- Email: [email protected]
The interaction of the VO2+ cation with oxidized glutathione
The Interaction of the V02+ Cation with Oxidized Glutathione Evelina G. Ferrer, Patricia A. M. Williams, and Enrique J. Baran Qubnica Inorghaica (QUINO@, Facultad de Ciencius Exactas, UniverskiadNaciorud de La Pliata,Argentina ABSTRACT The interaction of the vanadyl0V) cation with oxidized glutathione (GSSG) in solution has been investigated using spectrophotometric techniques. Tkvo complexes, stable at different metal-toligand ratios, could be identified at pH = 7. A solid 29 VO’+:GSSG complex could also be
isolated at ‘$H - 4.5 and characterized by chemical analysis and infrared and electronic spectroscopies. Its thermal behavior was investigated through TG and DTA measurements.
JNTRODUCTION
Glutathione (GSH) the tripeptide rcglutamyl-rccysteinyl-lglycine, has generally been regarded as the most abundant low-molecular weight thiol in a variety of cell types [1, 21 and it apparently plays an important role in the metabolism and biochemistry of vanadium [3, 41. In fact, in red cells vanadate(V) is almost quantitatively reduced to V02+ by glutathione, which can also act as a ligand for the generated vanadyI0V) cation [5-71. We have recently investigated this VO’+/GSH interaction in detail, using spectrophotometric techniques [8]. Oxidized glutathione (GSSG, Fig. 1) is produced during the biological reduction of vanadate(V) to vanadyl(IV). Therefore, it seems interesting to verify whether the V02+ cation interacts with GSSG and, eventually, to obtain more information about the generated complexes. In this paper, we present the first evidence on the formation of two different V02+/GSSG complexes in solution. Also, a solid complex was isolated and characterized.
Address reprint requests and correspondence to: Professor Dr. Enrique J. Baran, Qukica Inorgtica, Facultad de Ciencias Exactas, UNLP, Casilla de Correo %2,19CKM,a Plats, Argentina. 50,253-262 (1993) 253 0 1993Eilsevierscience Publishing Co., Inc., 655Avenue of the Americas, NY, NY 10010 0162-0134/93/$6.00
Joumal oftnogrnic Biocheti~,
254
E. G. Ferrer et al.
0 II
0 II
k02ct~c~2C~2’C~t4c~-I~~~c~2c0~ I NH2
CH2 4 5 I Cl-12
NH2 I
I
HOzCCHCHfiHzCNH .
CHCNHCHzCOzH
II 0
II 0
FIGURE 1. Sche&atic structure of oxidized glutathione (GSSG).
EXPERIMENTAL.
Oxidized glutathione and VOSO,SH,O were commercially available from Sigma and Merck, respectively. All experiments were carried out under anaerobic conditions in order to prevent oxidation phenomena. Electronic absorption spectra were obtained with a Hewlett-Packard 8452 Diode-Array spectrophotometer using quartz cells (1 cm optical path). The pH of the solutions was fixed using either 0.1 N HCl or 0.1 N NaOH. In order to avoid interactions of V02+ with other ligands, no buffers were used. The solid complex was obtained by mixing aqueous solutions, 1.25 X 10m3 M of GSSG, and 5 X 10e4 M of vanadyl(IV) sulfate in a 2.5:1 molar ratio. The pH value was adjusted to 4.5. The generated pale blue precipitate was filtered through a G4-fritted-glass funnel and immediately dried at room temperature. The infrared spectra of the solid complex and of the free ligand were obtained with a Perkin-Elmer 580 B spectrophotometer, using the KBr pellet technique. The reflectance spectra were measured with a Shimadzu UV-300 instrument, using MgO as a standard. The thermogravimetric study was performed with a Stanton 781 thermobalance under static air atmosphere. The heating rate was lO”C/min and Al,O, was used as a DTA standard. RESULTS AND DISCUSSION 1. Studies in Solution In the previous paper [8] we have shown that the interaction of reduced glutathione with V02+ strongly depends on the initial V02+:GSH ratio. Starting with a tenfold GSH excess, the coordination takes place through the two carboxylate groups of the ligand and at pH = 7 a blue 1:2 V02+/GSH complex is generated. Higher GSH concentrations produce a violet complex, which is the most stable species of this system. It can also be obtained by adding GSH to the blue species. In this violet complex, the cation apparently interacts with S and some of the N atoms of the peptide In order to verify the behavior of the V02+/GSSG system we have first investigated its dependence to the concentration ratio.
GLUTATHIONE
VO*+ CATION WITH OXIDIZED
255
0.04-
400 FIGURE
2.
VO*+/GSSG VO*+/GSSG
1
I
500
600
I ‘O” tnm I
I
_
*O”
Electronic spectra of an aqueous 8 X lo-’ M VO*+ solution (a); a (5 x 1O-3 M and 5 x lo- 2 M, respectively) solution (b); and a (5 x 10e3 M and 0.5 M, respectively) solution Cc).
Starting from 10e3M VOSO, and lo-* M GSSG solutions in a l&l GSSG:VO*+ ratio and at pH = 7, we obtained the spectrum depicted in Figure 2b (cf. Table 1 also). The higher energy band, which lies at 625 nm in [VO(H,O),]*+ (Fig. 2a), is clearly split in the VO*+/GSSG solution. Some noticeable changes are produced in some of the spectral bands by adding more ligand to the solution; increasing the GSSG:VO*+ ratio. At a 1oO:l ratio, the spectrum of Figure 2c is obtained. It can be seen that the ‘highest and lowest energy bands are clearly displaced to higher energies, whereas the third band remains practically unchanged (cf. Table 1 and Fig. 2). In ,order to obtain a wider insight into the stoichiometry of the blue complex formed at a l&l GSSG/VO*+ ratio, we have performed a spectrophotometric titration [lo], monitoring the absorbance changes as a function of the metal-toligand ratio at constant wavelength (818 nm). One of those experiences is reproduced in Figure 3. The results point clearly to the formation of a 21 GSSG/VO*+ complex. This complex seems to be more stable than the 2:l complex formed with reduced glutathione because in this case the ligand-to-metal ratio can be reduced to 0.5 without VO(OH), precipitation. In the case of GSH, such
TABLE 1. Electronic Spectral Data of [Vo(H~O)512+ and of Solutions Conta@ng Different VO*+/GSSG Ratios (pH = 7) VO*+:GSSG [VO(H@)J*+a lo:1 2&l So:1 loo:1 a cf. Ref. [9] also.
Band Positions (nm) 770 795 782 764 757
16.0
625 584 584 584 584
a(M- ‘.cm; ‘1
566 564 555 550
17.6 17.5 19.4 20.0
7.5 16.6 16.6 16.5 16.4
15.6 17.6 17.5 17.4
256
E. G: Ferrer et al.
0
2.5
0 FIGURE
3.
5
’
’
’
’
’
’
7.5 [GSSGl/[
’
’
’
10
V02*]
Spectrophotometric titration of V02+ with GSSG (5 X low2 M) at pH = 7,
under N,.
precipitation is observed for ratios around 1.5 181. The spectrum of Figure 2c is also directly generated from a lOOA GSSG:VO*+ ratio. Further addition of VO*+ produces an inverse band displacement as shown in Table 1 and, finally, the spectrum of Figure 2b is again observed. It is evident that at higher VO*+ concentrations (i.e., at lower GSSG:VO*+ ratios) coordination occurs through oxygen donors (probably carboxylate groups) as is suggested by the spectral shifts, which are typical for the coordination of vanadyl(IV) to carboxylate ligands [9, 11, 121. The displacement of the two extreme bands to higher energies, as the GSSG concentration increases, clearly suggests the participation of nitrogen donors in the coordination sphere of the metal. It is interesting to point out that in this case both complexes can be transformed into each other, by simple alteration of the ligand-&metal ratio. This is not possible in the case of reduced glutathione. The violet complex, generated at higher GSH concentrations, cannot be transformed into the blue form, stable only at lower ligand concentrations. This different behavior is probably due to the fact that in GSH the sulfhydril group participates in the formation and stabilization of the violet complex [6, 81 whereas in the present case this group is not available because the formation of the disulfide bridge (Fig. 1). The appearance of three bands in the electronic spectra of the VO’+/GSSG complexes instead of the two observed for [Vo
VO’+
CATION WITH OXIDIZED
GLUTATHIONE
257
corresponds to the 3d, + 3d,, 3d, (b, + e) and the second one (II) to the 3d, --) 3d,2_,2 (b, -B b,) transitions, respectively. Another band (III), usually located below 450 nm, has been assigned to the 3d, -+ 3d,2 (b, + a,) transition. VanadylUV) complexes of lower symmetries have been controversialy assigned [9, 14, 151. These disagreements refer to the relative positions of bands I and II and also the the energy of band III with respect to a charge transfer transition observed in the same range. A splitting of the e level may be expected as a consequence of symmetry lowering and a crossover between the e doublet and the b, singlet may even occur. On the other hand, Selbin and Morpurgo [16] have analyzed the electronic spectra of a number of low-symmetry VO*+ complexes with tartrate, malate, mandelate, and citrate as ligands, using the so-called “clustered-level” scheme [17]. In this model, band I involves the three transitions from 3d, to the 3d,, 3d, and 3d,2_,2 orbitals, whereas band II ‘would be the 3d, + 3d,2 transition. As our spectra are similar to those measured for the above mentioned carboxylate complexes, the same model can be used to assign the electronic transitions in this case. It can be assumed that a very strong bonding occurs in the equatorial plane around the VO*+ cation, due to the negative charge of the interacting carboxylate groups. So the energy of the 3d,2_,2 level is expected to increase quite strongly in relation to the 3d, and 3d, levels. Therefore, the three bands observed in the spectra of Figure 2b and 2c can be assigned as follows, from lower to higher energies: 3d, -+ 3d,, 3d,;
3d,, -+ 3d,2_,2;
and 3d, -+ 3d,2.
This assignment also shows that the 3d,2 orbital in our complexes is somewhat stabilized in comparison with its position in other vanadyl(IV) complexes with carboxylate ligands [16]. Alternatively, but less probable, the first two bands may represent transitions to the two components of the split e level (3d, and 3d,,). This would imply a rather unlikely splitting of 4000 cm-‘. 2. Characterization
of a Solid V02+/GSSG Complex
2.1. Synthesisand Chemical Chumcterization. The possibility of a solid vanadyl(IV) complex formation with GSSG is specially interesting because no solid GSSG complexes have been reported [2]. We have carried out a large number of experiments searching for such a solid complex in the VO*+/GSSG system. Finally, we have found that adjusting the pH value to 4.5 and using a GSSG:VO*+ ratio of 2.51, a solid compound precipitates (cf. Experimental). This solid is very unstable and redissolves quickly as the pH of the solution increases. Once isolated, it is highly hygroscopic and this behavior makes handling very difficult. The elemental analysis of a great number of samples provided different results, a fact which can be attributed to the formation of different hydrates or to a rapid absorption of humidity during manipulation. Notwithstanding, the analyses clearly confirm the formation of a 1:2 GSSG/VO*+ complex and suggest the presence of an octahydrate. Typical values were: V:10.7-11.7%; C:26.8-27.5%; H:4.8-5.5%;
250
E. G. Few
et al.
N:8.9-9.7% (calculated for C&,N,0,,H,(V0),.8Hz0 (MW&36S):V: 11.49%; C:27.10%; H:4.96%; N:9.48%). Vanadium was determined spectrophotometritally as tungstovanadophosphoric acid and the remaining elements by standard techniques of elemental organic analysis (UMYMFOR-Department0 de Quimica Orgtiica, UBA, Buenos Aires). 2.2. Thermul Be/m&r. The proposed stoichiometry and composition of the complex was further confirmed by its thermal behavior. A typical thermogram is shown in Figure. 4. As can be seen, the loss of water begins at a very low temperature. A first, weak and broad endothermic DTA-signal is observed at ca. 7o”C, and at 100°C the complex has given up nearly 7.7% of its mass, which corresponds to the loss of ca. 4 mols of water (calculated = 8.1%). A second step extends up to practically 260°C. At this temperature a very strong exothermic DTA-peak is observed. In this second step a weight loss of 5.6% is found (ca. 3 mols water; calculated = 6.09%). But, simultaneously with this loss of water, another and more important degradative process may occur as suggested by the important DTA-signal. The remaining water is then lost, together with other products, in the third step (260-320’0 The last step, which extends up to 5WC, is related to the two exothermic DTA-peaks located at 415°C and 44O”C, respectively. The total thermal degradation can be represented by the following equation:
/” GSSG(V0)2.8H,0a”‘V205
7
+ 8H,O + other products
I exo
I
endo
DTA ,---‘J
--_----,
TG I
1
100
I
I
300
1
I
1
500
1
a
700
T (‘Cl FIGURE 4.
Thermogram
of the solid
(VO),GSSG.&H,O
complex.
(1)
V02+
CATION WITH OXIDIZED
GLUTATHIONE
259
The residual mass found was of 20.6%, which is in excellent agreement with the theoretical value (20.5%) calculated from Eq. (1). The generation of V,O, as the only solid residue was confirmed by IR spectroscopy. On the other hand, the weak endothermic peak observed in the thermogram at ca. 640°C is in quite good agreement with the fusion temperature of V,O, (658°C) 1181. 2.3. Reflectance Spectrum. The reflectance spectrum of the solid complex shown in Figure 5, and compared with that of crystalline VOSO,,.SH,O, present two broad and not well-defined bands at 840 and 575 nm, respectively. This pattern suggests coordination of the metal through the carboxylate groups of the ligand. 2.4. I&wed Spectrum. The infrared spectra of free GSSG and of its vanadyl (IV) complex are shown in Figure 6. The’exact band positions, together with the assignment proposed for the principal bands based on general literature references [19-221, are given in Table 2. C-H and N-H stretching vibrations are observed in the high-frequency region. The broadening and intensification observed in the spectrum of the complex in this range is attributed to the additional appearance of O-H stretching modes due to the presence of the water molecules. In the middle region of the spectrum, various interesting and important changes are evident: the dissappearance of the 1730 cm-’ band, the shift of the 1530 cm-’ band, and the general broadening between 1700 and 1500 cm-‘. These changes confirm the participation of the carboxylate groups in the coordination. The general broadening is surely’ enhanced by the presence of S(H,O) vibrations, which are expected to lie’ between 1600-1650 cm-‘.
1
I
I
I
1
400
500
600
700
800
(nml FIGURE 5. Electronic reflectance spectra of VOSO.,SH# VO/GSSG complex (-1.
(-----I and of the solid
260
E. G. Ferrer et al.
,,,*..,r..l
0
3000
I
2000
,
,
,
,
,
,
,
,
1500
1
,
I
1000 [cm-l]
I
I
I
I
.i 5oo
FIGURE 6. Infrared spectra of GSSG (- - - - -1 and of the solid VO/GSSG complex (-1.
TABLE 2. Infrared Spectra of GSSG-and WO),GSSG.8H20 GSSG 3280 qbr 3060 m,br -2965s -2940s 1730 m 166OvS 1530 vs 1450 w 1405 s 1225 vs 108Ow 104Ow 875 w 838 w 808W
765 w 728 w _ 658 m,br 54Om 495 VW 422 w 331 w
(VO),GSSG.OH,O 3280 vs,br 3080 m,br -2970s -2945s I 1650 vs> 1540 m,br 1440 sh 1400s 1300 w,br 1250 sh 1155 w 1120 WJ 1074 vw 1045 vw 975 vs 885 w
Assignment v(CH) and VW-I) vacoo - 1 + v(C= 01, Amide I 6(NH), Amide II
v*oxm -) dcoo-)
v(V=O)
840W NVW
765 w 728w 670 m 605m 42Ow 327 VW
v(C-S) v(S--s)
V02+
CATION WITH OXIDIZED
GLUTATHIONE
261
The characteristic V=O stretching vibration can be clearly identified as a strong band at 975 cm-‘. The fact that a unique and rather symmetric band is observed, suggests that both V=O vibrators are equivalent. Another interesting aspect worth commenting is the position of the band assigned to the disulfide bond. In the free ligand this band lies at 540 cm-’ and it is displaced to 605 cm-’ in the complex. Its position in free GSSG suggests a truns-gauche-truns conformation of the -HC-CI-I,-S-S-CH,-CHlinkage [21]. Its shift to higher frequencies in the complex points to important structural changes of this linkage after complexation. Also, the band assigned to the C-S stretching mode suffers a small shift to higher frequencies after complexation. CONCLUSIONS This study demonstrates the existence of two different VO’+/GSSG complexes in solution, at pH = 7, which can be easily transformed into each other by simply changing the metal-to-ligand relations. The formation of these complexes suggest that GSSG might also participate in the stabilization and transport of V02+ immediately after the GSH mediated reduction of vanadate(V) to vanadyl(IV) in biological systems. On the other hand, it was possible to prepare and characterize a solid 21 V02+/GSSG complex, which apparently is the second example of a solid metallic complex of oxidized glutathione reported. The only solid metallic complex of GSSG previously reported was a C&I), species, which also presents a 2:l Cu:GSSG stoichiometry [2, 231. It was suggested that Cu(I1) ions should be bound to the carboxylate group and to the ammino nitrogen of both glutamyl moieties [23]. In our case, however, the electronic reflectance spectrum suggests the presence of just oxygen ligands in the coordination sphere of the V02+ cation. This work was supported by CONICET and CIC-PBA.
REFERENCES 1. A. L. Lehninger, ~ncipks of Biochem&y, Worth, New York, 1982. 2. D. L. Rabenau, R. Guevremont, and Ch. A. Evans, in &tal Ions in Biologicaf Svstems. H. Siael, Ed., M. Dekker, New York, Vol. 9, 1979, p. 103. 3. 6. D. Chastee~, k&t. Bonding 53,105 (1983). 4. D. Rehder, Angew. Chem. Zntemat. (english edition) 30,148 (1991). 5. I. G. Macara, K Kustin, and L. C. Can&y Jr., Biuchim. Bio&s. Acta 629,25 (1980). 6. M. Degani, M. Go&in, S. J. D. KarIish, and Y. Schechter, Biochemistry Zo, 5795 (1981). 7. M. De&i, E. GaggeIli, A. Lepri, and G. VaIensin, Inotg. Chim. Acta 107,87 (1985). 8. E. G. Ferrer, P. A. M. Williams, and E. J. Baran, Biof. Tmce Ekm. Res. 30, 175 (1991). 9. A. B. P. Lever, Inorganic Electtvnk Specharcopy, Ekevier, Amsterdam, 2nd, Edition, 1984. 10. K. A. Connors, Binding Constants, Wiley, New York, 1987.
262
E. G. Ferrer et al.
11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
K. Wiihtrich, Helv. Chim. Acta 44, 779 (1965). E. J. Baran, A. H. Jubert, and A. L. Rocha, J. Raman S’ctr. 20,801 (1989). C. J. Ballhausen and H. B. Gray, Znorg. C/rem. 1, 111 (1962). J. Selbin, Coord. Chem. Rev. 1, 293 (1966). P. Amo& R. Ibaiiez, A. Belt&, and D. Beltrain, J. Chem. Sot. Dalton Tmns., 1665, (1988). J. Selbin and L. Morpurgo, J. Znorg. Nucl. Chem. 27, 673, (1965). T. R. Ortolano, J. Selbin, and S. P. MC Glynn, J. Chem. Phys. 41, 262 (1964). R. J. H. Clark, The Chemistryof Titaniumand Vanadium, Elsevier, Amsterdam, 1968. L. J. Bellamy, Ultrarot-S&&nun und Chemische Konstitution,Steinkopf, Darmstadt, 1955. F. S. Parker, Applications of Infrared Spectrosco~ in Biochemistry, Biology and Medicine, Adam Hilger, London, 1971. A. T. Tu, Raman S’ctroscopy in Biology, Wiley, New York, 1982. E. Pretsch, J. Seibl, W. Simon, and Th. Clerc, Tables of Spectml Data for Structure Determinationof Organic Compounds, Springer, Berlin, 2nd Edition, 1989. P. Kroneck, J. Am. Chem. Sot. 97,3839 (1975).
Received May 20, 1992; accepted September 17, 1992
isolated at ‘$H - 4.5 and characterized by chemical analysis and infrared and electronic spectroscopies. Its thermal behavior was investigated through TG and DTA measurements.
JNTRODUCTION
Glutathione (GSH) the tripeptide rcglutamyl-rccysteinyl-lglycine, has generally been regarded as the most abundant low-molecular weight thiol in a variety of cell types [1, 21 and it apparently plays an important role in the metabolism and biochemistry of vanadium [3, 41. In fact, in red cells vanadate(V) is almost quantitatively reduced to V02+ by glutathione, which can also act as a ligand for the generated vanadyI0V) cation [5-71. We have recently investigated this VO’+/GSH interaction in detail, using spectrophotometric techniques [8]. Oxidized glutathione (GSSG, Fig. 1) is produced during the biological reduction of vanadate(V) to vanadyl(IV). Therefore, it seems interesting to verify whether the V02+ cation interacts with GSSG and, eventually, to obtain more information about the generated complexes. In this paper, we present the first evidence on the formation of two different V02+/GSSG complexes in solution. Also, a solid complex was isolated and characterized.
Address reprint requests and correspondence to: Professor Dr. Enrique J. Baran, Qukica Inorgtica, Facultad de Ciencias Exactas, UNLP, Casilla de Correo %2,19CKM,a Plats, Argentina. 50,253-262 (1993) 253 0 1993Eilsevierscience Publishing Co., Inc., 655Avenue of the Americas, NY, NY 10010 0162-0134/93/$6.00
Joumal oftnogrnic Biocheti~,
254
E. G. Ferrer et al.
0 II
0 II
k02ct~c~2C~2’C~t4c~-I~~~c~2c0~ I NH2
CH2 4 5 I Cl-12
NH2 I
I
HOzCCHCHfiHzCNH .
CHCNHCHzCOzH
II 0
II 0
FIGURE 1. Sche&atic structure of oxidized glutathione (GSSG).
EXPERIMENTAL.
Oxidized glutathione and VOSO,SH,O were commercially available from Sigma and Merck, respectively. All experiments were carried out under anaerobic conditions in order to prevent oxidation phenomena. Electronic absorption spectra were obtained with a Hewlett-Packard 8452 Diode-Array spectrophotometer using quartz cells (1 cm optical path). The pH of the solutions was fixed using either 0.1 N HCl or 0.1 N NaOH. In order to avoid interactions of V02+ with other ligands, no buffers were used. The solid complex was obtained by mixing aqueous solutions, 1.25 X 10m3 M of GSSG, and 5 X 10e4 M of vanadyl(IV) sulfate in a 2.5:1 molar ratio. The pH value was adjusted to 4.5. The generated pale blue precipitate was filtered through a G4-fritted-glass funnel and immediately dried at room temperature. The infrared spectra of the solid complex and of the free ligand were obtained with a Perkin-Elmer 580 B spectrophotometer, using the KBr pellet technique. The reflectance spectra were measured with a Shimadzu UV-300 instrument, using MgO as a standard. The thermogravimetric study was performed with a Stanton 781 thermobalance under static air atmosphere. The heating rate was lO”C/min and Al,O, was used as a DTA standard. RESULTS AND DISCUSSION 1. Studies in Solution In the previous paper [8] we have shown that the interaction of reduced glutathione with V02+ strongly depends on the initial V02+:GSH ratio. Starting with a tenfold GSH excess, the coordination takes place through the two carboxylate groups of the ligand and at pH = 7 a blue 1:2 V02+/GSH complex is generated. Higher GSH concentrations produce a violet complex, which is the most stable species of this system. It can also be obtained by adding GSH to the blue species. In this violet complex, the cation apparently interacts with S and some of the N atoms of the peptide In order to verify the behavior of the V02+/GSSG system we have first investigated its dependence to the concentration ratio.
GLUTATHIONE
VO*+ CATION WITH OXIDIZED
255
0.04-
400 FIGURE
2.
VO*+/GSSG VO*+/GSSG
1
I
500
600
I ‘O” tnm I
I
_
*O”
Electronic spectra of an aqueous 8 X lo-’ M VO*+ solution (a); a (5 x 1O-3 M and 5 x lo- 2 M, respectively) solution (b); and a (5 x 10e3 M and 0.5 M, respectively) solution Cc).
Starting from 10e3M VOSO, and lo-* M GSSG solutions in a l&l GSSG:VO*+ ratio and at pH = 7, we obtained the spectrum depicted in Figure 2b (cf. Table 1 also). The higher energy band, which lies at 625 nm in [VO(H,O),]*+ (Fig. 2a), is clearly split in the VO*+/GSSG solution. Some noticeable changes are produced in some of the spectral bands by adding more ligand to the solution; increasing the GSSG:VO*+ ratio. At a 1oO:l ratio, the spectrum of Figure 2c is obtained. It can be seen that the ‘highest and lowest energy bands are clearly displaced to higher energies, whereas the third band remains practically unchanged (cf. Table 1 and Fig. 2). In ,order to obtain a wider insight into the stoichiometry of the blue complex formed at a l&l GSSG/VO*+ ratio, we have performed a spectrophotometric titration [lo], monitoring the absorbance changes as a function of the metal-toligand ratio at constant wavelength (818 nm). One of those experiences is reproduced in Figure 3. The results point clearly to the formation of a 21 GSSG/VO*+ complex. This complex seems to be more stable than the 2:l complex formed with reduced glutathione because in this case the ligand-to-metal ratio can be reduced to 0.5 without VO(OH), precipitation. In the case of GSH, such
TABLE 1. Electronic Spectral Data of [Vo(H~O)512+ and of Solutions Conta@ng Different VO*+/GSSG Ratios (pH = 7) VO*+:GSSG [VO(H@)J*+a lo:1 2&l So:1 loo:1 a cf. Ref. [9] also.
Band Positions (nm) 770 795 782 764 757
16.0
625 584 584 584 584
a(M- ‘.cm; ‘1
566 564 555 550
17.6 17.5 19.4 20.0
7.5 16.6 16.6 16.5 16.4
15.6 17.6 17.5 17.4
256
E. G: Ferrer et al.
0
2.5
0 FIGURE
3.
5
’
’
’
’
’
’
7.5 [GSSGl/[
’
’
’
10
V02*]
Spectrophotometric titration of V02+ with GSSG (5 X low2 M) at pH = 7,
under N,.
precipitation is observed for ratios around 1.5 181. The spectrum of Figure 2c is also directly generated from a lOOA GSSG:VO*+ ratio. Further addition of VO*+ produces an inverse band displacement as shown in Table 1 and, finally, the spectrum of Figure 2b is again observed. It is evident that at higher VO*+ concentrations (i.e., at lower GSSG:VO*+ ratios) coordination occurs through oxygen donors (probably carboxylate groups) as is suggested by the spectral shifts, which are typical for the coordination of vanadyl(IV) to carboxylate ligands [9, 11, 121. The displacement of the two extreme bands to higher energies, as the GSSG concentration increases, clearly suggests the participation of nitrogen donors in the coordination sphere of the metal. It is interesting to point out that in this case both complexes can be transformed into each other, by simple alteration of the ligand-&metal ratio. This is not possible in the case of reduced glutathione. The violet complex, generated at higher GSH concentrations, cannot be transformed into the blue form, stable only at lower ligand concentrations. This different behavior is probably due to the fact that in GSH the sulfhydril group participates in the formation and stabilization of the violet complex [6, 81 whereas in the present case this group is not available because the formation of the disulfide bridge (Fig. 1). The appearance of three bands in the electronic spectra of the VO’+/GSSG complexes instead of the two observed for [Vo
VO’+
CATION WITH OXIDIZED
GLUTATHIONE
257
corresponds to the 3d, + 3d,, 3d, (b, + e) and the second one (II) to the 3d, --) 3d,2_,2 (b, -B b,) transitions, respectively. Another band (III), usually located below 450 nm, has been assigned to the 3d, -+ 3d,2 (b, + a,) transition. VanadylUV) complexes of lower symmetries have been controversialy assigned [9, 14, 151. These disagreements refer to the relative positions of bands I and II and also the the energy of band III with respect to a charge transfer transition observed in the same range. A splitting of the e level may be expected as a consequence of symmetry lowering and a crossover between the e doublet and the b, singlet may even occur. On the other hand, Selbin and Morpurgo [16] have analyzed the electronic spectra of a number of low-symmetry VO*+ complexes with tartrate, malate, mandelate, and citrate as ligands, using the so-called “clustered-level” scheme [17]. In this model, band I involves the three transitions from 3d, to the 3d,, 3d, and 3d,2_,2 orbitals, whereas band II ‘would be the 3d, + 3d,2 transition. As our spectra are similar to those measured for the above mentioned carboxylate complexes, the same model can be used to assign the electronic transitions in this case. It can be assumed that a very strong bonding occurs in the equatorial plane around the VO*+ cation, due to the negative charge of the interacting carboxylate groups. So the energy of the 3d,2_,2 level is expected to increase quite strongly in relation to the 3d, and 3d, levels. Therefore, the three bands observed in the spectra of Figure 2b and 2c can be assigned as follows, from lower to higher energies: 3d, -+ 3d,, 3d,;
3d,, -+ 3d,2_,2;
and 3d, -+ 3d,2.
This assignment also shows that the 3d,2 orbital in our complexes is somewhat stabilized in comparison with its position in other vanadyl(IV) complexes with carboxylate ligands [16]. Alternatively, but less probable, the first two bands may represent transitions to the two components of the split e level (3d, and 3d,,). This would imply a rather unlikely splitting of 4000 cm-‘. 2. Characterization
of a Solid V02+/GSSG Complex
2.1. Synthesisand Chemical Chumcterization. The possibility of a solid vanadyl(IV) complex formation with GSSG is specially interesting because no solid GSSG complexes have been reported [2]. We have carried out a large number of experiments searching for such a solid complex in the VO*+/GSSG system. Finally, we have found that adjusting the pH value to 4.5 and using a GSSG:VO*+ ratio of 2.51, a solid compound precipitates (cf. Experimental). This solid is very unstable and redissolves quickly as the pH of the solution increases. Once isolated, it is highly hygroscopic and this behavior makes handling very difficult. The elemental analysis of a great number of samples provided different results, a fact which can be attributed to the formation of different hydrates or to a rapid absorption of humidity during manipulation. Notwithstanding, the analyses clearly confirm the formation of a 1:2 GSSG/VO*+ complex and suggest the presence of an octahydrate. Typical values were: V:10.7-11.7%; C:26.8-27.5%; H:4.8-5.5%;
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et al.
N:8.9-9.7% (calculated for C&,N,0,,H,(V0),.8Hz0 (MW&36S):V: 11.49%; C:27.10%; H:4.96%; N:9.48%). Vanadium was determined spectrophotometritally as tungstovanadophosphoric acid and the remaining elements by standard techniques of elemental organic analysis (UMYMFOR-Department0 de Quimica Orgtiica, UBA, Buenos Aires). 2.2. Thermul Be/m&r. The proposed stoichiometry and composition of the complex was further confirmed by its thermal behavior. A typical thermogram is shown in Figure. 4. As can be seen, the loss of water begins at a very low temperature. A first, weak and broad endothermic DTA-signal is observed at ca. 7o”C, and at 100°C the complex has given up nearly 7.7% of its mass, which corresponds to the loss of ca. 4 mols of water (calculated = 8.1%). A second step extends up to practically 260°C. At this temperature a very strong exothermic DTA-peak is observed. In this second step a weight loss of 5.6% is found (ca. 3 mols water; calculated = 6.09%). But, simultaneously with this loss of water, another and more important degradative process may occur as suggested by the important DTA-signal. The remaining water is then lost, together with other products, in the third step (260-320’0 The last step, which extends up to 5WC, is related to the two exothermic DTA-peaks located at 415°C and 44O”C, respectively. The total thermal degradation can be represented by the following equation:
/” GSSG(V0)2.8H,0a”‘V205
7
+ 8H,O + other products
I exo
I
endo
DTA ,---‘J
--_----,
TG I
1
100
I
I
300
1
I
1
500
1
a
700
T (‘Cl FIGURE 4.
Thermogram
of the solid
(VO),GSSG.&H,O
complex.
(1)
V02+
CATION WITH OXIDIZED
GLUTATHIONE
259
The residual mass found was of 20.6%, which is in excellent agreement with the theoretical value (20.5%) calculated from Eq. (1). The generation of V,O, as the only solid residue was confirmed by IR spectroscopy. On the other hand, the weak endothermic peak observed in the thermogram at ca. 640°C is in quite good agreement with the fusion temperature of V,O, (658°C) 1181. 2.3. Reflectance Spectrum. The reflectance spectrum of the solid complex shown in Figure 5, and compared with that of crystalline VOSO,,.SH,O, present two broad and not well-defined bands at 840 and 575 nm, respectively. This pattern suggests coordination of the metal through the carboxylate groups of the ligand. 2.4. I&wed Spectrum. The infrared spectra of free GSSG and of its vanadyl (IV) complex are shown in Figure 6. The’exact band positions, together with the assignment proposed for the principal bands based on general literature references [19-221, are given in Table 2. C-H and N-H stretching vibrations are observed in the high-frequency region. The broadening and intensification observed in the spectrum of the complex in this range is attributed to the additional appearance of O-H stretching modes due to the presence of the water molecules. In the middle region of the spectrum, various interesting and important changes are evident: the dissappearance of the 1730 cm-’ band, the shift of the 1530 cm-’ band, and the general broadening between 1700 and 1500 cm-‘. These changes confirm the participation of the carboxylate groups in the coordination. The general broadening is surely’ enhanced by the presence of S(H,O) vibrations, which are expected to lie’ between 1600-1650 cm-‘.
1
I
I
I
1
400
500
600
700
800
(nml FIGURE 5. Electronic reflectance spectra of VOSO.,SH# VO/GSSG complex (-1.
(-----I and of the solid
260
E. G. Ferrer et al.
,,,*..,r..l
0
3000
I
2000
,
,
,
,
,
,
,
,
1500
1
,
I
1000 [cm-l]
I
I
I
I
.i 5oo
FIGURE 6. Infrared spectra of GSSG (- - - - -1 and of the solid VO/GSSG complex (-1.
TABLE 2. Infrared Spectra of GSSG-and WO),GSSG.8H20 GSSG 3280 qbr 3060 m,br -2965s -2940s 1730 m 166OvS 1530 vs 1450 w 1405 s 1225 vs 108Ow 104Ow 875 w 838 w 808W
765 w 728 w _ 658 m,br 54Om 495 VW 422 w 331 w
(VO),GSSG.OH,O 3280 vs,br 3080 m,br -2970s -2945s I 1650 vs> 1540 m,br 1440 sh 1400s 1300 w,br 1250 sh 1155 w 1120 WJ 1074 vw 1045 vw 975 vs 885 w
Assignment v(CH) and VW-I) vacoo - 1 + v(C= 01, Amide I 6(NH), Amide II
v*oxm -) dcoo-)
v(V=O)
840W NVW
765 w 728w 670 m 605m 42Ow 327 VW
v(C-S) v(S--s)
V02+
CATION WITH OXIDIZED
GLUTATHIONE
261
The characteristic V=O stretching vibration can be clearly identified as a strong band at 975 cm-‘. The fact that a unique and rather symmetric band is observed, suggests that both V=O vibrators are equivalent. Another interesting aspect worth commenting is the position of the band assigned to the disulfide bond. In the free ligand this band lies at 540 cm-’ and it is displaced to 605 cm-’ in the complex. Its position in free GSSG suggests a truns-gauche-truns conformation of the -HC-CI-I,-S-S-CH,-CHlinkage [21]. Its shift to higher frequencies in the complex points to important structural changes of this linkage after complexation. Also, the band assigned to the C-S stretching mode suffers a small shift to higher frequencies after complexation. CONCLUSIONS This study demonstrates the existence of two different VO’+/GSSG complexes in solution, at pH = 7, which can be easily transformed into each other by simply changing the metal-to-ligand relations. The formation of these complexes suggest that GSSG might also participate in the stabilization and transport of V02+ immediately after the GSH mediated reduction of vanadate(V) to vanadyl(IV) in biological systems. On the other hand, it was possible to prepare and characterize a solid 21 V02+/GSSG complex, which apparently is the second example of a solid metallic complex of oxidized glutathione reported. The only solid metallic complex of GSSG previously reported was a C&I), species, which also presents a 2:l Cu:GSSG stoichiometry [2, 231. It was suggested that Cu(I1) ions should be bound to the carboxylate group and to the ammino nitrogen of both glutamyl moieties [23]. In our case, however, the electronic reflectance spectrum suggests the presence of just oxygen ligands in the coordination sphere of the V02+ cation. This work was supported by CONICET and CIC-PBA.
REFERENCES 1. A. L. Lehninger, ~ncipks of Biochem&y, Worth, New York, 1982. 2. D. L. Rabenau, R. Guevremont, and Ch. A. Evans, in &tal Ions in Biologicaf Svstems. H. Siael, Ed., M. Dekker, New York, Vol. 9, 1979, p. 103. 3. 6. D. Chastee~, k&t. Bonding 53,105 (1983). 4. D. Rehder, Angew. Chem. Zntemat. (english edition) 30,148 (1991). 5. I. G. Macara, K Kustin, and L. C. Can&y Jr., Biuchim. Bio&s. Acta 629,25 (1980). 6. M. Degani, M. Go&in, S. J. D. KarIish, and Y. Schechter, Biochemistry Zo, 5795 (1981). 7. M. De&i, E. GaggeIli, A. Lepri, and G. VaIensin, Inotg. Chim. Acta 107,87 (1985). 8. E. G. Ferrer, P. A. M. Williams, and E. J. Baran, Biof. Tmce Ekm. Res. 30, 175 (1991). 9. A. B. P. Lever, Inorganic Electtvnk Specharcopy, Ekevier, Amsterdam, 2nd, Edition, 1984. 10. K. A. Connors, Binding Constants, Wiley, New York, 1987.
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11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
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Received May 20, 1992; accepted September 17, 1992