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A Raman spectroscopy and theoretical study of zinc–cysteine complexation
Vibrational Spectroscopy 44 (2007) 256–265 www.elsevier.com/locate/vibspec
A Raman spectroscopy and theoretical study of zinc–cysteine complexation Sarah Foley, Mironel Enescu * University of Franche-Comte, Laboratoire de Microanalyses Nucleaires, UMR CEA E4,16 route de Gray, 25030 Besancon, France Received 27 September 2006; received in revised form 4 December 2006; accepted 8 December 2006 Available online 16 December 2006
Abstract The Raman spectrum of the 1:2 zinc–cysteine complex in aqueous solution is compared to those of cysteine zwitterion and cysteine anion in order to identify specific complexation effects. Band assignment is based on frequency calculation and normal modes analysis using the density functional theory (DFT) method. Two bands specific to the complex were detected in the 200–400 cm1 spectral range. It is shown that the corresponding vibrations are not pure metal–ligand modes but that they result from the coupling between the Zn–S and Zn–N stretching modes with some cysteine internal modes. Raman spectra analysis also provides direct evidence for the deprotonation of the SH and NH3+ groups of cysteine upon zinc binding. It is found in addition that complexation significantly affects the cysteine internal mode mixing in the 500–1500 cm1 spectral range. The results are considered in connection with the spectral characterization of zinc–protein complexes of biological interest. # 2006 Elsevier B.V. All rights reserved. Keywords: Cysteine; Zinc; Metal–ligand bands; DFT calculations; Potential energy distribution
1. Introduction Among the first row transition metals, zinc is second only to iron in terms of abundance and importance in biological systems [1]. The Zn2+ cation can play a structural as well as a catalytical role in proteins. The zinc finger family are the most well studied proteins in which zinc plays a structural role, since it is known that these proteins are involved in nucleic acid binding and gene regulation [2,3]. In addition to its structural role, zinc serves an essential role in many enzymes and virtually all aspects of metabolism. One of the most commonly found ligands in zinc catalytic binding sites is cysteine, with others being histidine, aspartic acid and glutamic acid [4–6]. The thiol or sulphydryl (S–H) groups of cysteine are the most chemically reactive sites in proteins at physiological conditions [7] and a preferred ligand for the Zn2+ cation. The interaction between zinc and cysteine residue is complex since the cysteine is potentially a multidentate ligand. The NH and CO groups of cysteine may be involved in complexation, as well as the COO and NH3+ groups in the case of terminal cysteines. Indeed,
* Corresponding author. Tel.: +33 3 81 66 65 21; fax: +33 3 81 66 65 22. E-mail address: [email protected] (M. Enescu). 0924-2031/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vibspec.2006.12.004
potentiometric measurements show that the most stable complex of Zn2+ cation with cysteine zwitterion HS–CH2– CH(NH3+)–COO is a 1:2 complex in which the metal is coordinated by the terminal S, N and O of both cysteines [8]. On the other hand, a recent theoretical study indicates the combination of the three coordination centers of the unpolar form of cysteine HS–CH2–CH(NH2)–COOH provides the formation of metallocomplexes of various types with monodentate, bidentate or tridentate configurations [9]. Raman spectroscopy has been demonstrated to be a valuable method for the study of the ligand–protein interactions in both solution [10] and the crystalline state [11]. Spectral problems arising from solvent water molecules are also much less restrictive in Raman spectroscopy than in IR absorption spectroscopy. Fortunately, the S–H stretching Raman band of cysteine has the advantage of occuring within the spectral range 2500–2600 cm1, which is far removed from other Raman bands of proteins. Many Raman spectroscopic studies of cysteine have concentrated on this spectral zone since the S–H stretching band is known to be an environmentally sensitive probe [12–14]. Although the S–H stretching band is an useful probe, the low frequency spectral range can provide interesting information about the structure of metalloprotein complexes [15–17]. In the case of multiple coordinated metal complexes
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band assignment is difficult since a specific metal–ligand band is expected to be sensitive to both the number and nature of all ligand groups participating in the complex. Moreover, the metal–ligand vibrational modes may be considerably mixed with the proper ligand modes. Hence, detailed analysis on zinc– cysteine model complexes is a prerequisite for studying zinc– protein complexes. The aim is to better understand the way zinc–ligand interactions affect the Raman spectrum of cysteine and to identify specific markers of these interactions. It is also preferential to perform this investigation in aqueous solution since the Raman spectra of a crystalline sample could be distorted by intermolecular interaction especially in the low frequency spectral range. In the present paper we report a detailed analysis of the Raman spectrum and the related metal–ligand interactions for the zinc–cysteine complex Zn(Cys)2 in aqueous solution. To the best of our knowledge, this is the first time that this Raman spectrum is reported. The complexation effects are identified by comparing the Raman spectrum of the complex with those of cysteine zwitterion and cysteine anion S–CH2–CH(NH2)– COO in the 200–3200 cm1 spectral range. Band assignments are based on vibrational frequency calculations using the density functional theory (DFT) method. The normal modes were further analyzed by calculating their potential energy distributions (PED) with respect to the internal coordinates. The complexity of the spectral changes induced par zinc– cysteine interaction is emphasised and carefully analysed. 2. Methods 2.1. Experimental L-Cysteine (>98%) and zinc chloride (98%) obtained from Sigma–Aldrich were used to record the Raman spectra of Zn(Cys)2 complex. The spectra here reported were obtained on aqueous solutions with a metal/ligand ratio of 1:2 prepared for cysteine concentrations of 50 and 100 mM. The pH of all solutions was adjusted to 7.0 by the dropwise addition of 2 M NaOH. A solution with the same metal/ligand ratio but with a cysteine concentration of 5 mM was also prepared in order to test the spectral stability with respect to the complex concentration. In this case, the weak Raman bands were less resolved but the strong bands showed the same position and relative intensity as for the more concentrated solutions. We concluded that the Zn(Cys)2 Raman spectrum was stable in the tested concentration range. The Raman spectra of cysteine zwitterion and cysteine anion were obtained by measuring 50 and 100 mM cysteine aqueous solutions prepared at pH 7.0 for zwitterion and at pH 11 for the anion. The Raman spectroscopy experimental set up includes a Spectra Physics Nd:YAG laser model LAB-170-10 delivering pulses with a duration of about 5 ns at a repetition rate of 10 Hz. The spectra were recorded using the second harmonic emission wavelength (532 nm). The samples were placed in a 1 cm 1 cm quartz cell and were irradiated with a 10 mm diameter laser beam at an equivalent power density of 35 mW mm2. The scattered light was detected at 908 using
257
a Roper Scientific spectroscopy system including a Spectra Pro 2500i monochromator with a maximum resolution of 0.035 nm and a PIMAX-1024-RB CCD camera (Princeton Instruments). The camera intensifier was synchronously gated over the laser pulse duration in order to maximize the signal to noise ratio. The Rayleigh scattered light was eliminated using a notch filter with a 300 cm1 bandwidth and an optical density at 532 nm of 6. The system wavelength calibration was tested by detecting the N2 Raman band at 2331 cm1 which was reproduced with an error of less than 1 cm1. All spectra were acquisitioned over 10,000 laser pulses. The sample stability with respect to laser exposure was tested by repeating the acquisition sequence two or three times. No spectral changes were detected. Given the fact that the monochromator free spectral range was only 360 cm1, the analyzed spectral range (200–3200 cm1) was explored using many acquisition steps with the solution being renewed every step. The intensity detected at one step was corrected with respect to that detected at the preceding step in order to provide continuity at the boundary between adjacent spectral intervals. Usually these corrections were of the order of 1–3%. For each spectral interval, the solvent scattered light was also determined by alternatively measuring a pure water reference. The global reference spectrum, also corrected for boundary discontinuities, was then subtracted from the global sample spectrum. The base line of the difference spectrum was corrected by slightly adjusting the intensity ratio of the sample and reference spectra. This normalization procedure provides very reproducible relative intensities of the Raman bands. The analyzed spectral range was mainly limited by the very intense water Raman bands below 200 and over 3200 cm1. The wavenumbers of Raman bands were reproducible within 1 cm1. 2.2. Computational method All calculations were performed using the Gaussian 03 program [18] on a Windows-XP operating PC. Full geometry optimization for the all molecules were carried out by the DFT method using Becke’s three parameter hybrid functional combined with the Lee–Yang–Parr correlation functional (B3LYP) [19,20] with the 6-31+G(d) basis set. Vibrational frequencies were then computed at the same level of theory. All calculations were performed in aqueous solution using the polarizable continuum model (PCM) [21]. For each normal mode, the potential energy distribution (PED) with respect to the internal coordinates was calculated as follows: PED ð%Þ ¼
k j j S2ji kii Q2i
100
(1)
where kjj is the force constant for the internal coordinates j and Sji is the projection on this internal coordinate of the normal mode i having the amplitude Qi and the force constant kii. The Gaussian program gave directly the Sji and kii values while the force constant kjj were calculated by numerical differentiation of the potential energy.
258
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calculations involve third order energy derivative calculations. Hence, accurate Raman intensity calculations require significantly larger basis set with respect to frequency calculations [22]. Obviously, in the present study the choice of the basis set was strongly limited by the size of the zinc–cysteine complex. 3. Results and discussion
Fig. 1. Optimized structure of the Zn(Cys)2 complex water at the B3LYP/631+G(d) level of theory. Atomic distances are given in Angstroms.
With respect to the calculated Raman intensities, their agreement with the experimental intensities in the very low and very high frequency domains was unsatisfactory. This result is not very surprising since it is known that Raman intensity
In aqueous solutions Zn2+ cation and cysteine form both 1:1 and 1:2 complexes. Using the corresponding stability constants as determined by potentiometric measurements [8] one finds that in the concentration range used here and at pH 7 more than 95% of cysteines are bound to zinc as the Zn(Cys)2 complex. Potentiometric measurements also showed that at pH 7 cysteine loses two protons upon zinc binding. Obviously, the two groups susceptible to release these protons are the SH and the NH3+ groups which are protonated in the case of free cysteine solutions at neutral pH. This result indicates that the S and N atoms of the two cysteines are bound to the metal. On the other
Table 1 Experimental and calculated (not scaled) frequencies and potential energy distribution of Raman active normal modes of the cysteine zwitterions in aqueous solution No. crt
Calculateda
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
75 81 135 177 260 312 331 484 505 576 666 770 790 850 905 977 1061 1074 1119 1238 1301 1330 1369 1398 1461 1475 1595 1644 1657 2412 3013 3074 3148 3262 3308 3348
(12) (8.9) (3.2) (3.4) (3.2) (3.3) (4.5) (23) (14) (29) (100) (55) (9.8) (34) (31) (34) (8.7) (12) (23) (90) (14) (17) (52) (50) (38) (38) (63) (49) (22) (1198) (934) (890) (448) (762) (102) (271)
Experimentala
283 (29) 345 475 529 622 684 780 814 875 935 994 1064 1115 1140 1213 1276 1311 1345 1399 1430 1513 1614 1647
(26) (19) (26) (39) (100) (36) (25) (34) (30) (19) (20) (16) (17) (21) (14) (26) (46) (43) (35) (9.7) (18) (14)
2581 2837 2959 3001
(61) (10) (58) (31)
The band intensities normalized to 100 for the C–S band are given in parentheses. a in cm1. b n: stretch; d: deformation; z: rocking; g: waging; t: torsion; a: antisymmetric; s: symmetric. c PED (%).
Assignment tb(CO2) (100c) t(CH2SH) (100) t(NH3+) (100) db(C–C–S) (42), d(C–C–C) (38) d(C–C–C) (50), gb(CO2) (16) d(C–C–N) (20), t(CSH) (25) d(C–C–N) (47), t(CH2) (20) d(C–C–N) (42), d(C–C–S) (27), d(C–C–H) (17) zb(CO2) (50), nb(CN) (10) n(CC) (25), d(CO2) (23), d(C–C–N) (16) n(CS) (70), d(C–C–S) (13) z1(CH2) (26), n(CH) (21), d(C–S–H) (16), z2(CH2) (13) d(C–S–H) (33), d(CO2) (30), z2(CH2) (16) d(CO2) (24), n(CC) (20), z(NH3+) (16) n(CC) (36), n(CN) (32), d(C–S–H) (17), z(NH3+) (14) z(NH3+) (45), d(C–S–H) (32), n(CC) (15), z2(CH2) (14) z(NH3+) (34), n(CN) (15), d(C–S–H) (14), n(CC) (10) n(CN) (56), z(NH3+) (32) z(NH3+) (44), d(C–C–H) (32) d(C–C–C) (48), z2(CH2) (41) g(CH2) (52), n(CC) (22), d(C–C–H) (18) d(C–C–H) (30), g(CH2) (30), nsb(CO2) (28) d(N–C–H) (71) ns(CO2) (34), d(N–C–H) (24), n(CC) (19), d(CO2) (11) d(CH2) (78), d(NH3+) (15) g(NH3+) (38), d(NH3+) (30), d(CH2) (28) d(NH3+) (77) nab(CO2) (95) d(NH3+) (95) d(SH) (77) n(CH) (100) ns(CH2) (100) na(CH2) (100) ns(NH3+) (100) na1(NH3+) (100) na2(NH3+) (100)
S. Foley, M. Enescu / Vibrational Spectroscopy 44 (2007) 256–265
hand, our DFT optimization of a system formed by a Zn2+ cation and two cysteine anions shows that the stable configuration corresponds to the hexacoordination of the zinc cation (Fig. 1) including the participation of two O atoms from the two carboxylate groups. To the best of our knowledge, no structural data have been reported for this complex, either in aqueous solution or in the solid state. The interest in performing a Raman spectroscopy study on this system is to provide direct evidence for this haxacoordination and to emphasize the nature of the related spectral features. In theory, Zn complexation to cysteine could induce three kinds of effects on the vibrational spectrum of cysteine: those due to the formation of new bonds between the metal atom and the ligand atoms (S, N or O), those due to the deprotonation of the cysteine and those due to the coupling of the internal vibrational modes of cysteine via metal–ligand interactions. In order to identify the above effects we have compared experimental Raman frequencies with the calculated Raman frequencies for cysteine zwitterion (Table 1), cysteine anion (Table 2) and the Zn(Cys)2 complex (Table 3). Regarding the choice of the molecular conformations used in frequency calculation, it is worth noting that in the case of the zinc– cysteine complex only one isomer (Fig. 1) is compatible with that of zinc hexacoordination. In the case of the cysteine
259
zwitterion we have reoptimized the isomer corresponding to the X-ray diffraction structure of L-cysteine [23]. The calculated structure (Fig. 2 and Table 4) was in good agreement with the experimental one. In the case of cysteine anion the only stable structure is that for which the negatively charged carboxylate and thiolate groups point in opposite directions (Fig. 3). Among the three systems analyzed here only Zn(Cys)2 possesses point group symmetry: it belongs to the Ci point group hence all Raman active normal modes are Ag modes while the IR active normal modes are Au modes. 3.1. S–Zn, N–Zn and O–Zn bond stretching The intrinsic vibrational frequencies of these stretching modes can be evaluated with the simple harmonic oscillator formula: sffiffiffiffi 1 k n¼ (2) 2p m where k is the force constant and m is the reduced mass of the biatomic system formed by the ligand atom and the Zn atom. The actual force constants were obtained for the Zn(Cys)2 complex by calculating the second order derivatives of the potential
Table 2 Experimental and calculated (not scaled) frequencies and potential energy distribution of Raman active normal modes of the cysteine anion in aqueous solution No. crt
Calculateda
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
32 126 195 221 273 350 423 524 641 680 801 843 880 933 1001 1112 1172 1248 1252 1342 1354 1408 1484 1545 1631 2922 3032 3109 3406 3485
(6.8) (1.9) (2.1) (4.8) (5.8) (7.1) (1.6) (12) (10) (100) (8.2) (15) (91) (34) (25) (17) (8.7) (50) (8.5) (59) (22) (46) (67) (44) (27) (919) (619) (390) (823) (273)
Experimentala
290 (8.5) 330 (12) 414 (14) 541 625 683 792 834 911 1037 1065 1090 1151 1203 1239 1307 1349 1413 1428 1567 1658 2836 2915 2942
(32) (33) (100) (25) (33) (39) (16) (54) (17) (7.8) (10) (8.6) (16) (33) (22) (28) (7.9) (5.2) (16) (39) (36)
The band intensities normalized to 100 for the C–S band are given in parentheses. a in cm1. b n: stretch; d: deformation; z: rocking; g: waging; t: torsion; a: antisymmetric; s: symmetric. c PED (%).
Assignment tb(CO2) (100c) t(CH2S) (100) Collective db(C–C–H) (48), d(C–C–S) (26), t(NH2) (26) t(NH2) (80), nb(CS) (10), n(CC) (10) t(CH2S) (39), d(C–C–H) (32), t(NH2) (28) t(NH2) (95) zb(CO2) (41), t(NH2) (15) d(CO2) (29), n(CC) (17), n(CS) (10) n(CS) (69), d(C–C–S) (15) d(CO2) (47), gb(CO2) (10) z1(CH2) (37), z2(CH2) (22), n(CN) (21) n(CC) (27), g(NH2) (20), d(CO2) (14) n(C–C–C) (47), g(NH2) (18) z1(CH2) (53), g(NH2) (23), n(CN) (23) n(CC) (34), n(CN) (33), d(C–N–H) (14) z2(CH2) (45) g(CH2) (100) d(C–C–H) (38), z(NH2) (30), d(C–CH2) (17) d(N–C–H) (21), d(C–C–H) (21), z(CH2) (21), z(NH2) (13) d(N–C–H) (49), d(C–C–H) (49) z(NH2) (28), ns(CO2) (27), n(CC) (20), d(C–C–H) (20) d(CH2) (95) na(CO2) (60), d(NH2) (40) na(CO2) (54), d(NH2) (20), g(NH2) (17) n(CH) (100) nsb(CH2) (100) nab(CH2) (100) ns(NH) (100) na(NH) (100)
260
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Table 3 Experimental and calculated (not scaled) frequencies and potential energy distribution of Raman active normal modes of the Zn(Cys)2 complex in aqueous solution No. crt
Calculateda
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
75 97 135 146 188 238 316 383 420 560 588 653 719 799 864 911 953 1035 1067 1162 1194 1280 1336 1345 1419 1492 1592 1639 3012 3036 3090 3350 3427
(4) (8) (4) (10) (13) (12) (14) (27) (14) (16) (6) (100) (22) (43) (15) (11) (26) (26) (25) (32) (53) (51) (44) (45) (72) (69) (41) (26) (1162) (1464) (662) (1207) (478)
Experimentala
296 334 399 532 569 685
(119) (118) (61) (38) (39) (100)
805 849 920 968
(28) (21) (3) (19)
1058 1190 1218 1256 1301 1355 1405 1433 1596 1659 2851 2930 2967
(20) (16) (13) (14) (23) (24) (31) (22) (15) (12) (10) (38) (26)
Assignment [nsb(S–Zn–S)–ns(O–Zn–O)](51), tb(NH2) (49c) t(CH2S) (49), t(CO2) (53), ts(S–Zn–S) (17) ns(N–Zn–N) (63), t(CO2) (34), t(CH2S) (29) ns(O–Zn–S) (52), ns(N–Zn–N) (29), t(CH2S) (18) ns(N–Zn–N) (70), t(CH2S) (17), ns(O–Zn–O) (12) db(C–C–C) (68), zb(NH2) (25) d(C–C–C) (50), ns(S–Zn–S) (20), ns(O–Zn–O) (20), ns(N–Zn–N) (20) d(N–C–C) (37), ns(N–Zn–N) (24), t(NH2) (18), d(S–C–C) (16) z(CO2) (53), t(NH2) (12) t(NH2) (29), n(CC) (25), d(CO2) (15) t(NH2) (95) n(CS) (62), d(C–C–O) (12) d(CO2) (15), n(CS) (14), d(N–C–C) (14) d(C–C–C) (16), d(CO2) (13), n(CC) (12), d(S–C–C) (11) d(CO2) (33), n(CC) (25), gb(NH2) (20) z1(CH2) (38), g(NH2) (21) n(CC) (70), d(C–N–H) (22), d(C–C–H) (13) g(NH2) (45), d(NH2) (45) n(CN) (57), n(CC) (18), d(S–C–H) (13) z(NH2) (54), d(C–C–H) (22), d(S–C–H) (27) d(C–C–C) (83), d(S–C–H) (27) g(CH2) (60), d(C–C–N) (25), z(NH2) (16) d(C–C–H) (48), ns(CO2) (19), z(NH2) (16) d(C–C–H) (67), g(CH2) (12) ns(CO2) (51), n(CC) (28), d(CO2) (16) d(CH2) (95) nab(CO2) (96) d(NH2) (73), g(NH2) (27) n(CH) (98) ns(CH2) (97) na(CH2) (96) ns(NH2) (95) na(NH2) (95)
The band intensities normalized to 100 for the C–S band are given in parentheses. a in cm1. b n: stretch; d: deformation; z: rocking; g: waging; t: torsion; a: antisymmetric; s: symmetric. c PED (%).
energy with respect to the corresponding bond lengths. Finally, we obtained the following intrinsic vibrational frequencies: n(SZn) = 150 cm1, n(OZn) = 282 cm1 and n(NZn) = 450 cm1. These are, of course, fictive frequencies because in the complex the reduced mass of the related normal modes will be modified. Indeed, the stretching of the ligand–metal bonds within the complex is expected to be associated with significant relative and/or internal motions of the two cysteines. Such motions are particularly important in the case of Raman active modes which are all symmetrical. During a symmetrical vibration the Zn is fixed, thus the Zn–S(N,O) bond stretching involves a significant displacement of the ligands. The effect is an increase in the reduced mass of the stretching modes and a lowering of the corresponding vibrational frequencies. Accordingly, the calculated Raman spectrum for the Zn(Cys)2 complex (Table 3) shows the participation of the Zn–S, Zn–O and Zn–N bond stretching in several modes between 75 and 383 cm1. Of these modes two were detected in the experimental spectrum at 296 and 334 cm1 (Fig. 4c). The participation of the metal–ligand bond stretching in these bands is supported by the theoretical assignment and the PED analysis in Table 3. Moreover, the experimental spectrum
for cysteine anion (Fig. 4c) shows only weak Raman bands within the same spectral region thus confirming that the bands situated at 296 and 334 cm1 are related to the ligands interaction with the zinc. An equivalent enhancement of the Raman bands in the 200–300 cm1 spectral range induced by metal binding has also been reported by Faget et al. [24] who investigated a cysteine dichloride cadmium complex. Zinc binding to a zinc finger peptide was found to give rise to a Raman band at 242 cm1 that was attributed to the Zn–N(His) bond stretching, another band situated at 311 cm1 was attributed to the Zn–S stretching [16]. A similar frequency (306 cm1) of the Zn–S stretching was observed for a Zn(dmit)2 complex with tetrahedral zinc coordination (here dmit is 1,3-dithiol-2-thione-4,5-dithiolate ligand) [25]. The present calculations predict significant differences between the Raman and the IR spectrum of the Zn(Cys)2 complex only in the frequency domain below 300 cm1 (see Table 5). Obviously, a comparison between Raman and IR experimental spectra in this spectral domain would be very useful for testing the reliability of our theoretical results. Unfortunately, to the best of our knowledge, no IR spectra in
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261
Fig. 3. Optimized structure of cysteine anion in water at the B3LYP/6-31+G(d) level of theory.
3.2. Cysteine deprotonation effects Fig. 2. Optimized structure of cysteine zwitterion in water at the B3LYP/631+G(d) level of theory.
this spectral range has been reported for Zn(Cys)2 complex in aqueous solution. For the solid-state complex, it appears that only two IR spectra have been reported so far [26,27]. However, since the reported IR bands are situated above 800 and 500 cm1, respectively, they are not particularly relevant with respect to the present discussion. Interestingly, the PED analysis in Table 3 indicates that the two experimental bands attributed to zinc–ligand interaction include significant contributions from the cysteine internal modes. With respect to the frequency domain below 200 cm1, our calculations predict highly mixed Zn–S, Zn–O and Zn–N stretching bands. One exception is the mode at 188 cm1 having a very dominant contribution from the Zn–N stretching. The present analysis suggests that in the case of metal– protein complexes it is rather improbable to identify metal– ligand Raman bands that are super-imposable from one complex to another without significant alterations due to mode mixing.
The spectacular reduction of the n(SH) band intensity at 2581 cm1 (Fig. 5c) directly demonstrates thiol deprotonation. Comparing the corresponding band intensities in Fig. 5a and c indicates for the measured zinc–cysteine solution the fraction of non-complexed cysteine molecules is negligible. One also notes the calculated n(SH) frequency is lower by about 169 cm1 with respect to the experimental value suggesting the
Table 4 Optimized geometries of the cysteine zwitterion, cysteine anion and Zn(Cys)2 complex: comparison between the main internal parameters
S–C(10) S–C(10)–C(8) S–C(10)–C(8)–C(7) N–C(8) N–C(8)–C(7) N–C(8)–C(10)–S O(2)–C(7) O(2)–C(7)–C(8) O(2)–C(7)–C(8)–C(10) S–Zn S–Zn–S0 N4–Zn N–Zn–N0 O(2)–Zn O(2)–Zn–O(2)0
Cysteine zwitterion
Cysteine anion
Zn(Cys)2
˚ 1.843 A 114.988 66.768 ˚ 1.505 A 108.288 56.858 ˚ 1.262 A 115.818 136.438
˚ 1.862 A 114.468 169.778 ˚ 1.480 A 108.908 65.648 ˚ 1.273 A 114.518 133.98
˚ 1.854 A 113.228 64.948 ˚ 1.478 A 109.298 55.078 ˚ 1.258 A 116.688 91.108 ˚ 2.620 A 180.008 ˚ 2.171 A 180.008 ˚ 2.221 A 180.008
The geometries were optimised at the B3LYP/6-31+G(d) level of theory in water.
Fig. 4. Raman spectra of aqueous solutions of cysteine zwitterion (a), cysteine anion (b) and Zn(Cys)2 complex (c) in the spectral range 200–850 cm1. Bands labeling as indicated in Tables 1–3, respectively. Every spectrum was normalized for a S–C stretching band intensity of unit. All spectra were obtained for an equivalent cysteine concentration of 100 mM.
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Table 5 Calculated (not scaled) frequencies and potential energy distribution of IR active normal modes of the Zn(Cys)2 complex in aqueous solution No. crt 0
Calculated a
1 20 30 40 50 60 70 80 90 100
25 70 81 106 122 156 169 233 274 318
(10) (26) (8) (103) (30) (247) (50) (5) (60) (96)
110
383 (22)
120 130 140 150 160 170
421 560 597 654 720 800
(24) (25) (192) (30) (37) (29)
180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360
865 912 953 1031 1065 1161 1194 1284 1337 1345 1419 1492 1589 1639 3012 3036 3090 3356 3427
(58) (28) (13) (466) (186) (46) (15) (74) (159) (78) (458) (35) (2598) (172) (34) (164) (38) (100) (133)
Assignment Dispersed Dispersed nab(S–Zn–S) (24)c, na(O–Zn–O) (24)c, tb(NH2) (40) na(S–Zn–S) (50), na(O–Zn–O) (40) t(CH2S) (48), t(CO2) (50), na(S–Zn–S) (18) na(S–Zn–S) (45), na(O–Zn–O) (50) na(O–Zn–O) (40), t(CO2) (20), t(CH2S) (20) dbC–C–C) (68), zb(NH2) (25) na(N–Zn–N) (70), d(C–C–C) (20) d(C–C–C) (50), na(S–Zn–S) (20), na(O–Zn–O) (20), na(N–Zn–N) (20) d(N–C–C) (37), na(N–Zn–N) (24), t(NH2) (18), d(S–C–C) (16) z(CO2) (53), t(NH2) (12) t(NH2) (29), n(CC) (25), d(CO2) (15) t(NH2) (95) n(CS) (62), d(C–C–O) (12) d(CO2) (15), n(CS) (14), d(N–C–C) (14) d(C–C–C) (16), d(CO2) (13), n(CC) (12), d(S–C–C) (11) d(CO2) (33), n(CC) (25), gb(NH2) (20) z1(CH2) (38), g(NH2) (21) n(CC) (70), d(C–N–H) (22), d(C–C–H) (13) g(NH2) (45), d(NH2) (45) n(CN) (57), n(CC) (18), d(S–C–H) (13) z(NH2) (54), d(C–C–H) (22), d(S–C–H) (27) d(C–C–C) (83), d(S–C–H) (27) g(CH2) (60), d(C–C–N) (25), z(NH2) (16), d(C–C–H) (48), ns(CO2) (19), z(NH2) (16) d(C–C–H) (67), g(CH2) (12) ns(CO2) (51), n(CC) (28), n(CO2) (16) d(CH2) (95) nab(CO2) (96) d(NH2) (73), g(NH2) (27) n(CH) (98) ns(CH2) (97) na(CH2) (96) ns(NH2) (95) na(NH2) (95)
The band intensities normalized to 100 for the NH2 symmetric stretch band are given in parentheses. a in cm1. b n: stretch; d: deformation; z: rocking; g: waging; t: torsion; a: antisymmetric; s: symmetric. c PED (%).
H atom coupling to the solvent is over estimated in the PCM solvation model. With respect to the N–H stretching modes (symmetric and anti-symmetric), the characteristic frequencies are greater than 3200 cm1 thus falling outside the spectral range accessible for our measurements. On the other hand, the PED analysis predicts the NH2 angular deformation participating in the normal modes in the spectral range 1450–1700 cm1 (Tables 1–3). Theoretically, the cysteine zwitterion which is protonated in N possesses three bands for which the NH2 angular vibrations are predominant, at 1475, 1595 and 1657 cm1 (Table 1). On the other hand, the Zn(Cys)2 which is supposed to be deprotonated in N possesses only one, at 1659 cm1 (Table 3). Due to the strong Raman bands of water
Fig. 5. Raman spectra of aqueous solutions of cysteine zwitterion (a), cysteine anion (b) and Zn(Cys)2 complex (c) in the spectral range 500–3200 cm1. Bands labeling as indicated in Tables 1–3, respectively. Every spectrum was normalized for a S–C stretching band intensity of unit. All spectra were obtained for an equivalent cysteine concentration of 50 mM.
which fall within the same spectral range, the experimental detection of these bands was not accurate enough when using a concentration of 50 mM cysteine (Fig. 5). We thus performed an additional analysis for a cysteine concentration of 500 mM (and, in the case of the complex, a zinc concentration of 250 mM). As can be seen in Fig. 6b the Zn(Cys)2 has only two Raman bands in the 1500–1700 cm1 spectral range in good agreement with our theoretical calculations. According to these calculations, one of these bands should be attributed to the CO2 asymmetrical stretching and the other to the NH2 angular deformation (Table 3). In addition, these bands appear to be resolved in the case of the cysteine anion even at lower concentrations (Fig. 5b). For the cysteine zwitterion we detected three bands in the 1500–1700 cm1 spectral range, at 1513, 1614 and 1647 cm1 (Fig. 6a). One of them was attributed to the CO2 asymmetrical stretching and the other two to NH2 angular vibrations (Table 1). The third NH2 angular deformation band was not resolved but it could contribute to the broad
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263
3.3. CO2 coordination effects
Fig. 6. Raman spectra of aqueous solutions of cysteine zwitterion (a) and Zn(Cys)2 complex (b) in the spectral range 1450–1750 cm1. Bands labeling as indicated in Tables 1 and 3, respectively. Spectra were obtained for an equivalent cysteine concentration of 500 mM.
According to our calculations, the stable configuration of the complex corresponds to a hexacoordination of zinc with CO2 groups bound to Zn2+. This binding should affect the specific modes of the CO2 group. We looked for these evidences in the Raman spectrum of the zinc–cysteine complex. The CO2 rocking is predicted at 420 cm1 (Table 3) and detected at 399 cm1 (Fig. 4c). The band at 399 cm1 appears as specific to the complex since no equivalent band is present in the cysteine zwitterion spectrum (Fig. 4a) while the anion spectrum shows only a very weak band at 414 cm1 (Fig. 4b) with a very different PED (Table 2). Instead, for the cysteine zwitterion the CO2 rocking is predicted at 505 cm1 and detected at 529 cm1 (Fig. 4a). For the anion an equivalent band is detected at 541 cm1 (Fig. 4b). Although the spectrum of the complex also presents a band in the same spectral range (at 532 cm1) this band is significantly weaker and has a different PED. We conclude that the spectral differences here identified support the assumption of a zinc–cysteine complex with zinc hexacoordination. 3.4. Mode coupling effects
band observed between 1600 and 1700 cm1. The present assignment is in good agreement with that previously reported by Li et al. [28] who studied the Raman spectrum of crystalline L-cysteine and have based their normal modes analysis on force constants obtained from structural data. The IR spectrum for the solid state of the Zn(Cys)2 complex reported by Ikram and Powell [27] also presents only two bands in the spectral domain 1500–1700 cm1, one at 1580 cm1, and the other at 1615 cm1, in satisfactory agreement with our results. On the other hand, the corresponding IR bands reported by Shindo and Brown [26] are red shifted to 1588 and 1547 cm1, respectively. This discrepancy could be due to the structural distortion induced by the two sodium cations included in their solid-state complex. Finally, the spectral differences in Fig. 6 between the cysteine zwitterion and the Zn(Cys)2 complex clearly demonstrate the deprotonation of the NH3+ group upon complexation of cysteine with zinc. An alternative marker for the deprotonation of the NH3+ group is the NH2 torsional frequency. The reason is the NH2 torsional frequency is significantly higher than that of the NH3+ group due to a stronger force constant. Quantum chemical calculations predict for the Zn(Cys)2 complex a Raman active mode at 588 cm1 that is almost a pure NH2 torsional oscillation (Table 3). A corresponding Raman band was identified in the experimental spectrum of the complex at 569 cm1. It is worth noting that no similar band was found for cysteine zwitterion: the experimental band at 529 cm1 was assigned to a mode dominated by the CO2 rocking. For the cysteine anion a mode having an important contribution from the NH2 torsional oscillation is predicted to be at 273 cm1 while the corresponding experimental band was detected at 330 cm1. The frequency shift with respect to the complex is probably a mode mixing effect (Table 2).
For the three molecular systems discussed here, PED analysis predicts the normal modes below 1500 cm1 to have a rather complex structure with the mixing of several internal coordinates. It is therefore not surprising that the intensities and positions of the corresponding experimental bands illustrate significant differences on going from one system to another despite the local structure similarities of the three systems. A remarkable exception to the above is the C–S bond stretching band whose position (682 cm1) are almost identical for the three systems. Another very reproducible band is that for CH2 deformation which appears at 1430 cm1 for the zwitterion, 1428 cm1 for the anion and 1433 cm1 for the complex. Whilst it seems possible to identify other band correspondences within the experimental spectra, PED analysis does not support such associations. A relevant example being the group of 4–5 bands between 1250 and 1500 cm1 which have a similar appearance in all three spectra. However, the structure of the corresponding modes, as revealed by PED analysis seems to vary considerably from one system to another. The main conclusion to be retained from the analysis of this spectral domain is that weak interactions have marked visible consequences on the related normal modes. For instance, the cysteine anion and zwitterion conformations differ only by the value of the dihedral angle of C(7)C(8)C(10)S (169.778 and 66.778, respectively) (Figs. 2 and 3 and Table 4). It is thus likely that the marked differences between their Raman bands below 1500 cm1 are due to this conformational change. The conformation of the cysteine ligand in the Zn(Cys)2 complex is very similar to that of cysteine zwitterion except for the rotation of the CO2 group around the C(7)–C(8) bond (Table 4). Hence, in this case the observed spectral differences should be attributed to cysteine–metal interactions rather than to geometrical modifications. It is thus interesting to note that
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for the normal modes situated beyond 500 cm1, the PED weights of metal–ligand coordinates are quite negligible compared to those of the internal coordinate of the cysteine. Nevertheless, it seems these contributions are important enough to affect the coupling of the internal mode of the cysteine in this spectral range. It is worth highlighting the fact that if the conformational and metal–ligand interaction effects were negligible small, the cysteine anion and the Zn(Cys)2 complex would have identical Raman spectra between 500 and 3200 cm1.
zwitterion and cysteine anion were also found in the 500– 1500 cm1 spectral range. They were interpreted as effects of metal–ligand interactions upon the internal mode coupling of cysteine. The present work reveals that the modifications on the Raman spectra of cysteine upon binding with zinc are complex, thus providing useful information with respect to zinc–cysteine interaction. This is an interesting result showing that the Raman spectroscopy analysis on metal–ligand interactions in a biological environment can go beyond the simple detection of a specific metal–ligand band.
3.5. CH and CH2 bond stretching Acknowledgments These modes are completely decoupled from other internal coordinates and appear in the experimental spectra as a three band group. The first one positioned at 2844 cm1 (cysteine zwitterion), 2851 cm1 (Zn(Cys)2) and 2837 cm1 (cysteine anion) exhibits a weak intensity and was assigned to CH bond stretching. The other two, which show increased intensity were assigned to the ns(CH2) and na(CH2) modes. Although in the case of the complex the calculations predict an inversion between the n(CH) and ns(CH2) bands, the close similarity between the experimental bands of the three systems seems to invalidate this prediction. Moreover, assigning the first band in the group to n(CH) is in agreement with the results of Faget et al. [24] reported for the [Cd(Cys)Cl2]2 complex. Despite their supposed complete decoupling with respect to the other internal modes, the C–H stretching Raman bands in Fig. 5 are affected by the complex formation as well as by the cysteine deprotonation: a slight broadening and a red shift of these bands are observed for both the complex and the cysteine anion. 4. Summary and conclusion An experimental technique based on a pulsed Nd-YAG laser and a gated CCD camera allows us to detect in the 200– 3200 cm1 spectral range almost all theoretically predicted Raman bands of Zn(Cys)2 complex, cysteine zwitterion and cysteine anion in aqueous solutions. Spectra analysis directly demonstrate the deprotonation of the SH and NH3+ cysteine groups upon zinc binding. Spectral evidence for the CO2 group participating in the zinc coordination is also found. These results are consistent with a hexacoordination of the metal in the zinc–cysteine complex. Two specific metal–ligand bands were identified at 296 and 334 cm1 in the Raman spectrum of the complex. PED analysis indicates the corresponding modes are not pure metal–ligand modes but they include significant contributions from the cysteine internal coordinates. On this basis, it is suggested that in the case of metal-protein complexes the metal–ligand Raman bands are very sensitive to the configuration of the complex. Not only is the mode coupling highly sensitive to the geometry of the complex but the force constant for a specific metal– ligand atom bond is dependent on both the number and nature of the remaining coordinating atoms. Significant differences between Raman spectra of Zn(Cys)2 complex, cysteine
The authors thank the Conseil Re´gional de Franche´ Comte´ for its financial support in developing this project. The authors also thank Dr. Christophe Mavon for his technical assistance. References [1] B.L. Vallee, in: I. Bertini, C. Luchinat, W. Maret, M. Zeppezauer (Eds.), Zinc Enzymes, Birkhauser, Boston, 1986. [2] J.M. Berg, Y. Shi, Science 271 (1996) 1081. [3] J.M. Berg, H.A. Goodwin, Annu. Rev. Biophys. Biomol. Struct. 26 (1997) 357. [4] R. Jernigan, G. Raghunathan, I. Bahar, Curr. Opin. Struct. Biol. 4 (1994) 256. [5] D.L. Rabenstein, S.A. Daignault, A.A. Isab, A.P. Arnold, M.M. Shoukry, J. Am. Chem. Soc. 107 (1985) 6435. [6] B.L. Vallee, K.H. Falchuk, Physiol. Rev. 73 (1993) 79. [7] T.E. Creighton, Proteins, 2nd ed., W.H. Freeman & Co., New York, 1983. [8] P. Gockel, H. Vahrenkamp, A.D. Zuberbu¨hler, Helv. Chim. Acta 76 (1993) 511. [9] M. Belcastro, M.N. Russo, M.J. Toscano, Mass Spectrom. 40 (2005) 300. [10] D.L. Rousseau, D. Li, S.-R. Yeh, J. Inorg. Biochem. 99 (2005) 306. [11] P.R. Carey, J. Dong, Biochemistry 43 (2004) 8885. [12] H. Li, G.J. Thomas Jr., J. Am. Chem. Soc. 113 (1991) 456. [13] S.W. Raso, P.L. Clark, C. Haase-Pettingell, J. King, G.J. Thomas Jr., J. Mol. Biol. 307 (2001) 899. [14] R. Tuma, S. Vohnik, H. Li, G.J. Thomas Jr., Biophys. J. 65 (1993) 1066. [15] S.I. Gorelsky, L. Basumallick, J. Vura-Weis, R. Sarangi, K.O. Hodgson, B. Hedman, K. Fujisawa, E.I. Solomon, Inorg. Chem. 44 (2005) 4947. [16] T. Miura, T. Satoh, H. Takeuchi, Biochim. Biophys. Acta 171 (1998) 1384. [17] M. Vargek, X. Zhao, Z. Lai, G.L. McLendon, T.G. Spiro, Inorg. Chem. 38 (1999) 1372. [18] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukada, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Know, H.P. Hratchian, J.B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenburg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian 03, Revision B.04, Gaussian, Inc., Pittsburgh, PA, 2003. [19] A.D. Becke, Chem. Phys. 98 (1993) 5648. [20] C. Lee, W. Yank, R.G. Parr, Phys. Rev. 98 (1988) 785. [21] M. Cossi, G. Scalmani, N. Rega, V. Barone, J. Chem. Phys. 117 (2002) 43.
S. Foley, M. Enescu / Vibrational Spectroscopy 44 (2007) 256–265 [22] M. Halls, B. Schlegel, J. Chem. Phys. 111 (1999) 8819. [23] C.M. Gorbitz, B. Dalhus, Acta Crystallogr. Sect. C, Cryst. Struct. Commun. 52 (1996) 1756. [24] O.G Faget, J. Felcman, T. Giannerini, S.C.A. Te´llez, Spectrochim. Acta A 61 (2005) 2121.
265
[25] G.B. Ferreira, N.M. Comerlato, J.L. Wardell, E. Hollauer, J. Braz. Chem. Soc. 15 (2004) 951. [26] H. Shindo, T.L. Brown, J. Am. Chem. Soc. 87 (1965) 1940. [27] M. Ikram, D.B. Powell, Pakistan J. Scientific Res. 25 (1973) 53. [28] H. Li, C.J. Wurrey, G.J. Thomas Jr., J. Am. Chem. Soc. 114 (1992) 7463.
A Raman spectroscopy and theoretical study of zinc–cysteine complexation Sarah Foley, Mironel Enescu * University of Franche-Comte, Laboratoire de Microanalyses Nucleaires, UMR CEA E4,16 route de Gray, 25030 Besancon, France Received 27 September 2006; received in revised form 4 December 2006; accepted 8 December 2006 Available online 16 December 2006
Abstract The Raman spectrum of the 1:2 zinc–cysteine complex in aqueous solution is compared to those of cysteine zwitterion and cysteine anion in order to identify specific complexation effects. Band assignment is based on frequency calculation and normal modes analysis using the density functional theory (DFT) method. Two bands specific to the complex were detected in the 200–400 cm1 spectral range. It is shown that the corresponding vibrations are not pure metal–ligand modes but that they result from the coupling between the Zn–S and Zn–N stretching modes with some cysteine internal modes. Raman spectra analysis also provides direct evidence for the deprotonation of the SH and NH3+ groups of cysteine upon zinc binding. It is found in addition that complexation significantly affects the cysteine internal mode mixing in the 500–1500 cm1 spectral range. The results are considered in connection with the spectral characterization of zinc–protein complexes of biological interest. # 2006 Elsevier B.V. All rights reserved. Keywords: Cysteine; Zinc; Metal–ligand bands; DFT calculations; Potential energy distribution
1. Introduction Among the first row transition metals, zinc is second only to iron in terms of abundance and importance in biological systems [1]. The Zn2+ cation can play a structural as well as a catalytical role in proteins. The zinc finger family are the most well studied proteins in which zinc plays a structural role, since it is known that these proteins are involved in nucleic acid binding and gene regulation [2,3]. In addition to its structural role, zinc serves an essential role in many enzymes and virtually all aspects of metabolism. One of the most commonly found ligands in zinc catalytic binding sites is cysteine, with others being histidine, aspartic acid and glutamic acid [4–6]. The thiol or sulphydryl (S–H) groups of cysteine are the most chemically reactive sites in proteins at physiological conditions [7] and a preferred ligand for the Zn2+ cation. The interaction between zinc and cysteine residue is complex since the cysteine is potentially a multidentate ligand. The NH and CO groups of cysteine may be involved in complexation, as well as the COO and NH3+ groups in the case of terminal cysteines. Indeed,
* Corresponding author. Tel.: +33 3 81 66 65 21; fax: +33 3 81 66 65 22. E-mail address: [email protected] (M. Enescu). 0924-2031/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vibspec.2006.12.004
potentiometric measurements show that the most stable complex of Zn2+ cation with cysteine zwitterion HS–CH2– CH(NH3+)–COO is a 1:2 complex in which the metal is coordinated by the terminal S, N and O of both cysteines [8]. On the other hand, a recent theoretical study indicates the combination of the three coordination centers of the unpolar form of cysteine HS–CH2–CH(NH2)–COOH provides the formation of metallocomplexes of various types with monodentate, bidentate or tridentate configurations [9]. Raman spectroscopy has been demonstrated to be a valuable method for the study of the ligand–protein interactions in both solution [10] and the crystalline state [11]. Spectral problems arising from solvent water molecules are also much less restrictive in Raman spectroscopy than in IR absorption spectroscopy. Fortunately, the S–H stretching Raman band of cysteine has the advantage of occuring within the spectral range 2500–2600 cm1, which is far removed from other Raman bands of proteins. Many Raman spectroscopic studies of cysteine have concentrated on this spectral zone since the S–H stretching band is known to be an environmentally sensitive probe [12–14]. Although the S–H stretching band is an useful probe, the low frequency spectral range can provide interesting information about the structure of metalloprotein complexes [15–17]. In the case of multiple coordinated metal complexes
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band assignment is difficult since a specific metal–ligand band is expected to be sensitive to both the number and nature of all ligand groups participating in the complex. Moreover, the metal–ligand vibrational modes may be considerably mixed with the proper ligand modes. Hence, detailed analysis on zinc– cysteine model complexes is a prerequisite for studying zinc– protein complexes. The aim is to better understand the way zinc–ligand interactions affect the Raman spectrum of cysteine and to identify specific markers of these interactions. It is also preferential to perform this investigation in aqueous solution since the Raman spectra of a crystalline sample could be distorted by intermolecular interaction especially in the low frequency spectral range. In the present paper we report a detailed analysis of the Raman spectrum and the related metal–ligand interactions for the zinc–cysteine complex Zn(Cys)2 in aqueous solution. To the best of our knowledge, this is the first time that this Raman spectrum is reported. The complexation effects are identified by comparing the Raman spectrum of the complex with those of cysteine zwitterion and cysteine anion S–CH2–CH(NH2)– COO in the 200–3200 cm1 spectral range. Band assignments are based on vibrational frequency calculations using the density functional theory (DFT) method. The normal modes were further analyzed by calculating their potential energy distributions (PED) with respect to the internal coordinates. The complexity of the spectral changes induced par zinc– cysteine interaction is emphasised and carefully analysed. 2. Methods 2.1. Experimental L-Cysteine (>98%) and zinc chloride (98%) obtained from Sigma–Aldrich were used to record the Raman spectra of Zn(Cys)2 complex. The spectra here reported were obtained on aqueous solutions with a metal/ligand ratio of 1:2 prepared for cysteine concentrations of 50 and 100 mM. The pH of all solutions was adjusted to 7.0 by the dropwise addition of 2 M NaOH. A solution with the same metal/ligand ratio but with a cysteine concentration of 5 mM was also prepared in order to test the spectral stability with respect to the complex concentration. In this case, the weak Raman bands were less resolved but the strong bands showed the same position and relative intensity as for the more concentrated solutions. We concluded that the Zn(Cys)2 Raman spectrum was stable in the tested concentration range. The Raman spectra of cysteine zwitterion and cysteine anion were obtained by measuring 50 and 100 mM cysteine aqueous solutions prepared at pH 7.0 for zwitterion and at pH 11 for the anion. The Raman spectroscopy experimental set up includes a Spectra Physics Nd:YAG laser model LAB-170-10 delivering pulses with a duration of about 5 ns at a repetition rate of 10 Hz. The spectra were recorded using the second harmonic emission wavelength (532 nm). The samples were placed in a 1 cm 1 cm quartz cell and were irradiated with a 10 mm diameter laser beam at an equivalent power density of 35 mW mm2. The scattered light was detected at 908 using
257
a Roper Scientific spectroscopy system including a Spectra Pro 2500i monochromator with a maximum resolution of 0.035 nm and a PIMAX-1024-RB CCD camera (Princeton Instruments). The camera intensifier was synchronously gated over the laser pulse duration in order to maximize the signal to noise ratio. The Rayleigh scattered light was eliminated using a notch filter with a 300 cm1 bandwidth and an optical density at 532 nm of 6. The system wavelength calibration was tested by detecting the N2 Raman band at 2331 cm1 which was reproduced with an error of less than 1 cm1. All spectra were acquisitioned over 10,000 laser pulses. The sample stability with respect to laser exposure was tested by repeating the acquisition sequence two or three times. No spectral changes were detected. Given the fact that the monochromator free spectral range was only 360 cm1, the analyzed spectral range (200–3200 cm1) was explored using many acquisition steps with the solution being renewed every step. The intensity detected at one step was corrected with respect to that detected at the preceding step in order to provide continuity at the boundary between adjacent spectral intervals. Usually these corrections were of the order of 1–3%. For each spectral interval, the solvent scattered light was also determined by alternatively measuring a pure water reference. The global reference spectrum, also corrected for boundary discontinuities, was then subtracted from the global sample spectrum. The base line of the difference spectrum was corrected by slightly adjusting the intensity ratio of the sample and reference spectra. This normalization procedure provides very reproducible relative intensities of the Raman bands. The analyzed spectral range was mainly limited by the very intense water Raman bands below 200 and over 3200 cm1. The wavenumbers of Raman bands were reproducible within 1 cm1. 2.2. Computational method All calculations were performed using the Gaussian 03 program [18] on a Windows-XP operating PC. Full geometry optimization for the all molecules were carried out by the DFT method using Becke’s three parameter hybrid functional combined with the Lee–Yang–Parr correlation functional (B3LYP) [19,20] with the 6-31+G(d) basis set. Vibrational frequencies were then computed at the same level of theory. All calculations were performed in aqueous solution using the polarizable continuum model (PCM) [21]. For each normal mode, the potential energy distribution (PED) with respect to the internal coordinates was calculated as follows: PED ð%Þ ¼
k j j S2ji kii Q2i
100
(1)
where kjj is the force constant for the internal coordinates j and Sji is the projection on this internal coordinate of the normal mode i having the amplitude Qi and the force constant kii. The Gaussian program gave directly the Sji and kii values while the force constant kjj were calculated by numerical differentiation of the potential energy.
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calculations involve third order energy derivative calculations. Hence, accurate Raman intensity calculations require significantly larger basis set with respect to frequency calculations [22]. Obviously, in the present study the choice of the basis set was strongly limited by the size of the zinc–cysteine complex. 3. Results and discussion
Fig. 1. Optimized structure of the Zn(Cys)2 complex water at the B3LYP/631+G(d) level of theory. Atomic distances are given in Angstroms.
With respect to the calculated Raman intensities, their agreement with the experimental intensities in the very low and very high frequency domains was unsatisfactory. This result is not very surprising since it is known that Raman intensity
In aqueous solutions Zn2+ cation and cysteine form both 1:1 and 1:2 complexes. Using the corresponding stability constants as determined by potentiometric measurements [8] one finds that in the concentration range used here and at pH 7 more than 95% of cysteines are bound to zinc as the Zn(Cys)2 complex. Potentiometric measurements also showed that at pH 7 cysteine loses two protons upon zinc binding. Obviously, the two groups susceptible to release these protons are the SH and the NH3+ groups which are protonated in the case of free cysteine solutions at neutral pH. This result indicates that the S and N atoms of the two cysteines are bound to the metal. On the other
Table 1 Experimental and calculated (not scaled) frequencies and potential energy distribution of Raman active normal modes of the cysteine zwitterions in aqueous solution No. crt
Calculateda
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
75 81 135 177 260 312 331 484 505 576 666 770 790 850 905 977 1061 1074 1119 1238 1301 1330 1369 1398 1461 1475 1595 1644 1657 2412 3013 3074 3148 3262 3308 3348
(12) (8.9) (3.2) (3.4) (3.2) (3.3) (4.5) (23) (14) (29) (100) (55) (9.8) (34) (31) (34) (8.7) (12) (23) (90) (14) (17) (52) (50) (38) (38) (63) (49) (22) (1198) (934) (890) (448) (762) (102) (271)
Experimentala
283 (29) 345 475 529 622 684 780 814 875 935 994 1064 1115 1140 1213 1276 1311 1345 1399 1430 1513 1614 1647
(26) (19) (26) (39) (100) (36) (25) (34) (30) (19) (20) (16) (17) (21) (14) (26) (46) (43) (35) (9.7) (18) (14)
2581 2837 2959 3001
(61) (10) (58) (31)
The band intensities normalized to 100 for the C–S band are given in parentheses. a in cm1. b n: stretch; d: deformation; z: rocking; g: waging; t: torsion; a: antisymmetric; s: symmetric. c PED (%).
Assignment tb(CO2) (100c) t(CH2SH) (100) t(NH3+) (100) db(C–C–S) (42), d(C–C–C) (38) d(C–C–C) (50), gb(CO2) (16) d(C–C–N) (20), t(CSH) (25) d(C–C–N) (47), t(CH2) (20) d(C–C–N) (42), d(C–C–S) (27), d(C–C–H) (17) zb(CO2) (50), nb(CN) (10) n(CC) (25), d(CO2) (23), d(C–C–N) (16) n(CS) (70), d(C–C–S) (13) z1(CH2) (26), n(CH) (21), d(C–S–H) (16), z2(CH2) (13) d(C–S–H) (33), d(CO2) (30), z2(CH2) (16) d(CO2) (24), n(CC) (20), z(NH3+) (16) n(CC) (36), n(CN) (32), d(C–S–H) (17), z(NH3+) (14) z(NH3+) (45), d(C–S–H) (32), n(CC) (15), z2(CH2) (14) z(NH3+) (34), n(CN) (15), d(C–S–H) (14), n(CC) (10) n(CN) (56), z(NH3+) (32) z(NH3+) (44), d(C–C–H) (32) d(C–C–C) (48), z2(CH2) (41) g(CH2) (52), n(CC) (22), d(C–C–H) (18) d(C–C–H) (30), g(CH2) (30), nsb(CO2) (28) d(N–C–H) (71) ns(CO2) (34), d(N–C–H) (24), n(CC) (19), d(CO2) (11) d(CH2) (78), d(NH3+) (15) g(NH3+) (38), d(NH3+) (30), d(CH2) (28) d(NH3+) (77) nab(CO2) (95) d(NH3+) (95) d(SH) (77) n(CH) (100) ns(CH2) (100) na(CH2) (100) ns(NH3+) (100) na1(NH3+) (100) na2(NH3+) (100)
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hand, our DFT optimization of a system formed by a Zn2+ cation and two cysteine anions shows that the stable configuration corresponds to the hexacoordination of the zinc cation (Fig. 1) including the participation of two O atoms from the two carboxylate groups. To the best of our knowledge, no structural data have been reported for this complex, either in aqueous solution or in the solid state. The interest in performing a Raman spectroscopy study on this system is to provide direct evidence for this haxacoordination and to emphasize the nature of the related spectral features. In theory, Zn complexation to cysteine could induce three kinds of effects on the vibrational spectrum of cysteine: those due to the formation of new bonds between the metal atom and the ligand atoms (S, N or O), those due to the deprotonation of the cysteine and those due to the coupling of the internal vibrational modes of cysteine via metal–ligand interactions. In order to identify the above effects we have compared experimental Raman frequencies with the calculated Raman frequencies for cysteine zwitterion (Table 1), cysteine anion (Table 2) and the Zn(Cys)2 complex (Table 3). Regarding the choice of the molecular conformations used in frequency calculation, it is worth noting that in the case of the zinc– cysteine complex only one isomer (Fig. 1) is compatible with that of zinc hexacoordination. In the case of the cysteine
259
zwitterion we have reoptimized the isomer corresponding to the X-ray diffraction structure of L-cysteine [23]. The calculated structure (Fig. 2 and Table 4) was in good agreement with the experimental one. In the case of cysteine anion the only stable structure is that for which the negatively charged carboxylate and thiolate groups point in opposite directions (Fig. 3). Among the three systems analyzed here only Zn(Cys)2 possesses point group symmetry: it belongs to the Ci point group hence all Raman active normal modes are Ag modes while the IR active normal modes are Au modes. 3.1. S–Zn, N–Zn and O–Zn bond stretching The intrinsic vibrational frequencies of these stretching modes can be evaluated with the simple harmonic oscillator formula: sffiffiffiffi 1 k n¼ (2) 2p m where k is the force constant and m is the reduced mass of the biatomic system formed by the ligand atom and the Zn atom. The actual force constants were obtained for the Zn(Cys)2 complex by calculating the second order derivatives of the potential
Table 2 Experimental and calculated (not scaled) frequencies and potential energy distribution of Raman active normal modes of the cysteine anion in aqueous solution No. crt
Calculateda
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
32 126 195 221 273 350 423 524 641 680 801 843 880 933 1001 1112 1172 1248 1252 1342 1354 1408 1484 1545 1631 2922 3032 3109 3406 3485
(6.8) (1.9) (2.1) (4.8) (5.8) (7.1) (1.6) (12) (10) (100) (8.2) (15) (91) (34) (25) (17) (8.7) (50) (8.5) (59) (22) (46) (67) (44) (27) (919) (619) (390) (823) (273)
Experimentala
290 (8.5) 330 (12) 414 (14) 541 625 683 792 834 911 1037 1065 1090 1151 1203 1239 1307 1349 1413 1428 1567 1658 2836 2915 2942
(32) (33) (100) (25) (33) (39) (16) (54) (17) (7.8) (10) (8.6) (16) (33) (22) (28) (7.9) (5.2) (16) (39) (36)
The band intensities normalized to 100 for the C–S band are given in parentheses. a in cm1. b n: stretch; d: deformation; z: rocking; g: waging; t: torsion; a: antisymmetric; s: symmetric. c PED (%).
Assignment tb(CO2) (100c) t(CH2S) (100) Collective db(C–C–H) (48), d(C–C–S) (26), t(NH2) (26) t(NH2) (80), nb(CS) (10), n(CC) (10) t(CH2S) (39), d(C–C–H) (32), t(NH2) (28) t(NH2) (95) zb(CO2) (41), t(NH2) (15) d(CO2) (29), n(CC) (17), n(CS) (10) n(CS) (69), d(C–C–S) (15) d(CO2) (47), gb(CO2) (10) z1(CH2) (37), z2(CH2) (22), n(CN) (21) n(CC) (27), g(NH2) (20), d(CO2) (14) n(C–C–C) (47), g(NH2) (18) z1(CH2) (53), g(NH2) (23), n(CN) (23) n(CC) (34), n(CN) (33), d(C–N–H) (14) z2(CH2) (45) g(CH2) (100) d(C–C–H) (38), z(NH2) (30), d(C–CH2) (17) d(N–C–H) (21), d(C–C–H) (21), z(CH2) (21), z(NH2) (13) d(N–C–H) (49), d(C–C–H) (49) z(NH2) (28), ns(CO2) (27), n(CC) (20), d(C–C–H) (20) d(CH2) (95) na(CO2) (60), d(NH2) (40) na(CO2) (54), d(NH2) (20), g(NH2) (17) n(CH) (100) nsb(CH2) (100) nab(CH2) (100) ns(NH) (100) na(NH) (100)
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Table 3 Experimental and calculated (not scaled) frequencies and potential energy distribution of Raman active normal modes of the Zn(Cys)2 complex in aqueous solution No. crt
Calculateda
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
75 97 135 146 188 238 316 383 420 560 588 653 719 799 864 911 953 1035 1067 1162 1194 1280 1336 1345 1419 1492 1592 1639 3012 3036 3090 3350 3427
(4) (8) (4) (10) (13) (12) (14) (27) (14) (16) (6) (100) (22) (43) (15) (11) (26) (26) (25) (32) (53) (51) (44) (45) (72) (69) (41) (26) (1162) (1464) (662) (1207) (478)
Experimentala
296 334 399 532 569 685
(119) (118) (61) (38) (39) (100)
805 849 920 968
(28) (21) (3) (19)
1058 1190 1218 1256 1301 1355 1405 1433 1596 1659 2851 2930 2967
(20) (16) (13) (14) (23) (24) (31) (22) (15) (12) (10) (38) (26)
Assignment [nsb(S–Zn–S)–ns(O–Zn–O)](51), tb(NH2) (49c) t(CH2S) (49), t(CO2) (53), ts(S–Zn–S) (17) ns(N–Zn–N) (63), t(CO2) (34), t(CH2S) (29) ns(O–Zn–S) (52), ns(N–Zn–N) (29), t(CH2S) (18) ns(N–Zn–N) (70), t(CH2S) (17), ns(O–Zn–O) (12) db(C–C–C) (68), zb(NH2) (25) d(C–C–C) (50), ns(S–Zn–S) (20), ns(O–Zn–O) (20), ns(N–Zn–N) (20) d(N–C–C) (37), ns(N–Zn–N) (24), t(NH2) (18), d(S–C–C) (16) z(CO2) (53), t(NH2) (12) t(NH2) (29), n(CC) (25), d(CO2) (15) t(NH2) (95) n(CS) (62), d(C–C–O) (12) d(CO2) (15), n(CS) (14), d(N–C–C) (14) d(C–C–C) (16), d(CO2) (13), n(CC) (12), d(S–C–C) (11) d(CO2) (33), n(CC) (25), gb(NH2) (20) z1(CH2) (38), g(NH2) (21) n(CC) (70), d(C–N–H) (22), d(C–C–H) (13) g(NH2) (45), d(NH2) (45) n(CN) (57), n(CC) (18), d(S–C–H) (13) z(NH2) (54), d(C–C–H) (22), d(S–C–H) (27) d(C–C–C) (83), d(S–C–H) (27) g(CH2) (60), d(C–C–N) (25), z(NH2) (16) d(C–C–H) (48), ns(CO2) (19), z(NH2) (16) d(C–C–H) (67), g(CH2) (12) ns(CO2) (51), n(CC) (28), d(CO2) (16) d(CH2) (95) nab(CO2) (96) d(NH2) (73), g(NH2) (27) n(CH) (98) ns(CH2) (97) na(CH2) (96) ns(NH2) (95) na(NH2) (95)
The band intensities normalized to 100 for the C–S band are given in parentheses. a in cm1. b n: stretch; d: deformation; z: rocking; g: waging; t: torsion; a: antisymmetric; s: symmetric. c PED (%).
energy with respect to the corresponding bond lengths. Finally, we obtained the following intrinsic vibrational frequencies: n(SZn) = 150 cm1, n(OZn) = 282 cm1 and n(NZn) = 450 cm1. These are, of course, fictive frequencies because in the complex the reduced mass of the related normal modes will be modified. Indeed, the stretching of the ligand–metal bonds within the complex is expected to be associated with significant relative and/or internal motions of the two cysteines. Such motions are particularly important in the case of Raman active modes which are all symmetrical. During a symmetrical vibration the Zn is fixed, thus the Zn–S(N,O) bond stretching involves a significant displacement of the ligands. The effect is an increase in the reduced mass of the stretching modes and a lowering of the corresponding vibrational frequencies. Accordingly, the calculated Raman spectrum for the Zn(Cys)2 complex (Table 3) shows the participation of the Zn–S, Zn–O and Zn–N bond stretching in several modes between 75 and 383 cm1. Of these modes two were detected in the experimental spectrum at 296 and 334 cm1 (Fig. 4c). The participation of the metal–ligand bond stretching in these bands is supported by the theoretical assignment and the PED analysis in Table 3. Moreover, the experimental spectrum
for cysteine anion (Fig. 4c) shows only weak Raman bands within the same spectral region thus confirming that the bands situated at 296 and 334 cm1 are related to the ligands interaction with the zinc. An equivalent enhancement of the Raman bands in the 200–300 cm1 spectral range induced by metal binding has also been reported by Faget et al. [24] who investigated a cysteine dichloride cadmium complex. Zinc binding to a zinc finger peptide was found to give rise to a Raman band at 242 cm1 that was attributed to the Zn–N(His) bond stretching, another band situated at 311 cm1 was attributed to the Zn–S stretching [16]. A similar frequency (306 cm1) of the Zn–S stretching was observed for a Zn(dmit)2 complex with tetrahedral zinc coordination (here dmit is 1,3-dithiol-2-thione-4,5-dithiolate ligand) [25]. The present calculations predict significant differences between the Raman and the IR spectrum of the Zn(Cys)2 complex only in the frequency domain below 300 cm1 (see Table 5). Obviously, a comparison between Raman and IR experimental spectra in this spectral domain would be very useful for testing the reliability of our theoretical results. Unfortunately, to the best of our knowledge, no IR spectra in
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Fig. 3. Optimized structure of cysteine anion in water at the B3LYP/6-31+G(d) level of theory.
3.2. Cysteine deprotonation effects Fig. 2. Optimized structure of cysteine zwitterion in water at the B3LYP/631+G(d) level of theory.
this spectral range has been reported for Zn(Cys)2 complex in aqueous solution. For the solid-state complex, it appears that only two IR spectra have been reported so far [26,27]. However, since the reported IR bands are situated above 800 and 500 cm1, respectively, they are not particularly relevant with respect to the present discussion. Interestingly, the PED analysis in Table 3 indicates that the two experimental bands attributed to zinc–ligand interaction include significant contributions from the cysteine internal modes. With respect to the frequency domain below 200 cm1, our calculations predict highly mixed Zn–S, Zn–O and Zn–N stretching bands. One exception is the mode at 188 cm1 having a very dominant contribution from the Zn–N stretching. The present analysis suggests that in the case of metal– protein complexes it is rather improbable to identify metal– ligand Raman bands that are super-imposable from one complex to another without significant alterations due to mode mixing.
The spectacular reduction of the n(SH) band intensity at 2581 cm1 (Fig. 5c) directly demonstrates thiol deprotonation. Comparing the corresponding band intensities in Fig. 5a and c indicates for the measured zinc–cysteine solution the fraction of non-complexed cysteine molecules is negligible. One also notes the calculated n(SH) frequency is lower by about 169 cm1 with respect to the experimental value suggesting the
Table 4 Optimized geometries of the cysteine zwitterion, cysteine anion and Zn(Cys)2 complex: comparison between the main internal parameters
S–C(10) S–C(10)–C(8) S–C(10)–C(8)–C(7) N–C(8) N–C(8)–C(7) N–C(8)–C(10)–S O(2)–C(7) O(2)–C(7)–C(8) O(2)–C(7)–C(8)–C(10) S–Zn S–Zn–S0 N4–Zn N–Zn–N0 O(2)–Zn O(2)–Zn–O(2)0
Cysteine zwitterion
Cysteine anion
Zn(Cys)2
˚ 1.843 A 114.988 66.768 ˚ 1.505 A 108.288 56.858 ˚ 1.262 A 115.818 136.438
˚ 1.862 A 114.468 169.778 ˚ 1.480 A 108.908 65.648 ˚ 1.273 A 114.518 133.98
˚ 1.854 A 113.228 64.948 ˚ 1.478 A 109.298 55.078 ˚ 1.258 A 116.688 91.108 ˚ 2.620 A 180.008 ˚ 2.171 A 180.008 ˚ 2.221 A 180.008
The geometries were optimised at the B3LYP/6-31+G(d) level of theory in water.
Fig. 4. Raman spectra of aqueous solutions of cysteine zwitterion (a), cysteine anion (b) and Zn(Cys)2 complex (c) in the spectral range 200–850 cm1. Bands labeling as indicated in Tables 1–3, respectively. Every spectrum was normalized for a S–C stretching band intensity of unit. All spectra were obtained for an equivalent cysteine concentration of 100 mM.
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Table 5 Calculated (not scaled) frequencies and potential energy distribution of IR active normal modes of the Zn(Cys)2 complex in aqueous solution No. crt 0
Calculated a
1 20 30 40 50 60 70 80 90 100
25 70 81 106 122 156 169 233 274 318
(10) (26) (8) (103) (30) (247) (50) (5) (60) (96)
110
383 (22)
120 130 140 150 160 170
421 560 597 654 720 800
(24) (25) (192) (30) (37) (29)
180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360
865 912 953 1031 1065 1161 1194 1284 1337 1345 1419 1492 1589 1639 3012 3036 3090 3356 3427
(58) (28) (13) (466) (186) (46) (15) (74) (159) (78) (458) (35) (2598) (172) (34) (164) (38) (100) (133)
Assignment Dispersed Dispersed nab(S–Zn–S) (24)c, na(O–Zn–O) (24)c, tb(NH2) (40) na(S–Zn–S) (50), na(O–Zn–O) (40) t(CH2S) (48), t(CO2) (50), na(S–Zn–S) (18) na(S–Zn–S) (45), na(O–Zn–O) (50) na(O–Zn–O) (40), t(CO2) (20), t(CH2S) (20) dbC–C–C) (68), zb(NH2) (25) na(N–Zn–N) (70), d(C–C–C) (20) d(C–C–C) (50), na(S–Zn–S) (20), na(O–Zn–O) (20), na(N–Zn–N) (20) d(N–C–C) (37), na(N–Zn–N) (24), t(NH2) (18), d(S–C–C) (16) z(CO2) (53), t(NH2) (12) t(NH2) (29), n(CC) (25), d(CO2) (15) t(NH2) (95) n(CS) (62), d(C–C–O) (12) d(CO2) (15), n(CS) (14), d(N–C–C) (14) d(C–C–C) (16), d(CO2) (13), n(CC) (12), d(S–C–C) (11) d(CO2) (33), n(CC) (25), gb(NH2) (20) z1(CH2) (38), g(NH2) (21) n(CC) (70), d(C–N–H) (22), d(C–C–H) (13) g(NH2) (45), d(NH2) (45) n(CN) (57), n(CC) (18), d(S–C–H) (13) z(NH2) (54), d(C–C–H) (22), d(S–C–H) (27) d(C–C–C) (83), d(S–C–H) (27) g(CH2) (60), d(C–C–N) (25), z(NH2) (16), d(C–C–H) (48), ns(CO2) (19), z(NH2) (16) d(C–C–H) (67), g(CH2) (12) ns(CO2) (51), n(CC) (28), n(CO2) (16) d(CH2) (95) nab(CO2) (96) d(NH2) (73), g(NH2) (27) n(CH) (98) ns(CH2) (97) na(CH2) (96) ns(NH2) (95) na(NH2) (95)
The band intensities normalized to 100 for the NH2 symmetric stretch band are given in parentheses. a in cm1. b n: stretch; d: deformation; z: rocking; g: waging; t: torsion; a: antisymmetric; s: symmetric. c PED (%).
H atom coupling to the solvent is over estimated in the PCM solvation model. With respect to the N–H stretching modes (symmetric and anti-symmetric), the characteristic frequencies are greater than 3200 cm1 thus falling outside the spectral range accessible for our measurements. On the other hand, the PED analysis predicts the NH2 angular deformation participating in the normal modes in the spectral range 1450–1700 cm1 (Tables 1–3). Theoretically, the cysteine zwitterion which is protonated in N possesses three bands for which the NH2 angular vibrations are predominant, at 1475, 1595 and 1657 cm1 (Table 1). On the other hand, the Zn(Cys)2 which is supposed to be deprotonated in N possesses only one, at 1659 cm1 (Table 3). Due to the strong Raman bands of water
Fig. 5. Raman spectra of aqueous solutions of cysteine zwitterion (a), cysteine anion (b) and Zn(Cys)2 complex (c) in the spectral range 500–3200 cm1. Bands labeling as indicated in Tables 1–3, respectively. Every spectrum was normalized for a S–C stretching band intensity of unit. All spectra were obtained for an equivalent cysteine concentration of 50 mM.
which fall within the same spectral range, the experimental detection of these bands was not accurate enough when using a concentration of 50 mM cysteine (Fig. 5). We thus performed an additional analysis for a cysteine concentration of 500 mM (and, in the case of the complex, a zinc concentration of 250 mM). As can be seen in Fig. 6b the Zn(Cys)2 has only two Raman bands in the 1500–1700 cm1 spectral range in good agreement with our theoretical calculations. According to these calculations, one of these bands should be attributed to the CO2 asymmetrical stretching and the other to the NH2 angular deformation (Table 3). In addition, these bands appear to be resolved in the case of the cysteine anion even at lower concentrations (Fig. 5b). For the cysteine zwitterion we detected three bands in the 1500–1700 cm1 spectral range, at 1513, 1614 and 1647 cm1 (Fig. 6a). One of them was attributed to the CO2 asymmetrical stretching and the other two to NH2 angular vibrations (Table 1). The third NH2 angular deformation band was not resolved but it could contribute to the broad
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3.3. CO2 coordination effects
Fig. 6. Raman spectra of aqueous solutions of cysteine zwitterion (a) and Zn(Cys)2 complex (b) in the spectral range 1450–1750 cm1. Bands labeling as indicated in Tables 1 and 3, respectively. Spectra were obtained for an equivalent cysteine concentration of 500 mM.
According to our calculations, the stable configuration of the complex corresponds to a hexacoordination of zinc with CO2 groups bound to Zn2+. This binding should affect the specific modes of the CO2 group. We looked for these evidences in the Raman spectrum of the zinc–cysteine complex. The CO2 rocking is predicted at 420 cm1 (Table 3) and detected at 399 cm1 (Fig. 4c). The band at 399 cm1 appears as specific to the complex since no equivalent band is present in the cysteine zwitterion spectrum (Fig. 4a) while the anion spectrum shows only a very weak band at 414 cm1 (Fig. 4b) with a very different PED (Table 2). Instead, for the cysteine zwitterion the CO2 rocking is predicted at 505 cm1 and detected at 529 cm1 (Fig. 4a). For the anion an equivalent band is detected at 541 cm1 (Fig. 4b). Although the spectrum of the complex also presents a band in the same spectral range (at 532 cm1) this band is significantly weaker and has a different PED. We conclude that the spectral differences here identified support the assumption of a zinc–cysteine complex with zinc hexacoordination. 3.4. Mode coupling effects
band observed between 1600 and 1700 cm1. The present assignment is in good agreement with that previously reported by Li et al. [28] who studied the Raman spectrum of crystalline L-cysteine and have based their normal modes analysis on force constants obtained from structural data. The IR spectrum for the solid state of the Zn(Cys)2 complex reported by Ikram and Powell [27] also presents only two bands in the spectral domain 1500–1700 cm1, one at 1580 cm1, and the other at 1615 cm1, in satisfactory agreement with our results. On the other hand, the corresponding IR bands reported by Shindo and Brown [26] are red shifted to 1588 and 1547 cm1, respectively. This discrepancy could be due to the structural distortion induced by the two sodium cations included in their solid-state complex. Finally, the spectral differences in Fig. 6 between the cysteine zwitterion and the Zn(Cys)2 complex clearly demonstrate the deprotonation of the NH3+ group upon complexation of cysteine with zinc. An alternative marker for the deprotonation of the NH3+ group is the NH2 torsional frequency. The reason is the NH2 torsional frequency is significantly higher than that of the NH3+ group due to a stronger force constant. Quantum chemical calculations predict for the Zn(Cys)2 complex a Raman active mode at 588 cm1 that is almost a pure NH2 torsional oscillation (Table 3). A corresponding Raman band was identified in the experimental spectrum of the complex at 569 cm1. It is worth noting that no similar band was found for cysteine zwitterion: the experimental band at 529 cm1 was assigned to a mode dominated by the CO2 rocking. For the cysteine anion a mode having an important contribution from the NH2 torsional oscillation is predicted to be at 273 cm1 while the corresponding experimental band was detected at 330 cm1. The frequency shift with respect to the complex is probably a mode mixing effect (Table 2).
For the three molecular systems discussed here, PED analysis predicts the normal modes below 1500 cm1 to have a rather complex structure with the mixing of several internal coordinates. It is therefore not surprising that the intensities and positions of the corresponding experimental bands illustrate significant differences on going from one system to another despite the local structure similarities of the three systems. A remarkable exception to the above is the C–S bond stretching band whose position (682 cm1) are almost identical for the three systems. Another very reproducible band is that for CH2 deformation which appears at 1430 cm1 for the zwitterion, 1428 cm1 for the anion and 1433 cm1 for the complex. Whilst it seems possible to identify other band correspondences within the experimental spectra, PED analysis does not support such associations. A relevant example being the group of 4–5 bands between 1250 and 1500 cm1 which have a similar appearance in all three spectra. However, the structure of the corresponding modes, as revealed by PED analysis seems to vary considerably from one system to another. The main conclusion to be retained from the analysis of this spectral domain is that weak interactions have marked visible consequences on the related normal modes. For instance, the cysteine anion and zwitterion conformations differ only by the value of the dihedral angle of C(7)C(8)C(10)S (169.778 and 66.778, respectively) (Figs. 2 and 3 and Table 4). It is thus likely that the marked differences between their Raman bands below 1500 cm1 are due to this conformational change. The conformation of the cysteine ligand in the Zn(Cys)2 complex is very similar to that of cysteine zwitterion except for the rotation of the CO2 group around the C(7)–C(8) bond (Table 4). Hence, in this case the observed spectral differences should be attributed to cysteine–metal interactions rather than to geometrical modifications. It is thus interesting to note that
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for the normal modes situated beyond 500 cm1, the PED weights of metal–ligand coordinates are quite negligible compared to those of the internal coordinate of the cysteine. Nevertheless, it seems these contributions are important enough to affect the coupling of the internal mode of the cysteine in this spectral range. It is worth highlighting the fact that if the conformational and metal–ligand interaction effects were negligible small, the cysteine anion and the Zn(Cys)2 complex would have identical Raman spectra between 500 and 3200 cm1.
zwitterion and cysteine anion were also found in the 500– 1500 cm1 spectral range. They were interpreted as effects of metal–ligand interactions upon the internal mode coupling of cysteine. The present work reveals that the modifications on the Raman spectra of cysteine upon binding with zinc are complex, thus providing useful information with respect to zinc–cysteine interaction. This is an interesting result showing that the Raman spectroscopy analysis on metal–ligand interactions in a biological environment can go beyond the simple detection of a specific metal–ligand band.
3.5. CH and CH2 bond stretching Acknowledgments These modes are completely decoupled from other internal coordinates and appear in the experimental spectra as a three band group. The first one positioned at 2844 cm1 (cysteine zwitterion), 2851 cm1 (Zn(Cys)2) and 2837 cm1 (cysteine anion) exhibits a weak intensity and was assigned to CH bond stretching. The other two, which show increased intensity were assigned to the ns(CH2) and na(CH2) modes. Although in the case of the complex the calculations predict an inversion between the n(CH) and ns(CH2) bands, the close similarity between the experimental bands of the three systems seems to invalidate this prediction. Moreover, assigning the first band in the group to n(CH) is in agreement with the results of Faget et al. [24] reported for the [Cd(Cys)Cl2]2 complex. Despite their supposed complete decoupling with respect to the other internal modes, the C–H stretching Raman bands in Fig. 5 are affected by the complex formation as well as by the cysteine deprotonation: a slight broadening and a red shift of these bands are observed for both the complex and the cysteine anion. 4. Summary and conclusion An experimental technique based on a pulsed Nd-YAG laser and a gated CCD camera allows us to detect in the 200– 3200 cm1 spectral range almost all theoretically predicted Raman bands of Zn(Cys)2 complex, cysteine zwitterion and cysteine anion in aqueous solutions. Spectra analysis directly demonstrate the deprotonation of the SH and NH3+ cysteine groups upon zinc binding. Spectral evidence for the CO2 group participating in the zinc coordination is also found. These results are consistent with a hexacoordination of the metal in the zinc–cysteine complex. Two specific metal–ligand bands were identified at 296 and 334 cm1 in the Raman spectrum of the complex. PED analysis indicates the corresponding modes are not pure metal–ligand modes but they include significant contributions from the cysteine internal coordinates. On this basis, it is suggested that in the case of metal-protein complexes the metal–ligand Raman bands are very sensitive to the configuration of the complex. Not only is the mode coupling highly sensitive to the geometry of the complex but the force constant for a specific metal– ligand atom bond is dependent on both the number and nature of the remaining coordinating atoms. Significant differences between Raman spectra of Zn(Cys)2 complex, cysteine
The authors thank the Conseil Re´gional de Franche´ Comte´ for its financial support in developing this project. The authors also thank Dr. Christophe Mavon for his technical assistance. References [1] B.L. Vallee, in: I. Bertini, C. Luchinat, W. Maret, M. Zeppezauer (Eds.), Zinc Enzymes, Birkhauser, Boston, 1986. [2] J.M. Berg, Y. Shi, Science 271 (1996) 1081. [3] J.M. Berg, H.A. Goodwin, Annu. Rev. Biophys. Biomol. Struct. 26 (1997) 357. [4] R. Jernigan, G. Raghunathan, I. Bahar, Curr. Opin. Struct. Biol. 4 (1994) 256. [5] D.L. Rabenstein, S.A. Daignault, A.A. Isab, A.P. Arnold, M.M. Shoukry, J. Am. Chem. Soc. 107 (1985) 6435. [6] B.L. Vallee, K.H. Falchuk, Physiol. Rev. 73 (1993) 79. [7] T.E. Creighton, Proteins, 2nd ed., W.H. Freeman & Co., New York, 1983. [8] P. Gockel, H. Vahrenkamp, A.D. Zuberbu¨hler, Helv. Chim. Acta 76 (1993) 511. [9] M. Belcastro, M.N. Russo, M.J. Toscano, Mass Spectrom. 40 (2005) 300. [10] D.L. Rousseau, D. Li, S.-R. Yeh, J. Inorg. Biochem. 99 (2005) 306. [11] P.R. Carey, J. Dong, Biochemistry 43 (2004) 8885. [12] H. Li, G.J. Thomas Jr., J. Am. Chem. Soc. 113 (1991) 456. [13] S.W. Raso, P.L. Clark, C. Haase-Pettingell, J. King, G.J. Thomas Jr., J. Mol. Biol. 307 (2001) 899. [14] R. Tuma, S. Vohnik, H. Li, G.J. Thomas Jr., Biophys. J. 65 (1993) 1066. [15] S.I. Gorelsky, L. Basumallick, J. Vura-Weis, R. Sarangi, K.O. Hodgson, B. Hedman, K. Fujisawa, E.I. Solomon, Inorg. Chem. 44 (2005) 4947. [16] T. Miura, T. Satoh, H. Takeuchi, Biochim. Biophys. Acta 171 (1998) 1384. [17] M. Vargek, X. Zhao, Z. Lai, G.L. McLendon, T.G. Spiro, Inorg. Chem. 38 (1999) 1372. [18] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukada, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Know, H.P. Hratchian, J.B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenburg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian 03, Revision B.04, Gaussian, Inc., Pittsburgh, PA, 2003. [19] A.D. Becke, Chem. Phys. 98 (1993) 5648. [20] C. Lee, W. Yank, R.G. Parr, Phys. Rev. 98 (1988) 785. [21] M. Cossi, G. Scalmani, N. Rega, V. Barone, J. Chem. Phys. 117 (2002) 43.
S. Foley, M. Enescu / Vibrational Spectroscopy 44 (2007) 256–265 [22] M. Halls, B. Schlegel, J. Chem. Phys. 111 (1999) 8819. [23] C.M. Gorbitz, B. Dalhus, Acta Crystallogr. Sect. C, Cryst. Struct. Commun. 52 (1996) 1756. [24] O.G Faget, J. Felcman, T. Giannerini, S.C.A. Te´llez, Spectrochim. Acta A 61 (2005) 2121.
265
[25] G.B. Ferreira, N.M. Comerlato, J.L. Wardell, E. Hollauer, J. Braz. Chem. Soc. 15 (2004) 951. [26] H. Shindo, T.L. Brown, J. Am. Chem. Soc. 87 (1965) 1940. [27] M. Ikram, D.B. Powell, Pakistan J. Scientific Res. 25 (1973) 53. [28] H. Li, C.J. Wurrey, G.J. Thomas Jr., J. Am. Chem. Soc. 114 (1992) 7463.