A Raman spectroscopy and theoretical study of zinc–cysteine complexation

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Vibrational Spectroscopy 44 (2007) 256–265

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 cm�1 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 cm�1

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 mono-

dentate, 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 cm�1, 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

mailto:[email protected]

http://dx.doi.org/10.1016/j.vibspec.2006.12.004

S. Foley, M. Enescu / Vibrational Spectroscopy 44 (2007) 256–265 257

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 cm�1 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 mm�2. The scattered light was detected at 908 using

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 cm�1 bandwidth and an optical density at 532 nm of

6. The system wavelength calibration was tested by detecting

the N2 Raman band at 2331 cm�1 which was reproduced with

an error of less than 1 cm�1. 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 cm�1, the analyzed spectral range (200–3200 cm�1) 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 cm�1.

The wavenumbers of Raman bands were reproducible within

�1 cm�1.

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 correla-

tion 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 ¼k j jS

2ji

kiiQ2i

� 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.

Fig. 1. Optimized structure of the Zn(Cys)2 complex water at the B3LYP/6-

31+G(d) level of theory. Atomic distances are given in Angstroms.

S. Foley, M. Enescu / Vibrational Spectroscopy 44 (2007) 256–265258

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

Table 1

Experimental and calculated (not scaled) frequencies and potential energy distributio

No. crt Calculateda Experimentala

1 75 (12)

2 81 (8.9)

3 135 (3.2)

4 177 (3.4)

5 260 (3.2) 283 (29)

6 312 (3.3)

7 331 (4.5) 345 (26)

8 484 (23) 475 (19)

9 505 (14) 529 (26)

10 576 (29) 622 (39)

11 666 (100) 684 (100)

12 770 (55) 780 (36)

13 790 (9.8) 814 (25)

14 850 (34) 875 (34)

15 905 (31) 935 (30)

16 977 (34) 994 (19)

17 1061 (8.7) 1064 (20)

18 1074 (12) 1115 (16)

19 1119 (23) 1140 (17)

20 1238 (90) 1213 (21)

21 1301 (14) 1276 (14)

22 1330 (17) 1311 (26)

23 1369 (52) 1345 (46)

24 1398 (50) 1399 (43)

25 1461 (38) 1430 (35)

26 1475 (38) 1513 (9.7)

27 1595 (63) 1614 (18)

28 1644 (49) 1647 (14)

29 1657 (22)

30 2412 (1198) 2581 (61)

31 3013 (934) 2837 (10)

32 3074 (890) 2959 (58)

33 3148 (448) 3001 (31)

34 3262 (762)

35 3308 (102)

36 3348 (271)

The band intensities normalized to 100 for the C–S band are given in parenthesesa in cm�1.b n: stretch; d: deformation; z: rocking; g: waging; t: torsion; a: antisymmetric;c PED (%).

calculations involve third order energy derivative calculations.

Hence, accurate Raman intensity calculations require sig-

nificantly 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

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

n of Raman active normal modes of the cysteine zwitterions in aqueous solution

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: symmetric.


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

Table 2

Experimental and calculated (not scaled) frequencies and potential energy distribu

No. crt Calculateda Experimentala

1 32 (6.8)

2 126 (1.9)

3 195 (2.1)

4 221 (4.8) 290 (8.5)

5 273 (5.8) 330 (12)

6 350 (7.1) 414 (14)

7 423 (1.6)

8 524 (12) 541 (32)

9 641 (10) 625 (33)

10 680 (100) 683 (100)

11 801 (8.2) 792 (25)

12 843 (15) 834 (33)

13 880 (91) 911 (39)

14 933 (34) 1037 (16)

15 1001 (25) 1065 (54)

16 1112 (17) 1090 (17)

17 1172 (8.7) 1151 (7.8)

18 1248 (50) 1203 (10)

19 1252 (8.5) 1239 (8.6)

20 1342 (59) 1307 (16)

21 1354 (22) 1349 (33)

22 1408 (46) 1413 (22)

23 1484 (67) 1428 (28)

24 1545 (44) 1567 (7.9)

25 1631 (27) 1658 (5.2)

26 2922 (919) 2836 (16)

27 3032 (619) 2915 (39)

28 3109 (390) 2942 (36)

29 3406 (823)

30 3485 (273)

The band intensities normalized to 100 for the C–S band are given in parenthesesa in cm�1.b n: stretch; d: deformation; z: rocking; g: waging; t: torsion; a: antisymmetric;c PED (%).

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:

n ¼ 1

2p

ffiffiffiffik

m

s(2)

where k is the force constant and m is the reduced mass of the bi-

atomic 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

tion of Raman active normal modes of the cysteine anion in aqueous solution

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)

.

s: symmetric.

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 Experimentala Assignment

1 75 (4) [nsb(S–Zn–S)–ns(O–Zn–O)](51), tb(NH2) (49c)

2 97 (8) t(CH2S) (49), t(CO2�) (53), ts(S–Zn–S) (17)

3 135 (4) ns(N–Zn–N) (63), t(CO2�) (34), t(CH2S�) (29)

4 146 (10) ns(O–Zn–S) (52), ns(N–Zn–N) (29), t(CH2S�) (18)

5 188 (13) ns(N–Zn–N) (70), t(CH2S) (17), ns(O–Zn–O) (12)

6 238 (12) db(C–C–C) (68), zb(NH2) (25)

7 316 (14) 296 (119) d(C–C–C) (50), ns(S–Zn–S) (20), ns(O–Zn–O) (20), ns(N–Zn–N) (20)

8 383 (27) 334 (118) d(N–C–C) (37), ns(N–Zn–N) (24), t(NH2) (18), d(S–C–C) (16)

9 420 (14) 399 (61) z(CO2�) (53), t(NH2) (12)

10 560 (16) 532 (38) t(NH2) (29), n(CC) (25), d(CO2�) (15)

11 588 (6) 569 (39) t(NH2) (95)

12 653 (100) 685 (100) n(CS) (62), d(C–C–O) (12)

13 719 (22) d(CO2�) (15), n(CS) (14), d(N–C–C) (14)

14 799 (43) 805 (28) d(C–C–C) (16), d(CO2�) (13), n(CC) (12), d(S–C–C) (11)

15 864 (15) 849 (21) d(CO2�) (33), n(CC) (25), gb(NH2) (20)

16 911 (11) 920 (3) z1(CH2) (38), g(NH2) (21)

17 953 (26) 968 (19) n(CC) (70), d(C–N–H) (22), d(C–C–H) (13)

18 1035 (26) g(NH2) (45), d(NH2) (45)

19 1067 (25) 1058 (20) n(CN) (57), n(CC) (18), d(S–C–H) (13)

20 1162 (32) 1190 (16) z(NH2) (54), d(C–C–H) (22), d(S–C–H) (27)

21 1194 (53) 1218 (13) d(C–C–C) (83), d(S–C–H) (27)

22 1280 (51) 1256 (14) g(CH2) (60), d(C–C–N) (25), z(NH2) (16)

23 1336 (44) 1301 (23) d(C–C–H) (48), ns(CO2�) (19), z(NH2) (16)

24 1345 (45) 1355 (24) d(C–C–H) (67), g(CH2) (12)

25 1419 (72) 1405 (31) ns(CO2�) (51), n(CC) (28), d(CO2

�) (16)

26 1492 (69) 1433 (22) d(CH2) (95)

27 1592 (41) 1596 (15) nab(CO2

�) (96)

28 1639 (26) 1659 (12) d(NH2) (73), g(NH2) (27)

29 3012 (1162) 2851 (10) n(CH) (98)

30 3036 (1464) 2930 (38) ns(CH2) (97)

31 3090 (662) 2967 (26) na(CH2) (96)

32 3350 (1207) ns(NH2) (95)

33 3427 (478) na(NH2) (95)

The band intensities normalized to 100 for the C–S band are given in parentheses.a in cm�1.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 cm�1, n(OZn) = 282 cm�1 and n(NZn) = 450

cm�1. 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 calcu-

lated 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 cm�1. Of these modes two

were detected in the experimental spectrum at 296 and 334 cm�1

(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 cm�1 are related to the ligands interaction

with the zinc. An equivalent enhancement of the Raman bands in

the 200–300 cm�1 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 cm�1

that was attributed to the Zn–N(His) bond stretching, another

band situated at 311 cm�1 was attributed to the Zn–S stretching

[16]. A similar frequency (306 cm�1) of the Zn–S stretching was

observed for a Zn(dmit)2 complex with tetrahedral zinc coordi-

nation (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 cm�1 (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

Fig. 2. Optimized structure of cysteine zwitterion in water at the B3LYP/6-

31+G(d) level of theory.

Fig. 3. Optimized structure of cysteine anion in water at the B3LYP/6-31+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 cm�1, 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 cm�1,

our calculations predict highly mixed Zn–S, Zn–O and Zn–N

stretching bands. One exception is the mode at 188 cm�1

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.

Table 4

Optimized geometries of the cysteine zwitterion, cysteine anion and Zn(Cys)2

complex: comparison between the main internal parameters

Cysteine zwitterion Cysteine anion Zn(Cys)2

S–C(10) 1.843 A 1.862 A 1.854 A

S–C(10)–C(8) 114.988 114.468 113.228S–C(10)–C(8)–C(7) �66.768 169.778 �64.948N–C(8) 1.505 A 1.480 A 1.478 A

N–C(8)–C(7) 108.288 108.908 109.298N–C(8)–C(10)–S 56.858 �65.648 55.078O(2)–C(7) 1.262 A 1.273 A 1.258 A

O(2)–C(7)–C(8) 115.818 114.518 116.688O(2)–C(7)–C(8)–C(10) 136.438 133.98 91.108S–Zn 2.620 A

S–Zn–S0 180.008N4–Zn 2.171 A

N–Zn–N0 180.008O(2)–Zn 2.221 A

O(2)–Zn–O(2)0 180.008

The geometries were optimised at the B3LYP/6-31+G(d) level of theory in

water.

3.2. Cysteine deprotonation effects

The spectacular reduction of the n(SH) band intensity at

2581 cm�1 (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 cm�1 with respect to the experimental value suggesting the

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 cm�1. Bands

labeling as indicated in Tables 1–3, respectively. Every spectrum was normal-

ized for a S–C stretching band intensity of unit. All spectra were obtained for an

equivalent cysteine concentration of 100 mM.

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 Calculateda Assignment

10 25 (10) Dispersed

20 70 (26) Dispersed

30 81 (8) nab(S–Zn–S) (24)c, na(O–Zn–O) (24)c, tb(NH2) (40)

40 106 (103) na(S–Zn–S) (50), na(O–Zn–O) (40)

50 122 (30) t(CH2S�) (48), t(CO2�) (50), na(S–Zn–S) (18)

60 156 (247) na(S–Zn–S) (45), na(O–Zn–O) (50)

70 169 (50) na(O–Zn–O) (40), t(CO2�) (20), t(CH2S�) (20)

80 233 (5) dbC–C–C) (68), zb(NH2) (25)

90 274 (60) na(N–Zn–N) (70), d(C–C–C) (20)

100 318 (96) d(C–C–C) (50), na(S–Zn–S) (20), na(O–Zn–O) (20),

na(N–Zn–N) (20)

110 383 (22) d(N–C–C) (37), na(N–Zn–N) (24), t(NH2) (18),

d(S–C–C) (16)

120 421 (24) z(CO2�) (53), t(NH2) (12)

130 560 (25) t(NH2) (29), n(CC) (25), d(CO2�) (15)

140 597 (192) t(NH2) (95)

150 654 (30) n(CS) (62), d(C–C–O) (12)

160 720 (37) d(CO2�) (15), n(CS) (14), d(N–C–C) (14)

170 800 (29) d(C–C–C) (16), d(CO2�) (13), n(CC) (12),

d(S–C–C) (11)

180 865 (58) d(CO2�) (33), n(CC) (25), gb(NH2) (20)

190 912 (28) z1(CH2) (38), g(NH2) (21)

200 953 (13) n(CC) (70), d(C–N–H) (22), d(C–C–H) (13)

210 1031 (466) g(NH2) (45), d(NH2) (45)

220 1065 (186) n(CN) (57), n(CC) (18), d(S–C–H) (13)

230 1161 (46) z(NH2) (54), d(C–C–H) (22), d(S–C–H) (27)

240 1194 (15) d(C–C–C) (83), d(S–C–H) (27)

250 1284 (74) g(CH2) (60), d(C–C–N) (25), z(NH2) (16),

260 1337 (159) d(C–C–H) (48), ns(CO2�) (19), z(NH2) (16)

270 1345 (78) d(C–C–H) (67), g(CH2) (12)

280 1419 (458) ns(CO2�) (51), n(CC) (28), n(CO2

�) (16)

290 1492 (35) d(CH2) (95)

300 1589 (2598) nab(CO2

�) (96)

310 1639 (172) d(NH2) (73), g(NH2) (27)

320 3012 (34) n(CH) (98)

330 3036 (164) ns(CH2) (97)

340 3090 (38) na(CH2) (96)

350 3356 (100) ns(NH2) (95)

360 3427 (133) na(NH2) (95)

The band intensities normalized to 100 for the NH2 symmetric stretch band are

given in parentheses.a in cm�1.b n: stretch; d: deformation; z: rocking; g: waging; t: torsion; a: antisym-

metric; s: symmetric.c PED (%).

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 cm�1.

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.


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 cm�1 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 cm�1

(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 cm�1 (Table 1). On the other hand, the Zn(Cys)2 which

is supposed to be deprotonated in N possesses only one, at

1659 cm�1 (Table 3). Due to the strong Raman bands of water

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 cm�1

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 cm�1 spectral range, at 1513, 1614 and

1647 cm�1 (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

Fig. 6. Raman spectra of aqueous solutions of cysteine zwitterion (a) and

Zn(Cys)2 complex (b) in the spectral range 1450–1750 cm�1. Bands labeling as

indicated in Tables 1 and 3, respectively. Spectra were obtained for an

equivalent cysteine concentration of 500 mM.


band observed between 1600 and 1700 cm�1. 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 cm�1, one at 1580 cm�1, and

the other at 1615 cm�1, 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 cm�1, 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 cm�1 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 cm�1. It is worth noting that no similar band was found for

cysteine zwitterion: the experimental band at 529 cm�1 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 cm�1

while the corresponding experimental band was detected at

330 cm�1. The frequency shift with respect to the complex is

probably a mode mixing effect (Table 2).

3.3. CO2� coordination effects

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 cm�1 (Table 3) and detected at

399 cm�1 (Fig. 4c). The band at 399 cm�1 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 cm�1 (Fig. 4b) with a very

different PED (Table 2). Instead, for the cysteine zwitterion the

CO2� rocking is predicted at 505 cm�1 and detected at

529 cm�1 (Fig. 4a). For the anion an equivalent band is detected

at 541 cm�1 (Fig. 4b). Although the spectrum of the complex

also presents a band in the same spectral range (at 532 cm�1)

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 hexacoordi-

nation.

3.4. Mode coupling effects

For the three molecular systems discussed here, PED

analysis predicts the normal modes below 1500 cm�1 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 cm�1) are almost identical for the

three systems. Another very reproducible band is that for CH2

deformation which appears at 1430 cm�1 for the zwitterion,

1428 cm�1 for the anion and 1433 cm�1 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 cm�1 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 cm�1 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


for the normal modes situated beyond 500 cm�1, 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 cm�1.

3.5. CH and CH2 bond stretching

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 cm�1 (cysteine

zwitterion), 2851 cm�1 (Zn(Cys)2) and 2837 cm�1 (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 cm�1 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 cm�1 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

zwitterion and cysteine anion were also found in the 500–

1500 cm�1 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.

Acknowledgments

The authors thank the Conseil Regional 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. Zuberbuhler, 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.


[22] M. Halls, B. Schlegel, J. Chem. Phys. 111 (1999) 8819.

[23] C.M. Gorbitz, B. Dalhus, Acta Crystallogr. Sect. C, Cryst. Struct. Com-

mun. 52 (1996) 1756.

[24] O.G Faget, J. Felcman, T. Giannerini, S.C.A. Tellez, Spectrochim. Acta A

61 (2005) 2121.

[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

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Transcript of A Raman spectroscopy and theoretical study of zinc–cysteine complexation