational normal modes. Comparison of the observed and predicted isotope shifts results in a consistent assignment of Raman bands to vibrational normal modes that can be used for both assignment and identification of the Raman bands. For correct assignments, it is important to determine force field scaling factors for each molecule separately, and scaling factors of 0.9824, 0.9843, and 0.9857 are determined for indole, 3MI, and 3EI, respectively. It is also important to use more than one parameter to assign vibrational normal modes to Raman bands, for example, the inclusion of isotope shifts other than those obtained from H/D-exchange. Finally, the results indicate that the Fermi doublet of indole may consist of just two fundamentals, whereas one fundamental and one combination band are identified for the Fermi resonance that gives rise to the doublet in 3MI and tryptophan. The aromatic amino acids tyrosine and tryptophan play essential roles in important biochemical processes such as protein electron transfer and enzymatic catalysis.1 Their neutral and cationic radicals have been observed or proposed as intermediates in a variety of enzymes such as ribonucleotide reductase, photosystem II, catalase-peroxidase, cytochrome c peroxidase, and DNA photolyase.2-9 Both tyrosine and tryptophan have also been used in model compounds to study their role in electron transfer processes.10-12 Tryptophan is also used to probe its local protein environment by using fluorescence.13 Although fluorescence spectroscopy of tryptophan is a very important tool, it lacks the level of local structural information that can be provided by Raman spectroscopy. Raman and ultraviolet resonance Raman spectroscopy has been used extensively to probe the local environment and conformation of tryptophan in proteins.14-24 Raman bands (labeled W1-W19)25 have been shown to be sensitive to hydrogen-bonding (W4, W6, W17), the C2-C3-Cβ-CR torsional angle (W3), and hydrophobicity of the environment of the indole ring (W7) (see Figure 1 for structures and atomic numbering).15,16,18,23 Since tryptophan radicals are (transiently) formed during protein electron transfer and enzyme catalysis, the spectroscopic identification of tryptophan radicals is also of great interest.26-32 The first resonance Raman spectrum of a tryptophan neutral radical in a protein was obtained in DNA photolyase, and it was proposed that neutral radicals on different * To whom correspondence should be addressed. Phone: 973-655-3301. Fax: 973-655-7772. E-mail: [email protected].

Figure 1. Structures and atomic numbering of indole, 3-methylindole, 3-ethylindole, and L-tryptophan.

tryptophan residues could be identified on the basis of their Raman spectra due to unique interactions with the protein environment.28 Recently, this was demonstrated to be the case for tryptophan neutral radicals in modified azurin.29,30 For the proper interpretation of the Raman spectra of tryptophan and its radicals, it is important to assign the Raman bands to vibrational normal modes. Much experimental and computational work has been done to investigate the vibrational spectra of tryptophan, which includes analysis of the related molecules indole and 3-methylindole. In experiments, isotope substitution has been used to identify and assign certain Raman bands.15,18,21,31,32 For the assignment of Raman bands to specific vibrational normal modes, computational approaches have been used. These approaches include normal coordinate analysis and Hartree-Fock (HF) and density functional theory (DFT) calculations on indole, 3-methylindole (3MI), 3-ethylindole (3EI), and L-tryptophan (Trp).18,24,29,30,33-41 Although there is general agreement between experiments, there is still significant

10.1021/jp107295p  2010 American Chemical Society Published on Web 09/22/2010

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disagreement concerning the assignment of vibrational normal modes to the Raman bands of indole, 3MI, and Trp. For Trp radicals, some experimental data are available, and calculations have been performed on indole, 3MI, and 3EI radicals.29,30,37,42 However, there is disagreement concerning the identification and assignment of several important Raman bands of the Trp neutral radical.28-30 In most cases, assignments have been based on direct comparison of observed and (scaled) calculated frequencies or on the analysis of the effect of H/D-exchange. The reliance on only (scaled) calculated frequencies has resulted in discrepancies in the literature,34,37,40,41 and H/D-exchange causes significant changes in the Raman spectra. In the latter case, it is difficult to track Raman bands and vibrational normal modes in the 1050 to 1450 cm-1 region due to normal mode scrambling,31,32,36 complicating the assignment of those Raman bands. In this paper, we investigate the use of observed and predicted isotope shifts for the identification of Raman bands and their assignment to vibrational normal modes. The Raman spectra of indole, 3MI, Trp, and several isotopomers have been measured to determine isotope shifts of the Raman bands. The Raman spectra of indole, 3MI, 3EI, and several isotopomers have been calculated to predict isotope shifts of vibrational normal modes. A consistent assignment of the Raman bands of indole, 3MI, and Trp to these vibrational normal modes is presented. The strong correlation between observed and predicted isotope shifts of these molecules demonstrates that this is an excellent approach for the identification of Raman bands and their assignment to vibrational normal modes. Therefore, the isotope-shift method holds great promise for the identification of Raman bands of the neutral and cationic radicals of these molecules and their assignment to vibrational normal modes. Materials and Methods Materials. Tryptophan, indole, and 3MI were purchased from Sigma-Aldrich. Tryptophan-indole-d5, Trp-15N2, indole-13C2, methanol-d4, and D2O were purchased from Cambridge Isotope Laboratories. All chemicals were used without further purification. Sample Preparation. Saturated solutions of indole and trypophan and their isotopomers were prepared in distilled water, and 3MI was dissolved in methanol. The NH/D-exchange was carried out by preparing saturated solutions of indole and Trp in D2O, and of 3MI in methanol-d4. All saturated solutions were spun in a MiniSpin (Eppendorf) to precipitate and remove any undissolved material. Raman Spectroscopy. The samples were excited with a 532 nm, 300 mW diode-pumped solid-state laser (LambdaPro). The laser light was focused into the sample with a 50 mm lens, and the Raman scattered light was collected with a 50 mm, F/1.2 camera lens (Nikkor, Nikon) under a 90° angle. The light passed through a 532.0 nm holographic notch filter to remove Rayleigh scattering and spurious laser light. The Raman scattered light was focused into the spectrograph (TriAx 320, Horiba JY) by a 200 mm lens through a wedge depolarizer (CVI) to avoid polarization effects in the spectrograph. The Raman scattered light was dispersed by using a 2400 mm-1 grating and detected with a liquid-nitrogen cooled, open-electrode, front-illuminated CCD detector (Symphony, Horiba JY). About 600 µL of sample was used in a custom-made Raman spinning cell. Each spectrum was signal-averaged for 30 to 40 min. The Raman spectra of the solvents (water, methanol, methanol-d4, and D2O) were measured to remove their contributions from the Raman spectra of the molecules of interest. OriginLab 7.0 (Microcal) was used for data analysis, including calibration of the Raman spectrum

Dieng and Schelvis

Figure 2. Raman spectra of indole (a) and indole 2-13C (c) in H2O; indole (e), and indole 2-13C (g) in D2O; and the calculated Raman spectra of indole (b), indole 2-13C (d), indole-ND (f), and indole-ND 2-13C (h) after scaling with a factor of 0.9824. The numbers in spectrum (b) refer to the vibrational normal modes of indole (see also Table 1).

with toluene, subtraction of solvent contributions, and correction for a slightly sloping baseline. Calculations. Density functional theory (DFT) with the B3LYP functional and the 6-311+G(d,p) basis set was used to calculate the Raman spectra of indole, 3-metylindole, 3EI, and the specific isotopomers that were studied. Gaussian 03 was used to build the molecules, optimize their geometries, and calculate the frequencies and Raman intensities of their vibrational normal modes.43 The Gabedit 2.2.0 freeware written by A.-R. Allouche was used to analyze and visualize the vibrational normal modes as well as to create the calculated Raman spectra by using Lorentzian lineshapes with a half-width of 5 cm-1. Corresponding vibrational normal modes of the different isotopomers were matched on the basis of visual inspection of the normal modes. For each molecule, a scaling factor was determined for the calculated frequencies by minimizing the square of the differences between measured Raman shifts and calculated frequencies. Only those pairs of measured Raman bands and calculated normal modes were used that were unambiguously assigned by comparison of measured and calculated isotope shifts. The quality of the assignment of calculated normal modes to measured Raman bands was determined by the root-mean-square of the difference (rmsd) between their frequencies. Results and Discussion For the remainder of the paper, we will refer to the calculated normal modes of indole, 3MI, and 3EI as I#, MI#, and EI#, respectively, and the numeric sign is followed by the normal mode number that results from the Gaussian output file. Vector diagrams of the assigned normal modes, Cartesian coordinates of the calculated structures, and calculated frequencies and Raman intensities are provided in the Supporting Information. Indole. The experimental Raman spectra of indole, indole13 C2 in H2O and D2O are shown in Figure 2 together with the corresponding calculated Raman spectra. The Raman spectra of indole and indole-ND are very similar to those reported previously in the literature.32,33,40 We have used the W-labeling of Trp to identify similar Raman bands in the spectrum of indole. We have extended the labeling to include the bands at 609 and 544 cm-1 as W20 and W21, respectively. No Raman bands of indole were found to correspond to W4, W10, and W19. As

Isotope Analysis of Indoles and Tryptophan

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TABLE 1: Assignment of Observed Raman Bands of Indole to Calculated Vibrational Normal Modes of Indolea

W1 W2 W3 W4d W5 W6 W7 W8 W9 W11 W12 W13 W14 W15 W16 W17 W18 W20 W21

Raman shift (cm-1) (13C2; ND; 13C2, ND)

I# b

scaled normal mode frequency (cm-1)c (13C2; ND; 13C2, ND)

1618 (-1, -1, -2) 1579 (-1, -7, -7) 1510 (-19, -5, -16) - (-, 1478, -7) 1459 (-5, -4, -11) 1424 (-8, -39, -44) 1356 (-2, -8, -9) 1339 (-3, -8, -12) 1283 (-1, -15, -17) 1252 (-1, -25, -30)e 1209 (-3, -37, -)e,f 1153 (-1, -4, -4) 1123 (-2, -5, -7) 1107 (-3, -162, -165)e 1069 (-3, +2, +1) 1011 (0, 0, -1) 900 (-7, -15, -19)e 878 (0, -19, -24)e 761 (-4, -5, -8) 609 (-1, -1, -1) 544 (-2, -1,-4)

35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 17 16 12 9 6

1627 (-1, -4, -4) 1586 (-1, -6, -6) 1519 (-16, -2, -16) - (-, 1483, -6) 1452 (-6, -2, -9) 1416 (-7, -34, -39) 1356 (-1, -8, -10) 1339 (-5, -12, -18) 1271 (-1, -7, -9) 1244 (-1, -24, -29) 1199 (-3, -34, -35) 1153 (0, -3, -5) 1120 (-1, -4, -5) 1088 (-4, -157, -159)g 1067 (-4, +5, +3) 1013 (0, 0, 0) 894 (-8, -19, -21) 870 (0, -32, -40) 760 (-4, -6, -8) 607 (-1, -2, -3) 541 (-2, -3, -5)

a The observed and predicted isotope shifts are between parentheses as indicated. b I#: number of the vibrational normal mode in the Gaussian output file. c Scaling factor is 0.9824. d W4 is only observed in indole-ND and indole-13C2-ND. e The ND and 13 C2-ND isotope shifts of these Raman bands were identified with the help of the predicted isotope shifts. f Band not observed in 13C2-ND isotopomer. g Composition of normal mode changes completely upon NH/D-exchange.

expected, the W3 band shows the largest sensitivity to the 13C2 isotope substitution and shifts 20 cm-1 from 1511 to 1491 cm-1. The normal mode associated with W3 has a strong contribution from ν(C2-C3).16 The W5, W6, W18, and 900 cm-1 bands also show considerable sensitivity to the 13C2 isotope. The Trp hydrogen bond markers W6 and W17 as well as the W8, W9, W11, W14, W17, and 900 cm-1 bands show strong sensitivity to NH/D exchange in indole. The W3 band shifts by 6 cm-1 to a lower frequency due to δ(NH) contribution to its normal mode in indole. The spectra of indole-ND and of indole13 C2-ND are very similar to each other. Only the W3 band and the bands at 1479 and 1455 cm-1 undergo significant changes in the double isotope compared to the indole-ND isotopomer. The calculated spectra resemble the measured spectra very well, and the strong correlation between observed and predicted isotope shifts is shown in Table 1. The frequencies of the calculated Raman spectra have been corrected with a scaling factor of 0.9824. The calculated normal modes were mainly assigned to Raman bands on the basis of the observed and calculated indole-13C2 isotope shifts. These assignments were further confirmed by observed and calculated shifts due to NH/ D-exchange of indole and indole-13C2. The agreement between observed and calculated shifts is striking for most Raman bands and normal modes. The assignment of isotope shifts due to NH/ D-exchange was difficult for the W9, W14, W17, and 900 cm-1 bands. Their corresponding Raman bands for the ND- and ND/ 13 C2-isotopomers could only be identified by using the calculated NH/D isotope shifts. The new band at 1478 cm-1 in the Raman spectrum of indole-ND corresponds to normal mode I#32, which does not align with any band in the Raman spectrum of indole. The composition of this normal mode is similar for all four isotopomers and is nearly identical to that of 3MI normal

Figure 3. Raman spectra of 3MI in methanol (a) and in methanol-d4 (c), and the calculated Raman spectrum of 3MI (b) and of 3MI-ND (d) after scaling with a factor of 0.9843. The numbers in spectrum (b) refer to the vibrational normal modes of 3MI (see also Table 2).

mode MI#39 and 3EI normal mode EI#46, which correspond to the W4 band of 3MI and Trp, respectively (see below). This suggests that normal mode I#32 could be the equivalent of W4 in indole. Even though the calculations predict a reasonable Raman intensity for W4, it is only observed in the experiment after NH/D-exchange. This NH/D-sensitivity and the agreement between observed and calculated shift for indole-13C2-ND support the assignment of normal mode I#32 to W4 in indole. Finally, the composition of normal modes I#16 and I#17 is changed into a linear combination of these modes, while the composition of normal mode I#22 is completely changed following NH/D-exchange. The assignments as listed in Table 1 yield and rmsd ) 6.3 cm-1 for naturally abundant indole, underscoring the excellent agreement of the assignments. Our calculations and assignments are in good qualitative agreement with the work by Walden and Wheeler, who used B3LYP and BLYP with the 6-31G(d) basis set and compared their results to infrared spectra of indole.35 Although our calculated frequencies are identical to those reported by Sundaragenesan et al.,40 we only agree with their assignments for W1, W2, W20, and W21 but not for the other 16 Raman bands in Table 1. This large discrepancy seems due to their use of the general scaling factor of 0.96,44 which overcorrects the harmonic frequencies in this case, and to the fact that their assignments are solely based on comparison of scaled calculated frequencies to experimentally observed ones. We believe that our use of measured and calculated isotope shifts for assignments as well as the determination of a scaling factor specific for the 1700 to 500 cm-1 frequency region of indole results in assignments with a much higher degree of confidence. 3-Methylindole. The experimental and calculated Raman spectra of 3MI in methanol and methanol-d4 are shown in Figure 3. The 3MI Raman bands are identified with the W-label of Trp,16 and no Raman bands were found to correspond to W11, W12, and W14. Similar to indole, the NH/D exchange causes significant changes in the 1450-1050 cm-1 range due to scrambling of the vibrational normal modes with contributions from δNH and of those that gain contributions from δND. It was possible to correlate the 3MI-ND normal modes to those of 3MI-NH. Only the normal mode associated with W17 changes completely upon NH/D exchange with a large δND contribution compared to a minor δNH contribution in the original normal mode. Although only the measured and calculated NH/D isotope shifts were used for the assignment

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TABLE 2: Assignment of Observed Raman Bands of 3-Methylindole to Calculated Vibrational Normal Modes of 3-Methylindolea

W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W13 W15 W16 W17 W18 W19 W20 W21

Raman shift (cm-1)

MI# b

scaled normal mode frequency (cm-1)c

1622 (-1) 1581 (-6) 1559 (-2) 1496 (-12) 1458 (-3) 1434 (-61) 1348 (0) ∼1339d (-8) 1305 (-18) 1254 (-14) 1233 (-48) 1128 (-2) 1078 (0) 1010 (0) 982 (+16) 878 (-19) 759 (-3) 708 (-5) 565 (0) 530 (+1) 465 (-10)

42 41 40 39 36 35 33 32 31 30 29 27 25 23 22 19 15 13 10 9 8

1632 (-5) 1591 (-4) 1569 (-1) 1497 (-8) 1456 (-2) 1422 (-48) 1350 (-1) 1343 (-9) 1298 (-14) 1249 (-11) 1222 (-43) 1130 (-2) 1072 (+4) 1017 (0) 983 (+14) 874 (-28)e 760 (-4) 706 (-5) 561 (-1) 530 (0) 463 (-13)

a The observed and predicted NH/D isotope shifts are between parentheses. b MI#: number of the vibrational normal mode in the Gaussian output file. c Scaling factor is 0.9843. d Raman band observed in methanol-d4, position in methanol estimated on predicted isotope shift. e Composition of normal mode changes completely upon NH/D-exchange.

of normal modes to the Raman bands, the correlation between experiment and calculation is very strong as shown in Table 2. Furthermore, the calculated spectra show considerable similarity to the measured spectra. The calculated frequencies have been corrected with a scaling factor of 0.9843, and we find an rmsd of 6.2 cm-1 for the observed and scaled calculated frequencies listed in Table 2, which underscores the quality of the assignments. Our Raman spectra and assignments of 3MI are in good agreement with those reported and proposed by others.16,37,38,45 Although our calculations and assignments are very similar to those recently reported by Combs et al and by Bunte et al.,37,38 we disagree with a few assignments made in the former paper. In that paper, normal modes MI#21 and MI#16 are assigned to the 982 cm-1 band and W18, respectively.38 We find that normal mode MI#22 and the 982 cm-1 band have the same NH/D shift (∼+15 cm-1), whereas no isotope sensitivity is predicted for normal mode MI#21. The predicted NH/D isotope shift of normal mode MI#15 (-4 cm-1) is very similar to that of W18 (-3 cm-1), whereas normal mode MI#16 has a much larger predicted isotope shift (-14 cm-1). Furthermore, normal modes MI#21 and MI#16 are OOP modes and are unlikely to have significant Raman intensity. Combs et al. describe normal mode MI#21 as a CH3 rock, similar to our normal mode MI#22, and normal mode MI#16 as a benzene C-C stretch, similar to our normal mode MI#15. Our assignments are the same as theirs on the basis of calculated frequencies and normal mode descriptions but have a different normal mode number. The origin of this discrepancy remains unclear. We disagree with one of the assignments made by Bunte et al.37 We assign W15 at 1078 cm-1 to normal mode MI#25, and they assign it to normal mode MI#26. Visual inspection shows that both normal modes are nearly identical. We favor normal mode MI#25 because it has a larger predicted Raman intensity

Figure 4. Experimental Raman spectra of Trp (a) and 15N2-Trp (c) in H2O and of Trp (e) and 15N2-Trp (g) in D2O. The gray spectra are the calculated Raman spectra of 3EI (b), 15N-3EI (d), 3EI-ND (f), and 15N3EI-ND (h) after scaling with a factor of 0.9857. The numbers in spectrum (b) refer to the vibrational normal modes of 3EI (see also Table 3).

and a predicted NH/D isotope shift of +4 cm-1 in agreement with the experiment, while normal mode MI#26 has a predicted NH/D isotope shift of -12 cm-1. Tryptophan. The Raman spectra of Trp and 15N2-Trp in H2O and D2O are shown in Figure 4 together with the calculated spectra of the corresponding 3EI isotopomers. The Raman spectra are very similar to those that have been reported before.15,16,18-21,31 The 15N substitution causes the largest effect on W6, W10, and W17, while W4 and W8 show modest sensitivity. The NH/D-exchange causes the hydrogen-bonding markers W4, W6, and W17 to shift significantly, whereas the Raman bands between 1450 and 1050 cm-1 undergo changes in their normal mode compositions with potentially large isotope shifts. The isotope shifts and the assignments of the Raman bands to normal modes of 3EI and 3MI are listed in Table 3. The assignments are largely based on comparison of the observed and calculated isotope shifts. The use of the NH/Dexchange data was limited, and the assignment of isotope shifts to W10 through W15 remains tentative due to normal mode scrambling. Comparison of the 15N effect on Trp and on TrpND facilitated the assignment of several NH/D shifts under the assumption that the 15N-effect is similar for corresponding Trp and Trp-ND modes. The assignment of the 3EI vibrational normal modes to the Raman bands listed in Table 3 has an rmsd ) 10.8 cm-1, and a scaling factor of 0.9857 is used for the calculated frequencies. Our assignment of experimental shifts of isotopically labeled Trp (15N, ND, and 15ND) is very similar to those proposed by Hirakawa et al. except for the NH/D shift of W9 at 1260 cm-1.31 On the basis of our combined analysis of observed and calculated isotope shifts, we prefer a smaller (-15 cm-1) shift, while they suggested a larger (-49 cm-1) shift to 1211 cm-1. We reserve the latter band for W11 of Trp-ND. Our NH/D assignments are in agreement with Hu and Spiro and largely in agreement with Shafaat et al.21,29,30 We disagree with the assignment of W19 to the 550 cm-1 band and that of W14 to the 1079 cm-1 band and the 1083 cm-1 band in H2O and D2O, respectively, by the latter group. Historically, the 711 cm-1 band has been assigned to W19 and the 1079 cm-1 band to W15.18,25 Concerning the differences in assignments of NH/D shifts, we do point out that this remains difficult for the Raman bands in the 1450 to 1050 cm-1 region and that we are only proposing

Isotope Analysis of Indoles and Tryptophan

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TABLE 3: Assignment of Observed Raman Bands of Tryptophan to Calculated Vibrational Normal Modes of 3-Ethylindole (3EI) and 3-Methylindole (3MI)a observed Raman shift (cm-1) W1 W2 W3 W4 W5 W6 W7e

W8 W9 W10 W11 W12 W13 W14 W15 W16 W17 W18 W19 W20 W21

3EI scaled normal mode frequency (cm-1)b

3MI scaled normal mode frequency (cm-1)c

N.A. (15N, ND, 15ND)

EI# d

N.A. (15N, ND, 15ND)

MI# d

N.A. (15N, ND, 15ND)

1621 (-2, -2, -3) 1578 (-1, -4, -5) 1551 (-1, -1, -1) 1494 (-3, -10, -11) 1461 (-1, -4, -5) 1434 (-9, -50, -56) 1361 (-1, -9, -11) 1344 (0, -9, -15) not observed 1333 (-3, -10, -13) 1305 (-5, -12, -17) 1260 (-1, -15, -19) 1236 (-4, -51, -53) 1206 (0, +10, +8) 1158 (-1, -7, -8) 1134 (-2, -5, -6) 1109 (0, -177, -172) 1076 (-2, +4, +3) 1012 (0, 0, 0) 880 (-10, -20, -27) 759 (-2, -4, -5) 711 (-3, -3, -5) 578 (-1, +1, -1) 539 (-1, +1, 0) 461 (-2, -9, -11)

49 48 47 46 42 41

1633 (-1, -5, -5) 1590 (-1, -4, -5) 1564 (-1, 0, -1) 1498 (-3, -9, -10) 1457 (-1, -2, -3) 1425 (-10, -46, -51)

42 41 40 39 36 35

1633 (-1, -5, -6) 1592 (-1, -4, -4) 1569 (-1, -1, -1) 1498 (-3, -8, -9) 1457 (-1, -2, -3) 1423 (-10, -48, -55)

39

1359 (0, -1, -1)

33

1351 (0, -1, -1)

38 37 36 35 34 33 32 31 29 28 26 22 17 15 12 11 10

1352 (-1, -9, -12) 1336 (-1, -4, -9) 1296 (-5, -8, -10) 1272 (-1, -15, -18) 1240 (-2, -60, -62) 1221 (-2, +16, +11) 1160 (0, -6, -8) 1134 (-1, -2, -2) 1087 (-4, -182, -185) 1069 (0, +10, +9) 1017 (0, 0, 0) 875 (-9, -28, -33) 751 (-1, -4, -5) 694 (-4, -4, -8) 564 (-1, -2, -4) 528 (0, -1, -2) 463 (-2, -13, -15)

32 31 30 29

1344 (-1,-9, -16) 1298 (-6, -14, -18) 1250 (-1, -11, -18) 1222 (-3, -43, -45)

28 27 26 25 23 19 15 13 10 9 8

1158 (0, -6, -8) 1130 (-1, -2, -2) 1088 (-6, -189, -193) 1073 (-1, +4, +3) 1017 (0, 0, 0) 874 (-9, -28, -33) 761 (-1, -4, -5) 706 (-5, -5, -10) 561 (-2, -1, -2) 530 (0, 0, 0) 463 (-2, -13, -15)

a The observed and predicted isotope shifts are between parentheses as indicated. b Scaling factor is 0.9857. c Scaling factor is 0.9847. d EI# and MI#: number of the vibrational normal mode in the Gaussian output file. e W7 is the Fermi doublet with two bands identified in the experiment and only one fundamental normal mode is calculated.

an assignment for those bands that give consistent observed and predicted isotope shifts. Although comparison of normal mode assignments from the literature is complicated by the use of different levels of calculation, various models, and scaling factors, the description of the calculated vibrational normal modes does not differ much. Therefore, the focus will be on the assignment of the vibrational normal modes from ab initio calculations to Raman bands of Trp. Indole, 3MI, 3EI, and L-Trp have been used for the assignment of vibrational normal modes to Trp Raman bands. The use of indole as a model for Trp is limited because indole lacks corresponding normal modes for W4, W10, and W19 (Table 1). Walden and Wheeler found good agreement between Trp Raman bands and indole normal modes.35 They assigned the 1492 cm-1 indole normal mode to W4. Our observation of a similar W4 band in indole-ND and indole-13C2,ND is in agreement with this assignment. Conform our analysis of indole, they did not report corresponding normal modes of indole for W10 and W19 of Trp. 3MI has been used as a model for Trp by several groups.33,34,37 In our analysis, we do not find a normal mode that corresponds to W11, in agreement with those groups. Our predicted 15N and ND isotope shifts of 3MI are in good agreement with those predicted by Takeuchi and Harada,33 but we disagree with several assignments of 3MI normal modes to Trp Raman bands made previously.34,37 In the one paper, W6 and W7 are incorrectly assigned to the 1361 and 1342 cm-1 Raman bands, respectively.34 Historically, W6 and W7 have been associated with the 1434 and 1361 cm-1 bands of Trp, respectively.16,25,46 Their proposed assignment of W6-W8 and W10 to 3MI vibrational normal modes also seems incorrect. In the other paper, a reversed order frequency assignment for W1(MI#41)

and W2(MI#42), for W4(MI#36) and W5(MI#39), and for W7(MI#32/MI#31) and W8(MI#33) was proposed, and normal mode MI#16 was assigned to W18.37 We disagree with these assignments. Our isotope shift analysis shows that all the calculated normal modes can be assigned to these Raman bands in the normal order from high to low frequency and that normal mode MI#15 corresponds to W18 (Table 3). In a recent paper on 3MI, Combs et al. provided a corrected analysis of those previously reported results.38 More recently, 3EI and L-Trp have been used to calculate vibrational normal modes for assignment to the Trp Raman spectrum.24,29,30,36,41 Addition of the ethyl group or the +H3N-CRCOO- main-chain introduces the importance of dihedral angles. Crystal structures of Trp analogs show that the Cβ-CR bond is not coplanar with the indole ring and that the dihedral angle about C2dC3sCβsCR (χ2,1) has values of 60° e |χ2,1| e 120°.16 Most calculations, including ours, have used optimized geometries that are in agreement with the crystal structures and have 75° e |χ2,1| e 105°.24,38,43,49 One group performed calculations with the ethyl group coplanar with the indole ring (χ2,1 ) 180°).29,30 This orientation may cause problems with the analysis of the vibrational normal modes. Jusczczak and Desamero showed that the calculated frequency of the normal mode corresponding to W3 varies with dihedral angle; a well-known experimental fact.24 They also found that addition of a water molecule to Trp changes χ2,1 to about 42°. The calculated Raman spectrum, and frequencies of the vibrational normal modes of 3EI are quite different for structures with χ2,1 ) 105° and χ2,1 ) 180° (Figure S1 and Table S2). Visual inspection of the normal modes show that some are also affected by the difference in dihedral angle, which is most likely due to different coupling of internal modes located on the indole ring with those located on the ethyl group (data not shown). We do want to point out

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that χ2,1 is a soft coordinate, and the χ2,1 ) 180° geometry may result in a shallow but local minimum. On the basis of the comparison of experimental data and 3EI calculations, we were able to assign 3EI vibrational normal modes to Trp Raman bands in a consistent way. Our assignments are in good agreement with those made by Gallouj et al., who used 3EI and 10 different scaling factors for the various internal coordinates.36 We disagree with their assignment of W8 (1310 cm-1) and W13 (1137 cm-1) to normal modes EI#37 and EI#32, respectively. Our isotope-shift analysis is consistent with the assignment of W8 and W13 to normal modes EI#36 and EI#31, respectively. We also propose different assignments for W5, W6, W14, and W18 of TrpND. We are very confident about our assignments of W5, W6, and W18, for which the NH/D isotope shift could easily be determined by matching the 3EINH and 3EI-ND modes through visual inspection. It is possible that their direct scaling of the internal force constants with multiple scaling factors in the earlier work has resulted in the calculation of slightly different frequencies and/or composition of the vibrational normal modes compared to our results. We largely agree with Shafaat et al., despite their use of a coplanar geometry of the ethyl and indole moieties for their calculations.29,30 Their assignment of W4 to a normal mode with a lower calculated frequency than W5 does not fit with our observed and predicted isotope shifts for the normal modes corresponding to W4 and W5. The origin of this discrepancy is not clear and may be due to their use of a smaller basis set, 6-31(d), for their calculations. Jusczczak and Desamero published DFT calculations on L-Trp and its 15N and d5 isotopomers for a few Raman bands.24 Our calculated isotope shifts for W3, W6, W7, and W17 are in agreement with theirs (Tables 3 and 4). Although they calculate an unscaled frequency for W6 that is significantly higher than ours (1524 cm-1 vs 1445 cm-1), they predict the same 15N isotope shift which confirms the proper assignment. The reason for the higher frequency is unclear and could be related to their use of a smaller basis set, 6-31G(d), or due to our use of 3EI instead of L-Trp for the calculations. The choice of computational method may matter because Cao and Fisher assigned a normal mode with an unscaled frequency of 1585 cm-1 to W6.47 In that work, SCRF/HF level calculations with 6-31G(d,p) were performed on L-Trp and compared to infrared spectra of Trp, and our assignments are in good agreement with theirs. Chuang and Chen performed similar calculations on L-Trp with DFT/B3LYP/6-311G and compared them to SERS, solution, and solid state Raman spectra of Trp.41 We strongly disagree with their assignments of, among others, the Raman bands at 1622 (W1), 1581 (W2), and 1463 cm-1 (W5) to asymmetric stretching of CO2-, NH3+- scissoring, and CH2- scissoring, respectively. Historically, these Raman bands have been assigned to normal modes that mainly consist of C-C stretching motions of the indole ring.16,33,35,38 It seems that these incorrect assignments are due to the fact that they seem solely based on matching observed and calculated frequencies, which were underestimated because of the use of a 0.97 scaling factor. Our work suggests that the use of isotope shifts and a scaling factor of 0.9857 is more appropriate for 3EI and, possibly, for L-Trp. Trp-Indole-d5. The Raman spectra of Trp-indole-d5 (Trpd5) in H2O and D2O together with the calculated spectra of the corresponding 3EI isotopomers are shown in Figure 5. The Raman spectrum of Trp-d5 is in good agreement with those reported before.18,21 Deuteration of the indole ring changes the Raman spectrum dramatically, which complicates the tracking of the Raman bands due to significant mode scrambling.

Dieng and Schelvis

Figure 5. Experimental Raman spectra of Trp (a) and Trp-d5 (c) in H2O and of Trp-d5 in D2O (e). The gray spectra are the calculated Raman spectra of 3EI (b), 3EI-d5 (d), and 3EI-d5-ND (f) after scaling with a factor of 0.9857.

Previous papers have used an alternative Wd-labeling for Trpd5 instead and have proposed some assignments.18,21 By using visual inspection of the normal modes and matching observed and predicted isotope shifts, the W1-W4, W16, W18-W21, and the 461 cm-1 bands in the Trp-d5 Raman spectrum can still be assigned with confidence as shown in Table 4. The assignments of W6 and W17 are tentative, and we did not find a good assignment for W5. For the assignment of W4, we agree with the smaller shift proposed by Hu and Spiro and not with the larger one previously proposed.18,21 Although the assignment of W6 is tentative, we agree with the smaller shift proposed previously18 and not with the larger one proposed more recently.24 The 3EI normal mode with the smaller shift had similar νC3-C9 and δNH contributions in Trp-d5 as in Trp. The normal mode corresponding to the larger shift would have a νC8-C9 and no δNH contribution in Trp-d5. Despite the differences in isotope shift, the assignments from the different groups for Trp-d5 W17 are all in agreement. Since only a subset of Raman bands could be identified for Trp-d5, this isotopomer has only limited use for the identification of Raman bands and their assignment to vibrational normal modes. W7 Doublet. The W7 band is arguably the most discussed Trp Raman band. The relative intensities of this doublet (1361 and 1344 cm-1) have been shown to be an indicator of the local environment of the indole ring and is still observed in the gas phase.15,38 The doublet is most likely due to a Fermi resonance between a fundamental in-plane mode around 1345 cm-1 and one or more combination bands of two out-of-plane (OOP) modes; bands at 920 and 420 cm-1 and at 745 and 600 cm-1 have been proposed to give rise to combination bands.15 The OOP modes are thought to be responsible for the sensitivity of the doublet to its environment.23,45 Most groups agree that the W7 doublets of Trp and of 3MI are due to a Fermi resonance, and it has been suggested to be the case for indole too. Two groups have argued that the doublet in Trp is due to two fundamentals on the basis of their calculations on indole and 3MI.35,37 Our calculations and those of others also show two or three fundamentals in the W7 region (1330-1370 cm-1).24,29,36,38 In the case of indole, we calculate two fundamentals in the W7 region, and the isotope shifts observed for the Raman bands at 1356 and 1339 cm-1 are very well predicted by indole normal

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TABLE 4: Assignment and Comparison of Observed and Predicted Shifts Due to the Indole-d5 Isotopomer of Tryptophana

W1 W2 W3 W4 W5 W6 W16 W17 W18 W19 W20 W21

Raman shift (cm-1)

EI# a

scaled normal mode frequency (cm-1)b

1621 (-20) 1578 (-23) 1551 (-27) 1494 (-50) 1461 (-) 1434 (-26) 1012 (-161) 880 (-56) 759 (-53) 711 (-25) 578 (-2) 539 (-18) 461 (-11)

49 48 47 46 42 41 26 22 17 15 12 11 10

1633 (-20) 1590 (-24) 1564 (-28) 1498 (-55) 1457 (-) 1425 (-22) 1017 (-168) 875 (-75) 751 (-53) 694 (-25) 564 (-6) 528 (-18) 463 (-24)

scaled normal mode frequency (cm-1)c

Raman shift (cm-1)d

Raman shift (cm-1)e

1617 (-18) 1577 (-24) 1557 (-28) 1487 (-666) 1458 (-24) 1424 (-17) 1009 (-164) 875 (-86) 756 (-53) 706 (-24)

1619 (-16)

1540 (-30) 1458 (-81) 857 (-76)

1552 (-27) 1493 (-49) 1460 (-78) 1012 (-163) 880 (-59) 757 (-53)

a

The observed and predicted indole-d5 isotope shifts are between parentheses. a EI#: number of the vibrational normal mode in the Gaussian output file. b This work with scaling factor is 0.9857. c Calculated frequencies from ref 24. d Observed Raman shifts from ref 18. e Observed Raman shifts from ref 21.

modes I#29 and I#28, respectively. Unlike 3MI and Trp, the NH/D-exchange has no strong effect on the W7 region. Therefore, we favor the idea that the W7 region of indole can be described properly by two fundamentals and that Fermi resonance may not occur. Although this is in agreement with Walden and Wheeler, we disagree with their proposal that the doublets in Trp and 3MI are also due to two fundamentals with no Fermi resonance.35 In the case of 3MI, we identify three Raman bands in the W7 region of 3MI-NH; a shoulder at ∼1354 cm-1, the main peak at 1348 cm-1, and a weak band around 1339 cm-1. We assign the normal mode at 1350 cm-1 (3MI#33) to the fundamental of the doublet, which is in agreement with Combs et al.38 In a recent study, normal mode MI#32 not MI#33 was favored as the fundamental on the basis of calculated Franck-Condon activity upon resonant excitation with ultraviolet light.45 In our analysis, normal mode MI#32 (1343 cm-1) has a predicted NH/D shift of -9 cm-1 and a 6-folder lower intensity. We assign it to the weak band at ∼1339 cm-1, which is clearly visible in the 3MI-ND spectrum at 1331 cm-1 and has a matching NH/D isotope shift of -8 cm-1. The ∼1354 cm-1 high frequency shoulder of W7 in 3MI-NH (separate peak in 3MI-ND) is still unaccounted for. Therefore, we assign the 1354 and 1348 cm-1 Raman bands to the Fermi doublet of 3MI and normal mode MI#33 to its fundamental. The analysis of W7 of Trp is more complicated. For the fundamental of the doublet, we find two possible vibrational normal modes at 1359 cm-1 (EI#39) and at 1352 cm-1 (EI#38). Normal mode EI#38 has the proper isotope shifts to be the fundamental with OOP normal modes EI#23 (924 cm-1) and EI#9 (423 cm-1) as the combination band (Table 5). However, its calculated Raman intensity is low, and it would lack the intensity to donate to the combination band in a Fermi resonance. Normal mode EI#39 has sufficient intensity to donate to a weak combination band in a Fermi resonance but it does not have the proper isotope shifts (Table 5), and the combination band would have to explain the observed isotope shifts. Our analysis shows that a combination band of OOP normal modes EI#16 (737 cm-1) and EI#13 (576 cm-1) would provide the correct isotope shifts for the Fermi doublet, while one consisting of OOP normal modes EI#23 (924 cm-1) and EI#8 (414 cm-1) would predict slightly larger isotope shifts (Table 5). In light of our calculations and the support for a 920/420 cm-1 combination band in the literature,15,45 we tentatively assign EI#39 to the W7 funda-

TABLE 5: Observed and Predicted Isotope Shifts of the Fundamental and the Two Candidates for the Combination Band of the W7 Fermi Doublet in Tryptophana EI# b

frequency (cm-1)c

W7

expd 39 38

1361 (-1, -9, -11)d 1359 (0, -1, -1) 1352 (-1, -9, -12)

920e

23

924 (0, 0, 0)

420e

9 8

423 (0, 0, 0,) 414 (0, -17, -18)

745f

18 16

764 (0, -1, -1) 737 (0, 0, 0)

600f

14 13

633 (-1, -3, -4) 576 (-2, -9, -11)

a The observed and predicted isotope shifts for the 15N, ND, and ND isotopomers are between parentheses. b EI#: number of the vibrational normal mode in the Gaussian output file. c Scaling factor is 0.9857. d Experimental data, not scaled. e Combination band due to bands at 420 and 920 cm-1. f Combination band due to bands at 600 and 745 cm-1. 15

mental and EI#23 and EI#8 to the combination band. Finally, we assign normal mode EI#37 (1336 cm-1) to the Raman band at the low-frequency shoulder of the Fermi doublet. In this picture, normal mode EI#38 may correlate to a weak Raman band that is obscured by the Fermi doublet. Our assignment of the fundamental to normal mode EI#39 at 1359 cm-1 is in agreement with Gallouj et al. and with Jusczczak and Desamero, who assign a normal mode at 1352 cm-1 (EI#39) and a Trp normal mode at 1360 cm-1 to the fundamental, respectively.24,36 The latter group also assigns a Trp normal mode at 1335 cm-1 to the W7 shoulder, which is similar to our assignment. More experimental and computational studies will be necessary to resolve the proper assignment of the W7 region. For the mean time, our proposed assignment, which is consistent with the observed isotope shift, will provide a good starting point for such an investigation. Conclusions The assignment of indole, 3MI, and Trp Raman bands to vibrational normal modes solely on the basis (scaled) calculated frequencies leads to incorrect assignments and inconsistencies

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in the literature. The current work shows that two things need to be considered when Raman bands are assigned to vibrational normal modes. First, a unique scaling factor of the calculated harmonic frequencies should be determined for the specific spectral region and molecule of interest. Second, two or more parameters should be compared before the assignment of a Raman band can be made. In the current work, scaling factors of 0.9824 and 0.9843 are determined for the 1750-450 cm-1 region of indole and 3MI, respectively, which result in an rmsd of 6.3 and 6.2 cm-1 for the observed Raman bands, respectively. The analysis of the Trp Raman spectra with 3MI and 3EI results in scaling factors of 0.9847 and 09857 and an rmsd of 10.5 and 10.8 cm-1, respectively. Although 3MI appears to be a slightly better model for Trp, 3EI is preferred because it provides sufficient normal modes to describe all observed Trp Raman bands. The use of observed and calculated isotope shifts as additional parameters results in a consistent assignment of Raman bands to vibrational normal modes. Given the significant changes in vibrational normal modes in the 1050-1450 cm-1 region, reliance on only H/D-exchange for normal mode assignment is discouraged. The use of 13C- and/or 15N-isotopomers is strongly recommended for this purpose. The assignment of Raman bands to vibrational normal modes by comparing isotope shifts largely eliminates the occurrence of incorrect assignments. Although the calculated Raman intensities and frequencies of some vibrational normal modes are not as accurate as may be obtained with methods using multiple scaling factors,36,48-50 we believe that it is more important that the observed Raman band follows the predicted isotope shifts of the calculated vibrational normal mode for the confirmation of the assignment. Our isotope-shift approach shows a strong correlation between observed and predicted isotope shifts for indole, 3MI, and Trp which results in a consistent assignment of the vibrational normal modes. The fact that the vibrational normal modes for W1 through W21 of indole, 3MI and Trp are basically identical (Supporting Information) provides additional support for the assignments. The success of this approach indicates that this methodology will also be very useful for the identification and assignment of the vibrational normal modes of the neutral and cationic radicals of Trp. Acknowledgment. J. P. M. S. gratefully acknowledges support from the National Science Foundation (MCB-0920013). The authors thank Dr. Marc L. Kasner for helpful discussions and for access to his computer to perform the calculations. Preliminary results were obtained by Mr. Morley Wang. Supporting Information Available: The calculated spectra of 3EI with different χ2,1 values, vector diagrams of all vibrational normal modes assigned to Raman bands, coordinates of optimized structures of indole, 3MI, and 3EI, and tables with the unscaled frequencies and Raman intensities of the normal modes of indole, 3MI, 3EI, and their isotopomers are available in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Stubbe, J.; van der Donk, W. A. Chem. ReV. 1998, 98, 705. (2) Bleifuss, G.; Kolberg, M.; Po¨tsch, S.; Hofbauer, W.; Bittl, R.; Lubitz, W.; Gra¨slund, A.; Lassmann, G.; lendzian, F. Biochemistry 2001, 40, 15362. (3) Baldwin, J.; Krebs, C.; Ley, B. A.; Edmondson, D. E.; Huynh, B. H.; Bollinger, J. M., Jr. J. Am. Chem. Soc. 2000, 122, 12195. (4) Barry, B. A.; Babcock, G. T. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7099.

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