Criteria for Determining the Hydrogen-Bond Structures of a Tyrosine

J. Phys. Chem. B 2007, 111, 13833-13844

13833

Criteria for Determining the Hydrogen-Bond Structures of a Tyrosine Side Chain by Fourier Transform Infrared Spectroscopy: Density Functional Theory Analyses of Model Hydrogen-Bonded Complexes of p-Cresol Ryouta Takahashi and Takumi Noguchi* Institute of Materials Science, UniVersity of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan ReceiVed: July 31, 2007; In Final Form: September 14, 2007

Fourier transform infrared (FTIR) spectroscopy is a powerful method to investigate the structures of key Tyr residues involved in various protein reactions. In this study, we have performed density functional theory (DFT) calculations for various hydrogen-bonded complexes of p-cresol, a simple model of a Tyr side chain, in different hydrogen-bond forms to develop explicit criteria for determining the hydrogen-bond structures of Tyr using FTIR spectroscopy. The CO stretching (νCO) and COH bending (δCOH) vibrations were focused as markers and calculated results were compared with experimental data of p-cresol and Tyr. The calculated and experimental νCO frequencies appeared at 1280-1260, 1260-1250, 1255-1235, and 1240-1220 cm-1 in the hydrogen-bond donor, free, donor-acceptor, and acceptor forms, respectively. These frequencies, which showed little overlap between the individual hydrogen-bond forms, had a negative linear correlation with the CO lengths in optimized geometries. The δCOH frequencies were found at 1255-1210 cm-1 in the donor form, while the free and acceptor forms showed relatively low δCOH frequencies at 1185-1165 and 11901160 cm-1, respectively. In the donor-acceptor form, the vibrational mode with a considerable δCOH contribution was found at 1280-1255 cm-1 with a weak IR intensity. This frequency and the νCO frequency in the donor-acceptor form are similar to the νCO and δCOH frequencies, respectively, of the donor form, making it difficult to discriminate the two forms. These two forms can be clearly distinguished by detecting a strong νCO(D) band in p-cresol-OD or Tyr-OD, in which the δCOD vibration largely downshifts to ∼1000 cm-1. The νCO(D) frequency of the donor-acceptor form was found at 1260-1240 cm-1, while that of the donor form was at 1270-1255 cm-1. Practically, plotting the frequency of the lower-frequency strong IR band (νCO of the donor-acceptor form or δCOH of the donor form) of undeuterated species against the νCO(D) frequency is convenient for accurate discrimination. Because the donor form shows a positive linear correlation between δCOH and νCO(D) frequencies, the two forms exhibited distinct areas in this plot. The effects of hydrogen-bond interactions on other potential IR and Raman markers are also discussed.

Introduction Tyr is an amino acid having a phenolic group, which shows versatile chemical properties. It can take four different hydrogenbond forms, i.e., free, hydrogen-bond donor, acceptor, and donor-acceptor forms (Figure 1). Tyr also becomes a tyrosinate anion upon deprotonation, while it can be oxidized to form a neutral radical by immediate proton release.1 Thus, Tyr often functions as a key amino acid in various protein reactions, such as signal transduction through a hydrogen-bond network, proton transduction, and proton-coupled electron transfer.1-6 Because the hydrogen-bond interactions of a Tyr side chain must play an essential role in such reactions, clarifying the hydrogenbonded structure of a key Tyr is an indispensable step in investigation of the molecular mechanism underlying the protein functions. Vibrational spectroscopy represented by infrared absorption and Raman scattering is a powerful method to obtain information about the structures and interactions of proteins. In particular, reaction-induced Fourier transform infrared (FTIR) difference spectroscopy has been extensively used to study the reactions and active-site structures of proteins.7-10 In this method, * Author to whom correspondence should be addressed. Phone: +8129-853-5126. Fax: +81-29-853-4490. E-mail: [email protected].

basically all of the structural changes in substrates, cofactors, polypeptide chains, and amino acid side chains are detected at the atomic level. Among numerous peaks in an FTIR difference spectrum, the signals of Tyr involved in the reaction can be identified by selective isotope labeling of Tyr side chains.2-6,11 UV resonance Raman spectroscopy is another powerful method to directly detect Tyr signals in proteins.12-19 Raman and IR markers to obtain structural information about Tyr have been argued by many authors. In Raman studies, the intensity ratio of a Fermi doublet at ∼850 and ∼830 cm-1,13,20,21 the ring CC vibrations (the 8a and 8b modes) at ∼1615 and ∼1600 cm-1,12,14,15,17 the CO stretch (7a′),16,22 and the CH bend (9a) at ∼1174 cm-1 16,19,22 have been proposed to reflect Tyr structures. In particular, Takeuchi et al.22 showed that a νCO Raman band can be a good marker to discriminate hydrogenbond donor and acceptor forms. Also, Gerothanassis et al.23 suggested in their FTIR study that upshifted δCOH bands can be markers of hydrogen-bond formation. However, in any of these studies, explicit criteria to discriminate all four hydrogenbond forms have not been provided. In this study, we investigated the effects of hydrogen-bond interactions on the Tyr vibrations using density functional theory (DFT) calculations, aiming at establishing the criteria for determining the hydrogen-bond forms of Tyr in FTIR spectra.

10.1021/jp0760556 CCC: $37.00 © 2007 American Chemical Society Published on Web 11/20/2007

13834 J. Phys. Chem. B, Vol. 111, No. 49, 2007

Takahashi and Noguchi FTIR spectra were measured on a Bruker IFS-66/S spectrophotometer equipped with a mercury cadmium telluride detector (D313-L). An attenuated total reflection (ATR) accessory (DuraSamp1IRII, Smiths Detection, Danbury, CT) with a three reflection silicon prism (3 mm in diameter) was used for measurements. FTIR spectra of solvents were subtracted from those of sample solutions to eliminate the contributions of solvents. For crystalline L-Tyr, powder samples were placed on the silicon prism to record spectra. All spectra were recorded at a resolution of 2 cm-1. Results

Figure 1. Hydrogen-bond forms of p-cresol as a model of a tyrosine side chain: (a) free form; (b) hydrogen-bond donor form; (c) hydrogenbond acceptor form; (d) hydrogen-bond donor-acceptor form.

DFT vibrational analyses were performed for various hydrogenbonded complexes of p-cresol, which is a simple model of a Tyr side chain. We have focused on the νCO and δCOH vibrations that should be most sensitive to hydrogen-bond interactions. The merit of quantum chemical calculations in such analyses is that any type of hydrogen-bonded complex can be assumed as model systems, which are often difficult to realize in experiments. In addition, quantum chemical calculations provide a theoretical background underlying the tendency of the hydrogen-bond effects. Vibrational analyses of p-cresol or Tyr in the free form have been performed by both the GF matrix method24 and quantum chemical calculations.21,25-28 The effect of hydrogen bonding was focused on in the DFT study by O’Malley,29 but only the complexes of p-cresol donating a hydrogen bond to imidazole were treated. We have recently performed DFT calculations for p-cresol complexes in all of the hydrogen-bond forms but only with acetamide or other similar amides as a hydrogen-bond partner.6 In this study, we have adopted various types of molecules as hydrogen-bond acceptors and donors and obtained general tendencies of hydrogen-bond effects from many examples of calculated results as well as experimental data for p-cresol and Tyr. Materials and Methods Molecular orbital calculations were performed using the Gaussian 03 program package.30 The B3LYP functional31,32 with the 6-31+G(d,p) basis set was used to optimize the geometries of model complexes and calculate the vibrational frequencies and IR intensities. All reagents other than p-cresol-OD and L-Tyr-OD were purchased from commercial sources. To obtain p-cresol-OD, p-cresol dissolved in hexane was mixed with methanol-OD, and then the solvents were evaporated. About 95% of p-cresol was deuterated, judging from a remaining OH stretching band of p-cresol. L-Tyr-OD and L-Tyr-OD‚DCl were prepared by recrystallization of L-Tyr in D2O and L-Tyr‚HCl in DCl/D2O solutions, respectively.

DFT Calculations of the νCO and δCOH Frequencies of Model Hydrogen-Bonded Complexes of p-Cresol. Table 1 shows the νCO (the 7a′ mode) and δCOH frequencies of free p-cresol and its hydrogen-bonded complexes calculated at the B3LYP/6-31+G (d,p) level. Various types of compounds were selected as hydrogen-bond partners. As hydrogen-bond acceptors in complexes of the donor form of p-cresol, oxygen atoms in OH (H2O, methanol, 2-propanol, or p-cresol), ether (diethyl ether or 1,4-dioxane), CdO (formaldehyde, acetone, carbonic acid, acetamide, or diformamide), and SdO (dimethyl sulfoxide (DMSO)) groups, nitrogen atoms in imidazole, pyridine, amines (methylamine and triethylamine), imine (methyleneimine), and nitrile (acetonitrile), and a sulfur atom in sulfide (dimethyl sulfide) were assumed. Also, as hydrogen-bond donors in complexes of the acceptor form of p-cresol, hydrogen halides (HF or HCl), OH groups (H2O, methanol, p-cresol, or trifluoroacetic acid), and NH groups (formamide, acetamide, methylformaide, or imidazole) were adopted. The optimized geometries of the model complexes are shown in Figures S1-S3 of the Supporting Information. When vibrations of hydrogen-bond partners were coupled to the νCO or δCOH vibration of p-cresol, such couplings were eliminated by appropriate isotope substitution of the partner molecules. The calculated νCO frequencies were scaled using a single scaling factor, 0.977, so as to adjust the frequency of free p-cresol to 1255 cm-1, the experimental value of p-cresol vapor.33 The same scaling factor was used also for the δCOH frequencies, because the two vibrations are more or less coupled with each other. The calculated δCOH frequency of free p-cresol, 1156 cm-1, was rather deviated from the experimental value of p-cresol vapor, 1178 cm-1,33 partly because of the scaling factor determined based on the νCO frequency. In complexes of the donor form of p-cresol, the νCO frequencies were calculated to be 1279-1265 cm-1, which is higher than the free value (1255 cm-1) by 10-24 cm-1, whereas the acceptor form of p-cresol showed the νCO frequencies at 1239-1222 cm-1, lower than the free value by 16-33 cm-1 (Table 1). The donor-acceptor form showed frequencies at 1253-1240 cm-1, which is located between the frequencies of the donor and acceptor forms. Thus, calculated νCO frequencies showed basically no overlap between these three forms. The plot of the νCO frequencies as a function of the CO lengths in the optimized geometries showed a negative linear correlation (Figure 2). This indicates that the above tendency of the νCO frequency depending on the hydrogen-bond forms are determined mainly by the changes in the CO lengths by hydrogenbond interactions; the CO length is shortened by hydrogen-bond donation and lengthened by hydrogen-bond acceptance, while the change is canceled by simultaneous hydrogen-bond formation. All of the νCO modes showed relatively strong IR intensities. In particular, the donor-acceptor form showed significantly stronger intensities (253-395 km/mol), while the

Hydrogen-Bond Structure of Tyr

J. Phys. Chem. B, Vol. 111, No. 49, 2007 13835

TABLE 1: Calculated Frequencies (cm-1) of the νCO and δCOH Vibrations of p-Cresol and Its Hydrogen-Bonded Complexes νCOc [IR int]d

p-cresol δCOHc [IR int]d

none

1255 [102]

Free Form 1156 [156]

H2O (1) methanol (2) 2-propanol (3) p-cresol (4) diethyl ether (5) 1,4-dioxane (6) formaldehyde (7) acetone (8) carbonic acid (9) formamide (10) acetamide (11) diformamide (12) dimethyl sulfoxide (13) 4-methylimidazole (14) pyridine (15) methylamine (16) triethylamine (17) methyleneimine (18) acetonitrile (19) dimethyl sulfide (20)

hydrogen-bond partnera (complex no.)b

p-cresol-OD νCOc (∆D)e [IR int]d δCODc [IR int]d 1248(-7) [145]

902 [83]

Hydrogen-Bond Donor Form 1268 [85] 1219 [178] 1269 [81] 1225 [181] 1269 [81] 1226 [198] 1265 [98] 1210 [118] 1268 [78] 1227 [176] 1269 [79] 1227 [202] 1268 [69] 1220 [256] 1272 [61] 1235 [279] 1269 [94] 1214 [156] 1273 [70] 1234 [268] 1274 [63] 1240 [321] 1266 [74] 1222 [189] 1274 [29] 1251 [307] 1277 [91] 1242 [191] 1275 [79] 1240 [183] 1279 [57] 1254 [206] 1279 [47] 1254 [219] 1274 [83] 1236 [191] 1270 [96] 1213 [162] 1265 [89] 1212 [208]

1260(-8) [155] 1261(-8) [155] 1260(-9) [163] 1257(-8) [165] 1260(-8) [156] 1260(-9) [163] 1258(-10) [163] 1261(-11) [185] 1261(-8) [157] 1263(-10) [178] 1264(-10) [194] 1257(-9) [167] 1262(-12) [194] 1268(-9) [171] 1266(-9) [160] 1268(-11) [163] 1266(-13) [165] 1265(-9) [163] 1262(-9) [159] 1255(-10) [180]

954 [84] 959 [99] 961 [90] 942 [88] 959 [87] 961 [86] 953 [132] 967 [112] 948 [100] 969 [83] 994 [132] 952 [165] 1009 [63] 994 [86] 991 [75] 1019 [160] 1011 [92] 989 [59] 947 [94] 944 [121]

HF (21) HCl (22) H2O (23) methanol (24) p-cresol (25) trifluoroacetic acid (26) formamide (27) acetamide (28) methylformamide (29) 4-methylimidazole (30)

Hydrogen-Bond Acceptor Form 1229 [145] 1186 [153] 1232 [134] 1169 [99] 1237 [114] 1166 [92] 1238 [118] 1165 [115] 1235 [140] 1169 [92] 1222 [119] 1188 [92] 1231 [103] 1168 [91] 1232 [104] 1167 [88] 1239 [125] 1168 [122] 1237 [132] 1168 [121]

1227(-2) [188] 1228(-4) [188] 1233(-4) [165] 1233(-5) [168] 1231(-4) [189] 1220(-2) [159] 1227(-4) [155] 1228(-4) [154] 1234(-5) [177] 1232(-5) [187]

937 [137] 913 [86] 911 [91] 911 [96] 913 [93] 919 [75] 912 [68] 912 [75] 913 [103] 913 [98]

H2O/H2O (31) methanol/methanol (32) acetone/H2O (33) carbonic acid/H2O (34) acetic acid/acetic acid (35) formamide/imidazole (36) acetamide/4-methylimidazole (37) 4-methylimidazole/formic acid (38) imidazole/acetamide (39)

Hydrogen-Bond Donor-Acceptor Form 1247 [262] 1266 [6] 1240 [262] 1264 [17] 1245 [305] 1264 [7] 1242 [395] 1256 [3] 1245 [342] 1268 [35] 1247 [338] 1261 [10] 1252 [353] 1267 [4] 1250 [272] 1277 [1] 1253 [256] 274 [18]

1247(0) [205] 1251(+11) [178] 1251(+6) [205] 1245(+3) [241] 1243(-2) [331] 1250(+3) [251] 1252(0) [268] 1249(-1) [239] 1259(+6) [198]

1018 [76] 990 [100] 998 [102] 967 [138] 1023 [140] 1000 [104] 1016 [141] 1051 [123] 1027 [113]

a

Hydrogen-bond acceptor, donor, and acceptor/donor in complexes of p-cresol in the donor, acceptor, and donor-acceptor forms, respectively. Figures in parentheses are model complex numbers. The optimized geometries of the complexes are shown in Figures S1 and S2 of the Supporting Information. c Calculated vibrational frequencies were scaled with a uniform scaling factor of 0.977. Coupling with vibrations of a hydrogen-bond partner was eliminated by appropriate isotope labeling of the partner molecule. d IR intensities in the brackets were calculated in km mol-1. e Shifts from the νCO frequencies of unlabeled p-cresol.

b

intensities of the donor form (29-98 km/mol) were a little lower than those of the free (102 km/mol) and acceptor (103-145 km/mol) forms (Table 1). The δCOH frequencies were calculated at 1254-1210 and 1188-1165 cm-1 in the donor and acceptor forms, respectively, with strong IR intensities (88-321 km/mol) (Table 1). These frequencies result from significant upshifts from the free-form value by 54-98 cm-1 in the donor form and slight upshifts by 9-32 cm-1 in the acceptor form. The donor-acceptor form showed a mode with a δCOH character at a very high frequency of 1277-1256 cm-1 (Table 1), which is even higher than the νCO frequency (1253-1240 cm-1) of this form. This mode includes relatively large contributions of the νCC and δCH vibrations of a phenolic ring. In complexes with a weak hydrogen bond at the oxygen such as complexes 32, 33, 36, and 39 (Figure S3 of the Supporting Information), the contribution of the νCO vibration is also large,

and instead, the δCOH vibration is significantly mixed into the lower-frequency νCO mode. This is reasonable because the extreme case of weak hydrogen-bond acceptance corresponds to a hydrogen-bond donor form, which shows the νCO and δCOH at higher (1279-1265 cm-1) and lower (1254-1210 cm-1) frequencies in the regions similar to the donor-acceptor form but with an opposite assignment. It is of particular interest that the δCOH-like mode of the donor-acceptor form showed a very weak IR intensity (1-35 km/mol) (Table 1). Table 1 also shows the νCO(D) and δCOD frequencies of p-cresol-OD and its complexes in which the phenolic OH group is deuterated. The δCOD frequencies largely downshift to 1051-902 cm-1. The νCO frequencies also downshift by 7, 8-13, and 2-5 cm-1 in the free, donor, and acceptor forms, respectively, indicative of a weak coupling with the δCOH vibration. The deuterium-induced shifts of the νCO of the donor-acceptor form range from -2 to +11, reflecting a


Takahashi and Noguchi

Figure 2. Calculated νCO frequencies of p-cresol in hydrogen-bond complexes as a function of the CO length: filled circle, free p-cresol; open circle, hydrogen-bond donor form; open square, hydrogen-bond acceptor form; open triangle, hydrogen-bond donor-acceptor form.

variable extent of coupling with the δCOH vibration depending on the strength relationship of the two hydrogen bonds. It is noted that the reason for the νCO upshifts of most of the donoracceptor complexes is that the νCO frequency is lowered by coupling with a higher-frequency δCOH vibration and hence upshifted in turn upon decoupling by deuteration. The effects of 13C labeling of the carbon atom (4-C) bearing the OH group on the νCO and δCOH frequencies are summarized in Table S1 of the Supporting Information. The νCO frequencies of p-cresol showed large downshifts by 25, 2023, 22-26, and 17-24 cm-1 in the free, donor, acceptor, and donor-acceptor forms, respectively. In contrast, the downshifts of the δCOH frequencies were relatively small in the free (4 cm-1) and acceptor (3-7 cm-1) forms, while medium δCOH downshifts were observed in the donor (9-21 cm-1) and donor-acceptor forms (15-19 cm-1). In p-cresol-OD, the downshift of νCO(D) by 4-13C labeling is also large (23-26 cm-1), whereas almost no isotope effect was observed in δCOD frequencies (0-1 cm-1). It should be noted that the oxygen atom of hydroxide can take two hydrogen bonds. Such a triple hydrogen-bonded complex (i.e., one hydrogen bond at the hydrogen and two hydrogen bonds at the oxygen) with water molecules as hydrogen-bond partners was examined by DFT calculation, which showed that one of the two hydrogen bonds at the oxygen was much weaker than the other. (The hydrogen-bond distances were 2.315 and 1.955 Å.) The pattern of the νCO and δCOH vibrations was similar to that of the double hydrogen-bonded complexes, that is, an IR-active νCO mode at 1237 cm-1 and an IR-inactive δCOH-like mode at 1256 cm-1. This νCO frequency is slightly lower than that of the νCO of the double hydrogen-bonded complex with water molecules (complex 31) (1247 cm-1) due probably to a stronger effect of hydrogenbond acceptance. Thus, the effect of two hydrogen bonds at the oxygen atom can be treated as a case of a stronger hydrogen bond at the oxygen, and hence further calculation was not performed for this type of model. FTIR Spectra of p-Cresol and Tyr. Figure 3 shows FTIR spectra in the νCO and δCOH region (1300-1150 cm-1) of p-cresol (black lines) and p-cresol-OD (red lines) in a hydrophobic solvent ((a) CCl4), nucleophilic solvents acting as hydrogen-bond acceptors ((b) diethyl ether, (c) DMSO, (d) tetraethylamine, and (e) acetonitrile), and a strong acid acting as a hydrogen-bond donor ((f) 200 mM trifluoroacetic acid/ CCl4). The spectra were measured using the ATR method

Figure 3. FTIR spectra of p-cresol (black lines) and p-cresol-OD (red lines) in the νCO and δCOH region in (a) CCl4, (b) diethyl ether, (c) DMSO, (d) triethylamine, (e) acetonitrile, and (f) 200 mM trifluoroacetic acid/CCl4. Spectra were measured using an ATR accessory, and the contribution of the solvent bands was eliminated by subtracting solvent spectra. Concentrations of p-cresol were 100 mM for a, 250 mM for b-e, and 30 mM for f.

because of its good performance in solvent subtraction. The spectral shapes in this region were virtually identical to those measured by the transmission method, and the peak frequencies obtained by the two methods agreed within 1 cm-1. In this spectral region, in addition to the νCO and δCOH bands, two other modes, 9a (δCH) and 7a (νCMe), with weak IR intensities (calculated to be 10 and 4 km/mol, respectively, in free p-cresol) are located at around 1172 and 1215 cm-1, respectively.24 In CCl4, in which p-cresol can exist as a free from, the prominent bands at 1256 and 1176 cm-1 (Figure 3a, black line) are assigned to νCO and δCOH, respectively. In fact, in p-cresol-OD (Figure 3a, red line), the latter peak

Hydrogen-Bond Structure of Tyr disappeared, while the former peak showed a downshift by 6 cm-1 as expected by calculations (7 cm-1; Table 1). The 1172 cm-1 peak is assigned to the 9a mode, because the weak band was left at the same frequency in p-cresol-OD. The 7a band was not observed in the spectra, reflecting its weak IR intensity. These assignments agree with previous Raman and IR studies.24,33 Band assignments in nucleophilic solvents (Figures 3b-e) are also clear. Relatively sharp bands at 1272-1262 cm-1 are assigned to νCO, and broad features at 1245-1210 cm-1 are attributed to δCOH, because the latter features disappeared in p-cresol-OD (Figures 3b-e, red lines). The νCO bands downshifted by 4-8 cm-1 upon deuteration, consistent with the predicted downshifts of ∼10 cm-1 by calculation (Table 1). The broad widths of the δCOH bands are also consistent with hydrogen-bond formation at the OH hydrogen, and the presence of two to three peaks on the broad feature might reflect different hydrogen-bond conformations with solvent molecules. The presence of a single CO(D) band in p-cresol-OD, however, indicates that p-cresol takes only a hydrogen-bond donor form and p-cresol aggregation does not exist in these solvents. Shoulders at 1212 and 1215 cm-1 in diethyl ether and DMSO, respectively, can arise from the 7a mode that obtains intensity by coupling with the δCOH vibration. Weak 9a peaks were observed at 1173-1168 cm-1. The complex of p-cresol with trifluroacetic acid (TFA) showed prominent bands at 1222 and 1179 cm-1 (Figure 3f). Although TFA is a strong hydrogen-bond donor (the acceptor number is 105 compared with 53 of acetic acid and 55 of water),34 it can also function as a hydrogen-bond acceptor. Hence, p-cresol (30 mM) was dissolved in 200 mM TFA/CCl4 rather than in pure TFA to avoid further complexation to form a donor-acceptor form. This condition was also advantageous to subtract strong CF stretching bands of TFA at 1231, 1185, and 1173 cm-1, superimposing the predicted νCO and δCOH region. The band frequencies of 1222 and 1179 cm-1 are in good agreement with the calculated frequencies of 1222 (νCO) and 1188 (δCOH) cm-1 in the corresponding p-cresol-TFA complex (Table 1); thus, the 1222 and 1179 cm-1 bands are assigned to νCO and δCOH vibrations, respectively. The relatively broad width of the νCO band compared with the corresponding bands in the free and donor forms is consistent with hydrogen-bond formation at the OH oxygen. The small peak at 1256 cm-1 is most probably due to νCO of residual free p-cresol in TFA/CCl4 solution, because the band position is identical to that in CCl4 (Figure 3a) and the intensity decreased in a solvent with a higher TFA/CCl4 ratio. The 9a band seems to appear at 1165 cm-1 as a shoulder of the δCOH band. The broad feature of this band may indicate that this mode is coupled with the δCOH vibration. Such coupling between the 9a and δCOH vibrations was indeed shown in calculations of some model complexes in an acceptor form. Alternatively, it could be possible that subtraction of a strong solvent band at 1173 cm-1 distorted the band shape around 1170 cm-1. Figure 4 shows FTIR spectra of p-cresol in (a) H(D)2O and (b) methanol (-OD), which have both hydrogen-bond donating and accepting characters, and (c) those of pure p-cresol (-OD) in a liquid state. In H2O, a broad band was observed around 1240 cm-1 with shoulders at about 1261 and 1217 cm-1 (Figure 4a, black line). p-Cresol-OD in D2O showed a single νCO(D) band at 1256 cm-1 (Figure 4a, red line), indicating that p-cresol in water mostly takes a single hydrogen-bond form, probably a donor-acceptor form. In fact, a strong band at 1240 cm-1 with a shoulder at 1261 cm-1 in H2O is in good agreement with the


Figure 4. FTIR spectra of p-cresol (black lines) and p-cresol-OD (red lines) in (a) H(D)2O, (b) methanol(-OD), and (c) pure liquid p-cresol. For p-cresol-OD in water and methanol, unlabeled p-cresol was dissolved in D2O and methanol-OD. Spectra were measured using an ATR accessory, and the contribution of the solvent bands was eliminated by subtracting solvent spectra. Concentrations of p-cresol were 125 mM for a and 250 mM for b.

calculated result of the double hydrogen-bonded complex with water molecules (complex 31; Figure S3 of the Supporting Information), which showed an IR-active νCO vibration at 1247 cm-1 and a δCOH-like vibration at 1266 cm-1 with a very weak intensity (Table 1). Another shoulder at 1217 cm-1 probably arises from the 7a mode, because the previous Raman spectrum of p-cresol in H2O showed a strong 7a band at the same frequency.22 In methanol, a relatively strong band at 1267 cm-1 and a broad feature with peaks at 1245, 1230, and 1216 cm-1 were observed (Figure 4b). This spectral feature is similar to that in nucleophilic solvents (Figures 3b-e). However, p-cresol-OD in methanol-OD showed two distinct νCO(D) peaks at 1262 and 1255 cm-1, indicating that two different hydrogen-bond forms coexist in methanol. The model complex of the donor form of p-cresol hydrogen-bonded with a methanol molecule (complex 2) showed calculated νCO and δCOH frequencies at 1269 and 1225 cm-1, respectively (Table 1). Thus, we interpreted that the 1267 and 1230 cm-1 bands in methanol arise from the νCO and δCOH modes, respectively, of p-cresol in a donor form. The νCO band may downshift to 1262 cm-1 by 5 cm-1 upon deuteration, which is consistent with the calculated downshift of 8 cm-1 in the model complex (Table 1) and the experimental shifts of 4-8 cm-1 in nucleophilic solvents (Figures 3b-e). Accordingly, the strong band at 1245 cm-1 can be assigned to the νCO vibration of the donor-acceptor form. The calculated νCO frequency of the corresponding complex



TABLE 2: Experimental Frequencies (cm-1) of the νCO and δCOH Vibrations of p-Cresol solvent

νCO

p-cresol δCOH

p-cresol-OD νCO (∆D)a

hydrogen-bond formb

vapor CCl4 CH2Cl2 CCl2dCCl2 CS2 diethyl ether 1,4-dioxane N,N′-dimethylformamide tetramethylurea/CS2 pyridine triethylamine dimethyl sulfoxide acetonitrile trifluoroacetic acid trifluoroacetic acid/CCl4 trichloroacetic acid H2O (D2O) methanol

1255 1256 1255 ∼1250 1254 1266 1265 1267 1266 1268 1272 1266 1262 1240 1222 1235 ∼1240 1267 1245 1265 ∼1236

1178 1176 1182 1171 1169 1222,c 1238 1224 1230 1222 1248 1230, 1245c 1233,c 1243 1211,c 1221 n.d.d 1179 n.d.d 1261she 1230 n.d.d 1226 n.d.d

n.d.d 1250 (-6) n.d.d n.d.d 1248 (-6) 1260 (-6) n.d.d n.d.d n.d.d 1265 (-3) 1264 (-8) 1262 (-4) 1257 (-5) n.d.d n.d.d n.d.d 1256 (+16) 1262 (-5) 1255 (+10) 1259 (-6) 1245 (+9)

F F F F F D D D D D D D D A A A D-A D D-A D D-A

pure p-cresol

reference 33 this work 22 23 24 this work 2 2 36 2 This work This work This work 22 This work 2 This work This work This work

a Shifts upon deuteration of the phenolic OH. b F, free; D, donor; A, acceptor; D-A, donor-acceptor. c The strongest peak in the δCOH bands is shown in bold. d Not determined. e Shoulder.

hydrogen-bonded with two methanol molecules (complex 32) was 1240 cm-1, in good agreement with the observed value. The 1255 cm-1 peak in p-cresol-OD may be due to the νCO(D) mode in this double hydrogen-bonded structure. These assignments were further supported by the observation that the intensities of the 1267 and 1230 cm-1 bands relative to the intensity of the 1245 cm-1 band changed together when CCl4 was mixed into the methanol solution. The shoulder at 1216 cm-1 is due to the 7a mode, because the previous Raman spectrum of p-cresol in methanol showed a strong 7a peak at the same frequency.22 The spectrum of liquid p-cresol showed a very broad feature at 1236 cm-1 with shoulders at 1265, 1226, and 1213 cm-1 (Figure 4c). Liquid p-cresol-OD showed a band at 1245 cm-1 with a shoulder at 1259 cm-1. Thus, similarly to the case of methanol, two different hydrogen-bond forms may coexist in liquid p-cresol. The calculated νCO and δCOH frequencies of the donor form of p-cresol hydrogen-bonded with another p-cresol (complex 4) were 1265 and 1210 cm-1, respectively; hence, the shoulders at 1265 and 1226 cm-1 can be assigned to the hydrogen-bond donor form. The peak at 1213 cm-1 may arise from the 7a mode that borrowed the intensity from the nearby δCOH vibration. The νCO band at 1265 cm-1 downshifted to 1259 cm-1 by 6 cm-1 upon deuteration, consistent with the downshifts (4-8 cm-1) in nucleophilic solvents (Figures 3b-e). Thus, the strong 1236 cm-1 band can be assigned to the νCO vibration in double hydrogen-bonded p-cresol. The weak peaks at 1176-1172 cm-1 in H2O, methanol, and pure p-cresol are assigned to the 9a vibration. The experimental frequencies of the νCO and δCOH vibrations are summarized in Table 2 together with those in the literature. FTIR spectra of L-Tyr are presented in Figure 5. Crystalline L-Tyr showed a strong band at 1247 cm-1 with a weak peak at 1267 cm-1 (Figure 5a, black line). Upon deuteration of OH, a single strong band assignable to νCO(D) was observed at 1259 cm-1 (Figure 5a, red line). According to the X-ray crystal structure of L-Tyr refined by neutron diffraction techniques,35 the phonolic OH of L-Tyr in a crystal acts as a strong hydrogenbond donor (the hydrogen-bond distance is 1.689 Å) and a very weak acceptor (the hydrogen-bond distance is 2.120 Å). Thus,

Figure 5. FTIR spectra of L-Tyr (black line) and L-Tyr-OD (red line) (a) in a crystalline state and (b) in DMSO. The samples for b were prepared by dissolving L-Tyr‚HCl or L-Tyr-OD‚DCl in DMSO at a concentration of 35 mM. The contribution of DMSO was eliminated by subtracting its spectrum.

the crystalline L-Tyr is an intermediate case between the donoracceptor form and the donor form. In fact, the intensity pattern of the two bands, i.e., weak and strong intensities for the higherand lower-frequency bands, respectively, is similar to that of the donor-acceptor form, while the νCO(D) frequency of TyrOD, 1259 cm-1, is more consistent with the calculated νCO(D) frequency of the donor form (1268-1255 cm-1) than that of the donor-acceptor form (1259-1243 cm-1) (Table 1).



TABLE 3: Experimental Frequencies (cm-1) of the νCO and δCOH Vibrations of Tyr sample/state

νCO

Tyr δCOH

Tyr-OD νCO (DD)a

hydrogen-bond formb

reference

Tyr/Ar matrix L-Tyr‚HCl/DMSO L-Tyr/crystal L-Tyr‚HCl/crystal GlyTyr‚HCl/crystal poly-L-Tyr/hydrated

1260 1271 1247 1236 1232 1243

1173 1242 1267wd n.d.c n.d.c 1266shd

n.d.c 1267 (-4) 1259 (+12) n.d.c n.d.c 1255 (+12)

F D D-wA A-wD A-wD D-A

27 this work this work 22 22 4

a Shifts upon deuteration of the phenolic OH of Tyr. b F, free; D, donor; A, acceptor; D-A, donor-acceptor; w, weak. c Not determined. d w, weak; sh, shoulder.

L-Tyr

as a hydrogen-bond donor was realized by dissolving in DMSO. The FTIR spectrum of a Tyr/DMSO solution (Figure 5b, black line) showed a typical donor-form spectrum, which exhibited a band assignable to νCO at 1271 cm-1 and a broader band at 1242 cm-1 due to δCOH. Upon deuteration, the 1242 cm-1 band diminished, and a νCO(D) band appeared at 1267 cm-1, which resulted from a downshift of νCO by 4 cm-1. The experimental frequencies of the νCO and δCOH vibrations of Tyr are summarized in Table 3 together with those in the literature. Frequencies of Other Potential Markers. Table 4 shows calculated frequencies of 8a (νCC), 8b (νCC), 19a (νCC + δCH), and 9a (δCH) vibrations of model hydrogen-bonded complexes. These modes have been argued as markers of Tyr structures.2,4,5,11,12,14-17,19,22 Experimental data obtained in FTIR measurements in this study (shown in Figure S4 of the Supporting Information for the 8a, 8b, and 19a bands) were also included in Table 4 in comparison with the calculated data for the corresponding hydrogen-bonded complexes. The calculated frequencies were scaled with different scaling factors, 0.971, 0.975, 0.976, and 0.9785, for the 8a, 8b, 19a, and 9a modes, respectively, to adjust the calculated frequencies of free p-cresol to those of experimental values in CCl4 obtained in the present study. Calculated frequencies of the 8a mode in the donor and donor-acceptor forms were found at 1619-1617 and 16181616 cm-1, respectively, which are similar to the value of the free form, 1618 cm-1. In contrast, the frequencies decreased by 2-4 cm-1 to 1614-1616 cm-1 in the acceptor form. The frequencies of the 8b mode in the donor form were calculated at 1596-1590 cm-1, which clearly downshifted from the free value of 1598 cm-1, whereas the frequencies upshifted to 1606-1601 cm-1 in the acceptor form. In the donor-acceptor form, the frequencies are mostly located at 1600-1597 cm-1 (complex 39 showed an exceptionally low frequency of 1594 cm-1, but it has a significantly weak hydrogen bond at the oxygen; Figure S3 of the Supporting Information) and thus are similar to the frequency of the free form. The 19a frequencies were calculated at 1519-1516 and 1518-1516 cm-1 in the donor and donor-acceptor forms, respectively. These values are slightly higher than the frequency of the free form, 1515 cm-1. In the acceptor form, the 19a frequencies were found at 1516-1513 cm-1, which was similar to or only slightly lower than the free value. The 9a frequencies of the donor form were calculated at 1172-1169 cm-1, which were only slightly lower than the free form (1172 cm-1), while the donor-acceptor form showed slightly higher frequencies at 1176-1172 cm-1. In the acceptor form, as mentioned above, the 9a mode was significantly coupled to the δCOH vibration, and hence the frequencies drastically change depending on the position of the δCOH vibration. Thus, the calculated frequencies were found in a relatively wide region of 1186-1174 cm-1. L-Tyr‚HCl

The tendency for hydrogen-bond dependence of the calculated frequencies is mostly consistent with the experimental data, although the scatter of the experimental data is larger than that of the calculated data (Table 4). Discussion Criteria for Determining the Hydrogen-Bond Forms of p-Cresol and Tyr Using the νCO and δCOH Vibrations. DFT calculations showed that the νCO (7a′) and δCOH vibrations of p-cresol occur in different frequency regions depending on the hydrogen-bond forms (Tables 1). Figure 6A shows the correlation between the νCO and the δCOH frequencies of the free (black marks), donor (blue marks), acceptor (green marks), and donor-acceptor (red marks) forms obtained by DFT calculations for various hydrogen-bonded complexes of p-cresol (solid circles) together with experimental data for p-cresol (open triangles) and Tyr (crosses). When more than one δCOH peak is observed in experimental FTIR spectra, the frequency of the most intense peak was adopted (Table 2, bold figures). The plot shows that calculated frequencies sufficiently agree with the experimental data, although the δCOH frequencies were estimated to be slightly lower than the experimental data in the free and donor forms. It is also seen that the frequencies of Tyr are close enough to the data of p-cresol, indicating that the relationship obtained for p-cresol is directly applicable to Tyr. In a free form, the experimental νCO and δCOH frequencies are observed at 1260-1250 and 1185-1165 cm-1, respectively (Figure 6A; Tables 2 and 3). In a hydrogen-bond donor form, the νCO and δCOH frequencies (calculated and experimental) upshift from the free values and are found in the regions of 1280-1260 and 1255-1210 cm-1, respectively (Figure 6A; Tables 1-3). It is noted that there is a linear correlation between the νCO and the δCOH frequencies (Figure 6A). The regression line for the experimental data for p-cresol (Figure 6A, blue dotted line) is expressed as

δCOH (cm-1) ) 3.692 × (νCO - 1255) + 1187 where (νCO - 1255) represents the shifts from the value of the p-cresol vapor. The δCOH value when the shift is zero is 1187 cm-1, which is slightly higher than the actual δCOH value of p-cresol vapor, 1178 cm-1. This deviation might originate from a coupling between νCO and δCOH, which increases when the frequency gap becomes smaller. In Figure 7, calculated νCO and δCOH frequencies are plotted against the hydrogen-bond distances in optimized geometries, showing that a shorter hydrogen-bond distance, i.e., a stronger hydrogen bond, gives rise to higher νCO and δCOH frequencies. It is notable that the hydrogen bond to a sulfur atom of dimethyl sulfide has a long hydrogen-bond distance of 2.356 Å and is rather deviated from the correlation by other complexes (Figure 7, open and filled squares). This specific hydrogen-bonding character of a sulfur atom should be kept in mind when a hydrogen-bond



TABLE 4: Calculated and Experimental Frequencies (cm-1) of the 8a, 8b, 19a, 9a Vibrations of p-Cresol and Its Hydrogen-Bonded Complexes hydrogen-bond partnera (complex no.)b

8a

8b

19a

9a

calcd.c

expt.d

calcd.c

expt.d

calcd.c

expt.d

calcd.c

expt.d

none

1618

1618e

Free Form 1598

1598e

1515

1515e

1172

1172e

H2O (1) methanol (2) 2-propanol (3) p-cresol (4) diethyl ether (5) 1,4-dioxane (6) formaldehyde (7) acetone (8) carbonic acid (9) formamide (10) acetamide (11) diformamide (12) dimethyl sulfoxide (13) 4-methyimidazole (14) pyridine (15) methylamine (16) triethylamine (17) methyleneimine (18) acetonitrile (19) dimethyl sulfide (20)

1617 1618 1618 1618 1618 1618 1618 1619 1618 1618 1618 1618 1618 1618 1618 1619 1618 1618 1618 1618

Hydrogen-Bond Donor Form 1594 1594 1594 1594 1620 1593 1597 1593 1595 1594 1594 1594 1594 1595 1615 1596 1595 1590 1591 1592 1618 1590 1593 1592 1619 1593 1597 1594

1517 1517 1517 1517 1517 1517 1517 1518 1518 1518 1519 1517 1518 1518 1518 1519 1519 1518 1518 1516

HF (21) HCl (22) H2O (23) methanol (24) p-cresol (25) trifluoroacetic acid (26) formamide (27) acetamide (28) methylformamide (29) 4-methylimidazole (30)

1615 1615 1616 1616 1616 1614 1615 1615 1616 1616

Hydrogen-Bond Acceptor Form 1606 1604 1602 1602 1604 1614 1606 1603 1601 1601 1603 1603

1514 1513 1514 1514 1514 1514 1515 1516 1514 1514

H2O/H2O (31) methanol/methanol (32)f acetone/H2O (33) carbonic acid/H2O (34) acetic acid/acetic acid (35) formamide/imidazole (36) acetamide/4-methylimidazole (37) 4-methylimidazole/formic acid (38) imidazole/acetamide (39)

1617 1617 1618 1617 1616 1617 1618 1616 1617

Hydrogen-Bond Donor-Acceptor Form 1614 1600 1599 1618 1597 1599 1598 1599 1600 1600 1600 1599 1594

1516 1517 1517 1516 1517 1517 1517 1517 1518

1517

1516

1517 1516

1171 1171 1171 1171 1171 1171 1172 1172 1171 1171 1171 1172 1171 1169 1170 1169 1170 1170 1171 1171

1171

1168 1173

1174 1175 1178 1178 1176 1177 1186 1186 1176 1176

1514

1516 1517

1171

1173 1172 1173 1173 1176

1176 1172

1174 1174 1172 1173

a Hydrogen-bond acceptor, donor, and acceptor/donor in complexes of p-cresol in the donor, acceptor, and donor-acceptor forms, respectively. Figures in parentheses are model complex numbers. The optimized geometries of the complexes are shown in Figures S1 and S2 of the Supporting Information. c Calculated vibrational frequencies were scaled with scaling factors of 0.971, 0.975, 0.976, and 0.9785 for the 8a, 8b, 19a, and 9a modes to adjust the frequencies of free p-cresol to the experimental ones in CCl4. d Experimental frequencies were obtained in this study by FTIR measurements. e Experimental frequencies in CCl4. fExperimental frequencies in methanol were assumed to mostly represent the donor-acceptor form.

b

interaction with methionine is considered in proteins. In contrast, no specific difference was observed between oxygen and nitrogen atoms in the relationship of the νCO and δCOH frequencies with the hydrogen-bond distances (Figure 7, circles and triangles), although hydrogen bonds with nitrogen atoms are generally stronger than those with oxygen atoms. In the hydrogen-bond acceptor form (Figure 6A, green marks), the calculated and experimental frequencies of νCO and δCOH are observed at 1240-1220 and 1190-1160 cm-1. Thus, the νCO frequency downshifts by 20-30 cm-1 from the free value, while the δCOH frequency does not change so much. The upshift and downshift of the νCO frequency of p-cresol or Tyr by hydrogen-bond donation and acceptance, respectively, were first proposed in the Raman study by Takeuchi et al.,22 and the correlation between the νCO frequency and the

hydrogen-bond strength was shown by Zhao and Spiro.16 Also, the upshift of δCOH by hydrogen-bond donation was detected in the FTIR study by Gerothanassis et al.23 These behaviors of νCO and δCOH were confirmed by Hienerwadel et al.2 in their FTIR study. In these studies, however, the criteria to discriminate between the donor and the donor-acceptor forms have not been explicitly presented. Although Gerothanassis et al.23 suggested that the donor and donor-acceptor forms showed δCOH bands at 1225-1210 and 1245-1235 cm-1, respectively, this criterion is clearly inaccurate because p-cresol in some hydrogen-bond donating solvents showed δCOH bands in the latter frequency region (Table 2, Figures 3 and 6A). Discriminating these two forms has been difficult because the frequency regions of the δCOH-like and νCO modes of the donor-acceptor form (1280-1255 and 1255-1235 cm-1,



Figure 7. Calculated νCO (filled marks) and δCOH (open marks) frequencies in model complexes of the donor form of p-cresol as a function of the hydrogen-bond distance. In the complexes, p-cresol is hydrogen-bonded with oxygen (circles), nitrogen (triangles), and sulfur (square) atoms.

Figure 6. (A) Relationship between the νCO and the δCOH frequencies of p-cresol or Tyr in free (black), hydrogen-bond donor (blue), acceptor (green), and donor-acceptor (red) forms. (B) Relationship between the νCO(D) frequency of p-cresol-OD or Tyr-OD and the frequency of lower-frequency νCO or δCOH mode with a strong IR intensity in hydrogen-bond donor (blue) and donor-acceptor (red) forms. Calculated data are expressed by filled circles tagged with model complex numbers. Experimental data for p-cresol are expressed by open triangles: a, vapor; b, CCl4; c, CH2Cl2; d, CCl2dCCl2; e, CS2; f, diethyl ether; g, 1,4-dioxiane; h, N,N′-dimethylformamide; i, tetramethylurea/ CS2; j, pyridine; k, triethylamine; l, DMSO; m, acetonitrile; n, trifluoroacetic acid/CCl4; o, H(D)2O; p, methanol(-OD); q, pure p-cresol. Experimental data for Tyr are expressed by crosses: r, Tyr/ Ar matrix; s, L-Tyr‚HCl/DMSO; t, crystalline L-Tyr; u, hydrated polyL-Tyr.

respectively) are similar to those of the νCO and δCOH modes of the donor form (1280-1260 and 1255-1210 cm-1, respectively) (Tables 1-3, Figure 6A). In both forms, the higherfrequency modes (the δCOH-like mode in the donor-acceptor form and the νCO mode of the donor form) showed strong Raman intensities, and hence the features of Raman spectra of p-cresol were similar between the two forms.22 Also, both of the lower-frequency modes (the νCO mode in the donor-

acceptor form and the δCOH mode in the donor form) showed similar intense IR bands (Figures 3-5). A weak IR intensity of the higher-frequency δCOH-like mode in the donor-acceptor form can be a potential marker but not a conclusive one. It should be noted that in these forms identification of the νCO and δCOH bands by deuteration of phenolic OH is not straightforward, because in most of the cases a new νCO(D) band appears between the νCO and the δCOH bands due to decoupling of the δCOH vibration (Tables 1-3). Only slightly larger downshifts of νCO frequencies upon 4-13C labeling (1724 cm-1) than those of δCOH frequencies (9-21 cm-1) in the donor and donor-acceptor forms (Table S1 of the Supporting Information) can be suggestive to the νCO and δCOH assignment. For definite discrimination between the donor and the donoracceptor forms, the νCO(D) band in p-cresol-OD or Tyr-OD is useful. It is relatively easy to identify a strong νCO(D) band in FTIR spectra, because a δCOD band largely downshifts to ∼1000 cm-1 and is absent in this region. Practically, it is convenient to make a plot of the frequencies of the lowerfrequency strong IR bands (the δCOH band of the donor form and the νCO band of the donor-acceptor form) of undeuterated species against the νCO(D) frequencies (Figure 6B). The calculated and experimental νCO(D) frequencies (Figure 6B, red marks) in the donor-acceptor form are found at 12601240 cm-1, while those in the donor form (Figure 6B, blue marks) are located at 1270-1255 cm-1. There is some overlap in the 1260-1255 cm-1 region. However, the plot areas of the two forms are sufficiently separated from each other (Figure 6B). In this plot, the calculated data for the donor-acceptor form (Figure 6B, red filled circles) seem to be slightly deviated from the experimental data (red open triangles and crosses), while the calculated data of the donor form (blue filled circles) are in good agreement with the experimental values (blue open triangles and a blue cross). The linear correlation of the experimental points of p-cresol (blue open triangles) is expressed by the regression line

δCOH (cm-1) ) 4.330 × (νCO(D) - 1250) + 1182 where 1250 cm-1 is the experimental value of free p-cresol in CCl4 (Figure 2a).



TABLE 5: Criteria for Determining the Hydrogen-Bond Forms of p-Cresol and Tyr by νCO and δCOH Vibrations νCO (cm-1) free acceptor donor donor-acceptor deprotonated (hydrogen-bonded) a

δCOH (cm-1)

1260-1250 1185-1165 1240-1220 1190-1160 1280-1260 1255-1210 linear correlation: δCOH (cm-1) ) 3.692 × (νCO - 1255) + 1187 ) 4.330 × (νCO(D) - 1250) + 1182 1255-1235 (strong IR intensity) 1280-1255 (weak IR intensity; Raman active) ∼1270 none

νCO(D)a (cm-1) ∼1250 1235-1220 1270-1255 1260-1240

νCO frequencies in p-cresol-OD or Tyr-OD.

TABLE 6: Experimental Frequencies (cm-1) of the νCO and δCOH Bands of Tyr in FTIR Spectra of Proteins and Polypeptides

a

proteins

Tyr νCO or δCOH

Tyr-OD νCO (∆D)a

hydrogen-bond formb

reference

TePixD(dark state) Tyr8 TePixD(light state) Tyr8 Leu-enkephalin/pH 5.5 photosystem II YD photosystem II YZ

1265, 1242 1273, 1235 1265, 1247 1275w, 1250 1279w, 1255

1262 (-3) 1270 (-3) n.d.c n.d.c n.d.c

D D D or D-A D or D-A D or D-A

6 6 23 2 and 11 3

Shifts upon deuteration of phenolic OH. b D, donor; A, acceptor; D-A, donor-acceptor. c Not determined.

It is worth noting that model complex 39, in which imidazole (acceptor) and acetamide (donor) are hydrogen-bonded (Figure S3 of the Supporting Information), has a weak hydrogen bond at the acceptor site. (The hydrogen-bond distance is 1.988 Å in contrast to 1.738 Å at the donor site.) In fact, it shows a νCO/ νCO(D) point near the area of the donor form (Figure 6B). A similar situation is shown in the experimental data for crystalline L-Tyr, which acts as a strong donor and a weak acceptor; the point t is found at the intermediate position between the area of the donor-acceptor form and that of the donor form. Thus, this type of plot can discriminate not only the distinct hydrogenbond forms but also intermediate cases. A deprotonated state of Tyr (tyrosinate) has been shown to have a νCO band at ∼1270 cm-1 in aqueous solution.2,4,5,11,12,23 This frequency is involved in the regions of the νCO and δCOHlike modes of the donor and donor-acceptor forms, respectively, of protonated Tyr. Thus, the νCO frequency itself cannot be a conclusive marker to determine the protonation state of Tyr. Additional evidence such as the absence of the δCOH band and the frequency of the 19a mode (see below) is necessary for the identification of tyrosinate. The criteria to determine the hydrogen-bond forms as well as the protonation state of p-cresol and Tyr using the νCO and δCOH bands in FTIR spectra are summarized in Table 5. Application to Biological Systems. Recently, we have obtained an FTIR difference spectrum of TePixD, a sensor protein related to phototaxis in cyanobacteria, upon its photoreaction and extracted the νCO and δCOH bands of a key Tyr residue using [4-13C]Tyr labeling.6 The dark state and lightsignaling state of TePixD showed νCO/δCOH frequencies at 1265/1242 and 1273/1235 cm-1, respectively (Table 6). In D2O, the δCOH bands disappeared, and νCO(D) bands were observed at 1262 and 1270 cm-1 in the dark and light states, respectively. These frequencies well fit the frequency regions of the hydrogenbond donor form (Table 5), as already concluded by DFT analysis using model complexes hydrogen-bonded with acetamide.6 The Tyr residue in Leu-enkephaliln, a peptide hormone, showed FTIR bands at 1265 and 1247 cm-1 (Table 6).23 It was reported that in D2O the latter band was eliminated, while the former band remained. (The frequency was not mentioned in the text.23) The authors originally assigned this Tyr to the donor-acceptor form from the δCOH position;23 however, if

the band position in the deuterated sample is located at 1265 cm-1, then this indicates that the Tyr in this hormone takes a donor form rather than a donor-acceptor form according to the criteria in Table 5 and Figure 6B. The accurate position of the νCO(D) band in the deuterated sample is necessary to draw a decisive conclusion. YZ and YD in photosystem II are Tyr residues that function as major and auxiliary electron donors, respectively, to the primary donor chlorophyll P680.1 Upon photooxidation, YZ and YD release protons to become neutral Tyr radicals, and thus, clarifying the hydrogen-bond structures of YZ and YD is essential for understanding their reaction mechanisms. YZ showed a major peak at 1255 cm-1 with a minor peak at 1279 cm-1,3 while YD had similar major and minor peaks at 1250 and 1275 cm-1, respectively (Table 6).2,11 The major peaks at 1255-1250 cm-1 and the minor peaks at 1279-1275 cm-1 have been assigned to the δCOH and νCO vibrations, respectively, and it has been proposed that YZ and YD donate a hydrogen bond to nearby His residues.2,3 DFT calculations for p-cresol complexes hydrogenbonded with imidazole supported this assignment.29 However, these frequencies are also located in the frequency range of the δCOH-like and νCO modes of a donor-acceptor form (Table 5). Weak intensities in the higher-frequency bands and relatively large 4-13C-induced downshifts by 21-25 cm-1 in the lowerfrequency bands2,3,11 are rather consistent with this form. Thus, there is a possibility that YZ and YD have another hydrogen bond with a nearby amino acid residue or a water molecule. Identification of the νCO(D) bands of dueterated YD and YZ is urgent to answer this question. Hydrogen-Bond Effects on Other Potential Markers. The 8a and 8b modes have been argued as Raman markers of hydrogen-bond structures.12,14,15,17,22 The reported effect of hydrogen-bonding on the 8a frequency has been rather controversial; the 8a band was suggested to downshift by hydrogenbond donation,15 to upshift in both donor and acceptor forms,17 and to show no clear tendency.22 DFT calculations in the present study showed that 8a frequencies in both the hydrogen-bond donor and the donor-acceptor forms are mostly identical to the free form (1618 cm-1), while the acceptor form showed a slight downshift by 2-4 cm-1 (Table 4). The experimental data were basically consistent with this tendency (Table 4). However, because the scatter of the experimental values was larger than the downshift (for example, the experimental 8a frequencies of

Hydrogen-Bond Structure of Tyr the donor form range between 1620 and 1615 cm-1), practically it might be difficult to judge the hydrogen-bond forms only from this mode. However, this mode can be a marker of deprotonation, because the band has been observed at ∼1600 cm-1 in tyrosinate.2,5,12 In contrast to 8a, the 8b mode exhibits a clearer hydrogenbond effect. The calculated frequencies downshift by 2-8 cm-1 and upshift by 3-8 cm-1 from the free value (1598 cm-1) in the donor and acceptor forms, respectively, while the donoracceptor form showed frequencies similar to the free value (Table 4). The experimental data in the present study followed the calculated tendency (Table 4). These results support the observations in previous Raman studies in which the 8b band downshifts and upshifts by hydrogen-bond donation and acceptance, respectively.14,15 The 8b mode was reported to undergo a large frequency decrease to ∼1558 cm-1 upon deprotonation.12 Although identification of the 8b band in FTIR spectra requires careful examination because of its weak intensity (Figure S4 of the Supporting Information), it can be a useful marker of both the hydrogen-bond forms and the protonation state. The 19a mode shows a very strong IR intensity (Figure S4 of the Supporting Information).2,4,5,11,24,27,33 The calculated and experimental frequencies of p-cresol are found in the narrow range of 1519-1513 cm-1 and are rather insensitive to hydrogen-bond forms, although it showed an only slight (by 1-4 cm-1) upshift in donor and donor-acceptor forms (Table 4). L-Tyr and poly-L-Tyr in H2O also showed peaks at 15191516 cm-1.2,4,5,11 Because tyrosinate in an aqueous solution has a peak at a much lower frequency of ∼1500 cm-1, the 19a band can be a good marker of the protonation state.2,4,5,11 Our preliminary calculation of the hydrogen-bonded complexes of p-cresolate showed that the 19a frequencies are higher by 4 and 15 cm-1 in singly hydrogen-bonded and free forms, respectively, than the frequency in a doubly hydrogen-bonded complex (data not shown). Thus, the above criterion for the 19a mode as a protonation marker can be applicable at least to hydrogenbonded tyrosinate. The 9a mode, which is located at 1172 cm-1 in a free form, shows only slightly lower (by 0-4 cm-1) and higher (by 0-4 cm-1) values in the donor and the donor-acceptor forms, respectively, in calculations and experiments (Table 4). The downshift tendency by hydrogen-bond donation has been suggested in Raman studies.16,19 In the acceptor form, the 9a mode strongly couples with the δCOH vibration, and hence accurate 9a frequencies are not identified. The 9a mode has been suggested to be sensitive to the dihedral angle between the COH and the benzene planes.22 Thus, application of this mode to biological systems as a hydrogen-bond marker requires caution. Conclusions The criteria to determine the hydrogen-bond forms of Tyr using νCO and δCOH bands in FTIR spectra were proposed. The free and hydrogen-bond acceptor forms can be easily discriminated from the hydrogen-bond donor and donoracceptor forms due to the relatively low νCO and δCOH frequencies (1260-1250 and 1185-1165 cm-1 in the free form and 1240-1220 and 1190-1160 cm-1 in the acceptor form). In the donor-acceptor form, the δCOH-like and νCO vibrations show weak and strong IR bands at 1280-1255 and 1255-1235 cm-1, respectively, which severely overlap the frequency regions of the νCO and δCOH bands at 1280-1260 and 1255-1210 cm-1 in the donor form. These two forms can be discriminated by detecting a νCO(D) band of Tyr-OD and plotting the frequency of the lower-frequency strong IR band (νCO of the

J. Phys. Chem. B, Vol. 111, No. 49, 2007 13843 donor-acceptor form or δCOH of the donor form) of undeuterated Tyr against the νCO(D) frequency. The νCO(D) frequencies are located at 1260-1240 and 1270-1255 cm-1 in the donor-acceptor and donor forms, respectively. Because of the positive linear correlation between δCOH and νCO(D) frequencies in the donor form, the plot areas of the two forms do not overlap with each other. The 19a mode (νCC + δCH), another prominent IR band found at 1519-1513 cm-1, is rather insensitive to hydrogen-bond interactions. Because tyrosinate shows this mode at ∼1500 cm-1, this band can be a good marker of the protonation state. The 8b mode (νCC) are sensitive to both the hydrogen-bond forms and the protonation state and thus can be a potential marker, although its weak IR intensity requires careful examination in band assignment. These IR markers and criteria to determine the hydrogen-bond and protonation structures of Tyr will be very useful in FTIR investigation of the molecular mechanisms of various protein reactions. Acknowledgment. This study was supported by Grants-inAid for Scientific Research (Grant Nos. 17GS0314 and 18570145) from the Ministry of Education, Science, Sports, Culture and Technology. Supporting Information Available: Effects of 4-13C labeling on the νCO and δCOH(D) frequencies in p-cresol and p-cresol-OD complexes, the optimized structures of the model complexes of p-cresol that acts as a hydrogen-bond donor, acceptor, and donor-acceptor, and the νCC region (1640-1505 cm-1) of the FTIR spectra of p-cresol in various solutions. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Berthomieu, C.; Hienerwadel, R. Biochim. Biphys. Acta 2005, 1707, 51. (2) Hienerwadel, R.; Boussac, A.; Breton, J.; Diner, B. A.; Berthomieu, C. Biochemistry 1997, 36, 14712. (3) Berthomieu, C.; Hienerwadel, R.; Boussac, A.; Breton, J.; Diner, B. A. Biochemistry 1998, 37, 10547. (4) Rothschild, K. J.; Roepe, P.; Ahl, P. L.; Earnest, T. N.; Bogomolni, R. A.; Das Gupta, S. K.; Mulliken, C. M.; Herzfeld, J. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 347. (5) Dollinger, G.; Eisenstein, L.; Lin, S. L.; Nakanishi, K.; Termini, J. Biochemistry 1986, 25, 6524. (6) Takahashi, R.; Okajima, K.; Suzuki, H.; Nakamura, H.; Ikeuchi, M.; Noguchi, T. Biochemistry 2007, 46, 6459. (7) Ma¨ntele, W. Trends Biochem. Sci. 1993, 18, 197. (8) Vogel, R.; Siebert, F. Curr. Opin. Chem. Biol. 2000, 4, 518. (9) Zscherp, C.; Barth, A. Biochemistry 2001, 40, 1875. (10) Noguchi T.; Berthomieu, C. In Photosystem II: The Light-DriVen Water:Plastoquinone Oxidoreductase; Wydrzynski, T., Satoh, K., Eds.; Springer: Dordrecht, The Netherlands, 2005; Chapter 16, p 367. (11) Noguchi, T.; Inoue, Y.; Tang, X.-S. Biochemistry 1997, 36, 14705. (12) Copeland, R. A.; Spiro, T. G. Biochemistry 1985, 24, 4960. (13) Rava, R. P.; Spiro, T. G. J. Phys. Chem. 1985, 89, 1856. (14) Hildebrandt, P. G.; Copeland, R. A.; Spiro, T. G.; Otlewski, J.; Laskowski, M.; Prendergast, F. G. Biochemistry 1988, 27, 5426. (15) Rodgers, K. R.; Su, C.; Subramaniam, S.; Spiro, T. G. J. Am. Chem. Soc. 1992, 114, 3697. (16) Zhao, X. J.; Spiro, T. G. J. Raman Spectrosc. 1998, 29, 49. (17) Nagai, M.; Imai, K.; Kaminaka, S.; Mizutani, Y.; Kitagawa, T. J. Mol. Struct. 1996, 379, 65. (18) Harada, I.; Yamagishi, T.; Uchida, K.; Takeuchi, H. J. Am. Chem. Soc. 1990, 112, 2443. (19) Ames, J. B.; Ros, M.; Raap, J.; Lugtenburg, J.; Mathies, R. A. Biochemistry 1992, 31, 5328. (20) Siamwiza, M. N.; Lord, R. C.; Chen, M. C.; Takamatsu, T.; Harada, I.; Matsuura, H.; Shimanouchi, T. Biochemistry 1975, 14, 4870. (21) Arp, Z.; Autrey, D.; Laane, J.; Overman, S. A.; Thomas, G. J. Biochemistry 2001, 40, 2522. (22) Takeuchi, H.; Watanabe, N.; Satoh, Y.; Harada, I. J. Raman Spectrosc. 1989, 20, 233.

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Criteria for Determining the Hydrogen-Bond Structures of a Tyrosine

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