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Electron paramagnetic resonance studies on VO(IV)-D-aspartic acid and VO(IV)-D-aspartic acid α-benzylester complexes
Electron Paramagnetic Resonance Studies on VO{IV}-D-Aspartic Acid and VO IIV I-D-Aspartic Acid a-Benzylester Complexes Rosa Pia Ferrari, Enzo Laurenti, Sonia Poli, and Luigi Casella RPF. Dipartimento di Chimica Inorganica, Chimica Fisica e Chimica dei Materiali, Universitb di Torino, Italy.--EL, SP, LC. Dipartimento di Chimica Inorganica e
Metallorganica, Universitit di Milano, Italy
ABSTRACT A detailed electron paramagnetic study has been done on oxovanadium(IV)-D-aspartic acid and oxovanadium(IV)-D-aspartic acid c~-benzylester complexes, molar ratio 1:1 and 1:2 in the physiological pH range. Their isotropic and anisotropic spin Hamiltonian parameters have been calculated to second order and the superimposed mixed species spectral patterns have been simulated. The complexes display an approximate C4v geometry whose relative hyperfine coupling constant values are connected with the ligand field strength on the equatorial plane. Furthermore it seems that aspartic acid, in some circumstances, binds oxovanadium(IV) acting as tridentate ligand, and aspartic acid a-benzylester can coordinate the metal-ion giving complexes with six-membered chelate rings (t~-NH 2/G-COO- donor groups). Our EPR results are in agreement with those of UV-visible and CD spectral measurements. ABBREVIATIONS Asp, aspartic acid; Asp-c~bz, aspartic acid t~-benzylester; Gly, glycine; O, H20 Oxygen; O - , carboxylate oxygen; NNH2, amino group nitrogen; S-, mercapto group sulphur.
INTRODUCTION Vanadium has been found in some biological systems and is believed to be essential for the life of cells. Little is known about its chemical forms and the physiological mechanisms in which it is involved. The metal in the oxidation state V is present in the metal site of some peroxidases and of recently isolated nitrogenases [1], and is an
Address reprint requests to: Professor Rosa Pia Ferrari, Dipartimento di Chimica Inorganica, Chimica Fisica e Chimica dei Materiali, via P. Giuria 7, 10125 Torino, Italy.
Journal oflnorganic Biochemistry, 45, 21-30 (1992)
21 © 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/92/$3.50
22
R. P. Ferrari et al.
inhibitor of N a + / K + ATPase activity [2]. Vanadium(IV) has been monitored in erythrocytes [3] and in some animal tissues [4, 5], resulting from vanadium(V) reduction. Since the coordination chemistry of vanadium(IV) can be conveniently and sensitively investigated by EPR techniques, also at room temperature, a great deal of effort has been addressed to study oxovanadium(IV)-small ligand model complexes [6, 71] and oxovanadium(IV)-protein complexes [6, 8, 9]~ In the framework of this general argument, and keeping in mind that an aspartic acid residue is the interaction site of vanadium in Na + / K + ATPase [10] and is probably present in the proximity of the peroxidases prosthetic group [It], we analyzed in detail the oxovanadium(IV) coordination to aspartic acid and aspartic acid ot-benzylester in order to evaluate the extent to which the metal coordination environment can be deduced from EPR results. In the last few years the problem was addressed [12] by potentiometric and spectroscopic measurements, with only qualita-tive EPR data. We think that an exhaustive investigation at room temperature and 77 K by EPR analysis is required for clarifying the oxovanadium(IV) coordination to aspartic acid close to the physiological pH range. Comparison with the systems VO(IV)-D-aspartic acid c~-benzylesters and VO(IV)-dicarboxyiic acids [13, 14} allows us to differentiate the chelate ring size and, in particular, to enlighten a selective interaction by the c~-COO and fi-COO groups.
EXPERIMENTAL
Materials D-aspartic acid and D-aspartic acid ct-benzylester were commercially available from Sigma. All other reagents were analytical grade from Merck. The metal-ligand complexes solutions have been prepared in a jacketed glass cell of 50 ml capacity; the cells were evacuated and purged with nitrogen. Then a gas flow was kept during all manipulations and transfers in order to avoid the V(IV) oxidation and the CO 2 side reactions.
Measurements The UV-visible and CD measurements were made at room temperature on a Kontron UVIKON 930 spectrophotometer and a JASCO J-500C spectropolarimeter, respectively. The EPR spectra were recorded at room temperature and 77 K on a Varian E 109 X band spectrometer equipped with a double cavity and an AQM Auriga/XT (STELAR) data system, using DPPH and VOSO4.5H20 as standards. Fhe aqueous solutions in the EPR tubes were added, before freezing of a microquantity of glycerol in order to form good glass samples and improve the spectral resolution.
Preparation of the VO(IV)-D-Aspartic Acid and VO(lV)-D-Aspartic Acid a-Benzylester Complexes Solutions Twenty ml of ligand aqueous solution 2.5 × 1 0 2 M were added of 0.5 and 1 ml respectively VOSO 4.5H20 1 M solution, to have a metal/ligand molar ratio 1:1 and 1:2, at pH = 3.2. Two solutions at pH = 4.5 and 6.5 were then obtained by adding NaOH 0.1 M to the initial solution. The preparation of the VO(IV)-D-aspartic acid
EPR O F V O ( I V ) - D - A S P A R T I C A C I D t~-BENZYLESTER
23
ot-benzylester complexes in aqueous solution was made by substituting the V O S O 4 . 5 H 2 0 solution with N a V O 3 solution in the presence o f a small excess of N a 2 S 2 0 4 because o f the lower stability o f the complexes.
RESULTS AND DISCUSSION In Table 1 we summarize the EPR spin Hamiltonian parameters o f the oxovanadiumD-aspartic acid and oxovanadium-D-aspartic acid tx-benzylester complexes. The EPR spectral patterns (Fig. l(a)) o f the aqueous solutions of the complexes kept at room temperature are typically isotropic, H = g ~HS + ASI, whereas those o f the same solutions frozen at 77 K (Fig. l(b)) are anisotropic with an^approximate C4v a x i ~ symmetry, I3t = / 3 [ g l l H z S z + g .L (HySy + HxSx)] + AiiSzIz + A ± (Syly + S x I x). The spin Hamiltonian parameters have been calculated to second order by an iterative procedure using the Bleaney perturbation equations [8, 15]. Furthermore, the parameters for the mixed species spectral patterns have been evaluated using a SIM14 Q C P E computer program for EPR spectra simulation [8]. The isotropic and anisotropic EPR parameters o f the metal-ligand solutions, 1:1 molar ratio at pH = 3, evidentiate the presence o f the ion [VO(H=O)s] 2+ as the dominant species. Starting from pH = 4 the oxovanadium(IV) appears, partially, to interact with aspartic acid to give EPR spectra constituted o f two patterns X and Y (Fig. 2, Fig. 3). In Figure 2 the pattern X parameters are analogous to those o f aquaion. For pattern Y, small variations in the A II and A o hyperfine values suggest the possible appearance of a monocoordinated species I in which VO(IV) is bound to the a - C O O - group in the equatorial plane. This follows the order o f dissociation constants o f the aspartic acid
TABLE 1. EPR Parameters of Oxovanadium(IV)-Ligand Complexes at r.t. and 77 K Ligand
VO2+/L a pH
Asparticacid
1:1
Asparticacidot-benzylester
1:1
Asparticacid
1:2
Asparticacidot-benzylester
1:2
Succinicacid
1:2
H20 a
gll
g.L
g0calc,b
go
Allc
A ± c A0calc.b'c
Aoc
3.2 1.937 1.978 1.964 1.961 183.28 4.0 1.938 1.982 1.967 183.35 1.944 1.984 1.971 174.96 3.2 1.937 1.978 1.964 1.961 183.28 4.0 1.929 1.973 1.958 182.58
69.67 68.73 68.79 69.67 68.85
107.54 106.16 106.94 104.18 107.54 106.16 106.76
3.1 1.927 1.971 4.5 1.932 1.969 7.0 1.934 1.969 1.941 1.974 3.3 1.927 1.970 4.6 1.936 1.972 7.0 1.944 1.979
67.45 61.88 61.56 58.04 67.63 64.26 55.56
105.59 106.45 9 9 . 1 0 99.20 9 8 . 3 6 99.54 91.87 87.18 105.77 106.78 100.63 100.70 90.77
3.3 4.6 5:1 5.7
1.920 1.925 1.927 1.927
1.956 1.957 1.957 1.963 1.956 1.961 1.967
1.957 1.959 1.959 1.963 1.956 1.961
181.87 173.55 171.97 159.53 182.06 173.36 161.19
1.964 1.949 1.950 182.89 68.18 1.968 1.953 1.951 180.72 66.12 1.968 1.954 1.952 179.38 66.11 1.968 1.954 178.47 64.64
1.933 1.978
1.963 1.964 182.60 70.70
106.42 104.32 103.87 102.58
106.36 104.66 104.19
108.00
106.30
a: VO2+/L = metal-to-ligand molar ratio b: gocat%= 1 / 3 ~ + 2g±); Aoca,c.= 1/3(A~ + 2A ±) c: x 10- ( c m - ) d: [VO(H20)5] 2+ EPR parameters in our experimental conditions and in agreement with those reported in literature [6].
24
R . P. F e r r a r i et al.
I _
<
i
t ,4
,,
0
C
0 L~
0
o. o.
m
0
---v---v---~ - - ~
5).
i
E
i 0°
~.
~
~
EPR OF VO(IV)-D-ASPARTIC ACID ct-BENZYLESTER
O .i.
t
(,.,.,.. O
4 II
O O
t:~
4
=-
O
o
O O
""
3," >
X-
o ,,4
~ ~
o°
-~
O
d
°O©
--.Ja
t
I
I
I
I
I
I
I
I
I
I
.c--
"~.
I
25
26
R. P. Ferrari et al.
0 0
T
E
0
4 -5
~q :r
>
a
i
~z
! 0 0
G
)
i I
~--~-l-
...... --T
l
io
C"
EPR OF VO(IV)-D-ASPARTIC ACID ol-BENZYLESTER
27
donor groups (pK ~COOH< pK ~coor~ < pK Nn3 +), in agreement with the literature data [16]. The corresponding experimental value A o = 104.18 × 10 -4 cm-~ is close to the theoretical estimate A 0 = 104.15 × 10 -4 c m - t for the monocoordinated model system [VO(O-O3)], obtained with the additivity calculations [8]. In these working experimental conditions, analogous behavior was displayed by bidentate and tridentate ligands, such as amino acids [17] and dicarboxylic acids [13, 14], in agreement with the successive ionizations of their protonated groups and relative stabilities of the metal-ligand chelate rings. By increasing the metal-to-ligand molar ratio 1:2, the metal-ion unprotected ligand interaction becomes more evident at pH = 4.5 and pH = 7.0, on the basis of the EPR parameters of the corresponding complexes (Table 1) as well as by their electronic and CD data (Table 2). One may notice the general trend for the g II values to decrease and for the A II values to increase with increasing the ligand field strength in the equatorial plane of the complexes, when the equatorial occupancy is by the NH 2 group of the aspartate. It then appears likely that formation of the chelate complex VO(Asp) II (at pH 4.5), in which VO(IV) binds an a - N H 2 and an a - C O O - to give a quite stable glycine-type five-atoms ring, can occur (Fig. 4 ). When the pH approaches 7.0 the EPR spectrum (Fig. 3) presents two mixed superimposed patterns: one has spin Hamiltonian parameters quite similar to those of species II, whereas the other possesses parameters with lower values, in particular A1~ = 159.53 × 10 -4 cm -1. We are in the presence of a third species VO(Asp) 2 III in which VO(IV) binds two ligand molecules to a - N H 2 and a - C O O - coordination sites to form two five-membered glycine-type [17] chelate rings (Fig. 411. Formation of six-atoms ( a - N H 2 / / ~ - C O O - ) and seven-atoms ( a - C O O / / 3 - C O O - ) chelate rings can be excluded on the basis of EPR data of VO(IV)-aspartic acid compared with those of VO(IV)-a-blocked aspartic acid and VO(IV)-succinic acid, respectively
TABLE 2. Electronic and Circular Dichroism d-d Absorption Maxima of Oxovanadium(IV)-Ligand (1:2) Complexes Ultraviolet-Visible a Ligand
pH
Aspartic acid
3.1 4.5 7.0
~km. . . . . (E, M - i cm- i)
766 604 760 582 758 534
(13.35) (sh) (13.68) (7.3) (5.77) (11.06)
Aspartic acida-benzylester
3.3
764 (12.39) 611 (sh)
Succinic acid
3.3
767 615 793 617 809 583 834 576
4.6 5.1 5.7 a: sh, shoulder
(18.79) (sh) (18.39) (8.67) (17.78) (13.13) (18.79) (sh)
CD km..... 808 561 784 561 791 556
28
R. P. Ferrari et al.
HOOC-CH= \ /CH
O
c-o<-.-ll o ----->V2 ." H20
......
~.... O H 2 OH=
OOC\ O
O C iF • - - O . . " ii "
>OHa
O O i C-~O "" 11 - " - H 2 N
CH2 /
.
HC
'!N~ ...... ',---'):OH, H2C-- C-,, O O II
FIGURE 4.
k~H, "/"-
......
H2C--C-O % O
~\
-'~o-;c " ' O
Ill
Structures of VO(IV)-Asp complex species 1, lI. III.
(Table 1). Confirmation of the structure of species II and lII could come also from the agreement of their A o experimental values with those of VO(GIy), VO(Gly) 2 [6, 17] and the theoretical estimates A o for model complexes VO(N~H2 O 0 2 ) and VO(N{NH2)20;), respectively [8]. Additionally, by taking into account that aspartic acid can act as tridentate ligand [11] and since the A i = 159.53 ~ 1 0 4 cm ~ value of the species III is lower than the A i! = 165 × 10 ~ cm " value for the VO{GIy) 2 equivalent species, one can conclude that lI and II1 also have an axial coordination of metal-ion to ~t-COO . Finally these structures are in agreement with the formation constant measurements recently reported [121. Another tridentate ligand, 2-mercapto succinic acid, exhibits behavior similar to that of aspartic acid: -SH is present here instead of the -NH2 group, and gives species type II and IIl with chelate rings (-COO /-S ) instead of ( - C O 0 /-NH2). The A o = 81 x 10 4 cm ~ for bis-chelate 2-mercapto succmate species Vl [141 is much lower than A 0 = 87.18 × 10 '* cm ~ for bis-chelate aspartate species III because the ligand fiekt strength due to the equatorial cluster ( 0 : $ 2 ) is higher than that of (O2 N 2 ). When the c~-COOH is blocked, as in D-aspartic acid ce-benzylester, it seems from EPR data reported in Table 1, that an alternative monocoordination of ~-COO to oxovanadium(IV) is not law)red at pH < 4. Monocoordinated species are generally not very stable and in these conditions /3-COOH is mostly protonated. Increasing pH to 4.6 and 7.0, the variations in the spin Hamiltonian parameters are indicative of interaction o f oxovanadium(IV) with the ligand. In particular, from their typical features we can argue the presence of the species VO(Asp-~bz) IV as chelate (ce-NH2/fl-COO ), and VO(Asp-c~bz)2V as bis-chelate ((~-NH~ 'i~-COO ) ( F i g , 5). The formation of a six-atom chelate ring structure is also confirmed by the noncoincidence o f its EPR parameters with those of a six-membered chelate ring (-COO / - C O O ) for VO(IV)-malonate species Vll [131 (Fig. 6). The electronic and CD data of 1:2 complexes are collected in Table 2, The data for 1 : 1 complexes are not reliable for their low stability with such a small amount of ligand. The same happens for 1: I and 1:2 oxovanadium(lV)--aspartic acid c,-benzylester complexes.
EPR OF VO(IV)-D-ASPARTIC ACID ct-BENZYLESTER
o
O
o
j&o----li\,, ...... . , N - . d H=C
~ _
/
~v--~. .
c-o--((
"~-J"
29
))
6H=
O~ ; H - N H , ......OH= =...... O-C O
V
IV
FIGURE 5. Structures of VO(IV)-Asp-c~bz complex species IV and V. Two d-d bands are observed in the visible spectra of oxovanadium(IV)-D-aspartic acid 1:2 complexes. On the basis of their Car geometry the corresponding transitions can be observed: 2B2(xy)-*2E(xz,yz )
and
2B2(xy)--*2Bn(x2-y2 )
in order of increasing energy. With increasing pH, both the electronic and CD bands in the visible region shift to higher energy. These changes are in agreement with the successive appearance of species VO(Asp) II and VO(Asp) 2 III. The CD spectra display a rather invariant pattern of bands with pH. The position of the CD bands roughly matches the absorption bands but their intensity is markedly dependent on pH. At acidic pH, where the monodentate species I is present, the optical activity is extremely low. It is possible that the CD spectrum observed depends on the small amount of chelate species II present. Species II maximizes near pH = 4.5 and corresponds to the most intense CD spectrum. The observed optical activity then decreases at higher pH but is due to the faster oxidation of the VO 2+ species in these conditions and the relatively long time required to record the CD spectra. The electronic absorption maxima of VO-succinate (Table 2) support and confirm the EPR results according to which monocoordinated species (type I) are formed at low pH and hydrolytic oxovanadium(IV) species dominate when pH is above 5. Conversely, the electronic spectra of VO(IV)-2-mercaptosuccinate support the EPR analysis of chelate species VI (type IV) formation [14]. CONCLUSIONS The detailed and quantitative EPR analysis, conducted at room temperature and 77 K, on aqueous solutions of VO(IV)-aspartic acid and VO(IV)-a-blocked aspartic acid allowed us to clearly distinguish: (a) the formation, close to physiological pH range, of predominant species VO(Asp) II, VO(Asp) 2 III, VO(Asp-abz) IV, and VO(Aspctbz) 2 V with an approximate C4v geometry; Co) the type of equatorial ligands and
O
O
-OOC.. CHa
O
........ -=s/
H2C--C%~O
O
O
O
......... o-'c
~O
OH2 O VII
Vl
FIGURE 6. Structures of VO(IV)-2-mercapto succinic acid (VI) and VO(IV)-malonic acid (VII) complex species.
30
R. P. Ferrari et aL
the size of chelate ring (nitrogen/oxygen donors groups); (c) the additional apical interaction of /3-COO- o f aspartic acid with the metal-ion which enhances the stability of complexes II and III; (d) the possibility to differentiate selectively a-COO-/~-COOmetal-ion interaction; (e) the formation of chelate species VI, VO(IV)-2-mercaptosuccinate and VII, VO(IV)-malonate, support the formation of V, VO(IV)-aspartate species as a six-atom chelate ring with (N20 ~ ) donor system on the equatorial plane. In conclusion, we can tentatively state that B-COO- group of aspartic acid residues, present in some proteins, can act as a binding site tbr vanadium (V) and (IV), particularly in proximity of amino acidic residues with nitrogen/sulphur donor groups [181. This work was supported by Ministero Italiano Pubblica lstruzione and by Centro di Calcolo CSI Piemonte.
REFERENCES 1. (a) E. De Boer, Y. Van Kooyk, M. G. M. Tromp, H. Platt, and R. Wever, Biochim. Biophys. Acta 869, 48 (1985); (b) H Platt, B. E. Krenn, and R. Wever, Biochem. J. 248, 277 (1987); (c) R. L. Robson, R. R. Eady, T. H. Richardson, R. W. Miller, M. Hawkins, and J. R. Postgate, Nature 322,388 (1986); (d) B. J. Holes, E~ E. Case, J. E Morningstar, M. F. Dzeda, and L. A. Monterer, Biochemistry 25, 7251 (1986). 2. L. Josephson and L. C. Cantley Jr., Biochemistry 16, 4572 (1977). 3. L. C. Cantley Jr. and P. Aisen, J. Biol. Chem. 254, 178t (1979). 4. J. L. Johnson, H. J. Cohen, and K. V. Rajagopalan. Biochem~ Biophys. Res. Commun. 59, 940 (1974). 5. H. Sakurai, S. Shimomura. K. Fukuzawa, and K~ Ishizu, Biochem. Biophys. Res. Commun. 96, 293 (1980). 6. N. D. Chasteen, in Biological Magnetic Resonance, L. J. Berliner and J. Reuben, Eds.. Plenum, New York, 1981, Vol. 3, Chap. 2. 7. L. F. Vilas Boas and L Costa Pessoa, in Comprehensive Coordination Chemistry, G Wilkinson, R. D. Gillard, and J. A. McCleverty, Eds., Pergamon, Oxfbrd, 1987. Vol. 3. pp, 453-583. 8. R. P. Ferrari, lnorg. Chim. Acta 176, 83 (t990). 9. R. P Ferrari, M. Finelli, C. Giunta, and M. Cavaletto, Congr. Naz. Rison. Magnet.. Pisa, October, 22-24, 1990. 10. L. C. Cantley Jr., L. G. Cantley, and L. Josephson, J. Biol. Chem. 253, 7361 (1978). 11. T, L. Poulos, A d w Inorg. Biochem. 7, 1 (1988). 12. J. Costa Pessoa, R. L. Marques, L. F. Vilas Boas, and R. D. Gillard, Polyhedron 9, 81 (1990). 13. D. B. McPhail and B. A. Goodman, J. Chem. Soc., Faraday Trans. 83, 3513 (1987). 14. G. Micera and A. Dess), J. lnorg. Biochem. 33, 99 (1988). 15. L. Casella, M. Gullotti. A. Pintar, S. Colonna, and A. Manfredi, lnorg. Chim. Acta 144, 89 (t988). 16. H. C. Freeman, in Inorganic Biochemistry, G. L. Eichorn, Ed., Elsevier, Amsterdam, New York, 1973, Vol. 1, p. 121. 17. C. R. Jonhson and R. E. Shepherd, Bioinorg. Chem~ 8, 115 (1978). 18, A. Breier, L. Tury-Nagy, A. Ziegelh6ffer, and R. Monosikova, Gen_ Phr'siol. Biophys. 8,283 (1989). Received March 5, 1991; accepted June 3, 1991
Metallorganica, Universitit di Milano, Italy
ABSTRACT A detailed electron paramagnetic study has been done on oxovanadium(IV)-D-aspartic acid and oxovanadium(IV)-D-aspartic acid c~-benzylester complexes, molar ratio 1:1 and 1:2 in the physiological pH range. Their isotropic and anisotropic spin Hamiltonian parameters have been calculated to second order and the superimposed mixed species spectral patterns have been simulated. The complexes display an approximate C4v geometry whose relative hyperfine coupling constant values are connected with the ligand field strength on the equatorial plane. Furthermore it seems that aspartic acid, in some circumstances, binds oxovanadium(IV) acting as tridentate ligand, and aspartic acid a-benzylester can coordinate the metal-ion giving complexes with six-membered chelate rings (t~-NH 2/G-COO- donor groups). Our EPR results are in agreement with those of UV-visible and CD spectral measurements. ABBREVIATIONS Asp, aspartic acid; Asp-c~bz, aspartic acid t~-benzylester; Gly, glycine; O, H20 Oxygen; O - , carboxylate oxygen; NNH2, amino group nitrogen; S-, mercapto group sulphur.
INTRODUCTION Vanadium has been found in some biological systems and is believed to be essential for the life of cells. Little is known about its chemical forms and the physiological mechanisms in which it is involved. The metal in the oxidation state V is present in the metal site of some peroxidases and of recently isolated nitrogenases [1], and is an
Address reprint requests to: Professor Rosa Pia Ferrari, Dipartimento di Chimica Inorganica, Chimica Fisica e Chimica dei Materiali, via P. Giuria 7, 10125 Torino, Italy.
Journal oflnorganic Biochemistry, 45, 21-30 (1992)
21 © 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/92/$3.50
22
R. P. Ferrari et al.
inhibitor of N a + / K + ATPase activity [2]. Vanadium(IV) has been monitored in erythrocytes [3] and in some animal tissues [4, 5], resulting from vanadium(V) reduction. Since the coordination chemistry of vanadium(IV) can be conveniently and sensitively investigated by EPR techniques, also at room temperature, a great deal of effort has been addressed to study oxovanadium(IV)-small ligand model complexes [6, 71] and oxovanadium(IV)-protein complexes [6, 8, 9]~ In the framework of this general argument, and keeping in mind that an aspartic acid residue is the interaction site of vanadium in Na + / K + ATPase [10] and is probably present in the proximity of the peroxidases prosthetic group [It], we analyzed in detail the oxovanadium(IV) coordination to aspartic acid and aspartic acid ot-benzylester in order to evaluate the extent to which the metal coordination environment can be deduced from EPR results. In the last few years the problem was addressed [12] by potentiometric and spectroscopic measurements, with only qualita-tive EPR data. We think that an exhaustive investigation at room temperature and 77 K by EPR analysis is required for clarifying the oxovanadium(IV) coordination to aspartic acid close to the physiological pH range. Comparison with the systems VO(IV)-D-aspartic acid c~-benzylesters and VO(IV)-dicarboxyiic acids [13, 14} allows us to differentiate the chelate ring size and, in particular, to enlighten a selective interaction by the c~-COO and fi-COO groups.
EXPERIMENTAL
Materials D-aspartic acid and D-aspartic acid ct-benzylester were commercially available from Sigma. All other reagents were analytical grade from Merck. The metal-ligand complexes solutions have been prepared in a jacketed glass cell of 50 ml capacity; the cells were evacuated and purged with nitrogen. Then a gas flow was kept during all manipulations and transfers in order to avoid the V(IV) oxidation and the CO 2 side reactions.
Measurements The UV-visible and CD measurements were made at room temperature on a Kontron UVIKON 930 spectrophotometer and a JASCO J-500C spectropolarimeter, respectively. The EPR spectra were recorded at room temperature and 77 K on a Varian E 109 X band spectrometer equipped with a double cavity and an AQM Auriga/XT (STELAR) data system, using DPPH and VOSO4.5H20 as standards. Fhe aqueous solutions in the EPR tubes were added, before freezing of a microquantity of glycerol in order to form good glass samples and improve the spectral resolution.
Preparation of the VO(IV)-D-Aspartic Acid and VO(lV)-D-Aspartic Acid a-Benzylester Complexes Solutions Twenty ml of ligand aqueous solution 2.5 × 1 0 2 M were added of 0.5 and 1 ml respectively VOSO 4.5H20 1 M solution, to have a metal/ligand molar ratio 1:1 and 1:2, at pH = 3.2. Two solutions at pH = 4.5 and 6.5 were then obtained by adding NaOH 0.1 M to the initial solution. The preparation of the VO(IV)-D-aspartic acid
EPR O F V O ( I V ) - D - A S P A R T I C A C I D t~-BENZYLESTER
23
ot-benzylester complexes in aqueous solution was made by substituting the V O S O 4 . 5 H 2 0 solution with N a V O 3 solution in the presence o f a small excess of N a 2 S 2 0 4 because o f the lower stability o f the complexes.
RESULTS AND DISCUSSION In Table 1 we summarize the EPR spin Hamiltonian parameters o f the oxovanadiumD-aspartic acid and oxovanadium-D-aspartic acid tx-benzylester complexes. The EPR spectral patterns (Fig. l(a)) o f the aqueous solutions of the complexes kept at room temperature are typically isotropic, H = g ~HS + ASI, whereas those o f the same solutions frozen at 77 K (Fig. l(b)) are anisotropic with an^approximate C4v a x i ~ symmetry, I3t = / 3 [ g l l H z S z + g .L (HySy + HxSx)] + AiiSzIz + A ± (Syly + S x I x). The spin Hamiltonian parameters have been calculated to second order by an iterative procedure using the Bleaney perturbation equations [8, 15]. Furthermore, the parameters for the mixed species spectral patterns have been evaluated using a SIM14 Q C P E computer program for EPR spectra simulation [8]. The isotropic and anisotropic EPR parameters o f the metal-ligand solutions, 1:1 molar ratio at pH = 3, evidentiate the presence o f the ion [VO(H=O)s] 2+ as the dominant species. Starting from pH = 4 the oxovanadium(IV) appears, partially, to interact with aspartic acid to give EPR spectra constituted o f two patterns X and Y (Fig. 2, Fig. 3). In Figure 2 the pattern X parameters are analogous to those o f aquaion. For pattern Y, small variations in the A II and A o hyperfine values suggest the possible appearance of a monocoordinated species I in which VO(IV) is bound to the a - C O O - group in the equatorial plane. This follows the order o f dissociation constants o f the aspartic acid
TABLE 1. EPR Parameters of Oxovanadium(IV)-Ligand Complexes at r.t. and 77 K Ligand
VO2+/L a pH
Asparticacid
1:1
Asparticacidot-benzylester
1:1
Asparticacid
1:2
Asparticacidot-benzylester
1:2
Succinicacid
1:2
H20 a
gll
g.L
g0calc,b
go
Allc
A ± c A0calc.b'c
Aoc
3.2 1.937 1.978 1.964 1.961 183.28 4.0 1.938 1.982 1.967 183.35 1.944 1.984 1.971 174.96 3.2 1.937 1.978 1.964 1.961 183.28 4.0 1.929 1.973 1.958 182.58
69.67 68.73 68.79 69.67 68.85
107.54 106.16 106.94 104.18 107.54 106.16 106.76
3.1 1.927 1.971 4.5 1.932 1.969 7.0 1.934 1.969 1.941 1.974 3.3 1.927 1.970 4.6 1.936 1.972 7.0 1.944 1.979
67.45 61.88 61.56 58.04 67.63 64.26 55.56
105.59 106.45 9 9 . 1 0 99.20 9 8 . 3 6 99.54 91.87 87.18 105.77 106.78 100.63 100.70 90.77
3.3 4.6 5:1 5.7
1.920 1.925 1.927 1.927
1.956 1.957 1.957 1.963 1.956 1.961 1.967
1.957 1.959 1.959 1.963 1.956 1.961
181.87 173.55 171.97 159.53 182.06 173.36 161.19
1.964 1.949 1.950 182.89 68.18 1.968 1.953 1.951 180.72 66.12 1.968 1.954 1.952 179.38 66.11 1.968 1.954 178.47 64.64
1.933 1.978
1.963 1.964 182.60 70.70
106.42 104.32 103.87 102.58
106.36 104.66 104.19
108.00
106.30
a: VO2+/L = metal-to-ligand molar ratio b: gocat%= 1 / 3 ~ + 2g±); Aoca,c.= 1/3(A~ + 2A ±) c: x 10- ( c m - ) d: [VO(H20)5] 2+ EPR parameters in our experimental conditions and in agreement with those reported in literature [6].
24
R . P. F e r r a r i et al.
I _
<
i
t ,4
,,
0
C
0 L~
0
o. o.
m
0
---v---v---~ - - ~
5).
i
E
i 0°
~.
~
~
EPR OF VO(IV)-D-ASPARTIC ACID ct-BENZYLESTER
O .i.
t
(,.,.,.. O
4 II
O O
t:~
4
=-
O
o
O O
""
3," >
X-
o ,,4
~ ~
o°
-~
O
d
°O©
--.Ja
t
I
I
I
I
I
I
I
I
I
I
.c--
"~.
I
25
26
R. P. Ferrari et al.
0 0
T
E
0
4 -5
~q :r
>
a
i
~z
! 0 0
G
)
i I
~--~-l-
...... --T
l
io
C"
EPR OF VO(IV)-D-ASPARTIC ACID ol-BENZYLESTER
27
donor groups (pK ~COOH< pK ~coor~ < pK Nn3 +), in agreement with the literature data [16]. The corresponding experimental value A o = 104.18 × 10 -4 cm-~ is close to the theoretical estimate A 0 = 104.15 × 10 -4 c m - t for the monocoordinated model system [VO(O-O3)], obtained with the additivity calculations [8]. In these working experimental conditions, analogous behavior was displayed by bidentate and tridentate ligands, such as amino acids [17] and dicarboxylic acids [13, 14], in agreement with the successive ionizations of their protonated groups and relative stabilities of the metal-ligand chelate rings. By increasing the metal-to-ligand molar ratio 1:2, the metal-ion unprotected ligand interaction becomes more evident at pH = 4.5 and pH = 7.0, on the basis of the EPR parameters of the corresponding complexes (Table 1) as well as by their electronic and CD data (Table 2). One may notice the general trend for the g II values to decrease and for the A II values to increase with increasing the ligand field strength in the equatorial plane of the complexes, when the equatorial occupancy is by the NH 2 group of the aspartate. It then appears likely that formation of the chelate complex VO(Asp) II (at pH 4.5), in which VO(IV) binds an a - N H 2 and an a - C O O - to give a quite stable glycine-type five-atoms ring, can occur (Fig. 4 ). When the pH approaches 7.0 the EPR spectrum (Fig. 3) presents two mixed superimposed patterns: one has spin Hamiltonian parameters quite similar to those of species II, whereas the other possesses parameters with lower values, in particular A1~ = 159.53 × 10 -4 cm -1. We are in the presence of a third species VO(Asp) 2 III in which VO(IV) binds two ligand molecules to a - N H 2 and a - C O O - coordination sites to form two five-membered glycine-type [17] chelate rings (Fig. 411. Formation of six-atoms ( a - N H 2 / / ~ - C O O - ) and seven-atoms ( a - C O O / / 3 - C O O - ) chelate rings can be excluded on the basis of EPR data of VO(IV)-aspartic acid compared with those of VO(IV)-a-blocked aspartic acid and VO(IV)-succinic acid, respectively
TABLE 2. Electronic and Circular Dichroism d-d Absorption Maxima of Oxovanadium(IV)-Ligand (1:2) Complexes Ultraviolet-Visible a Ligand
pH
Aspartic acid
3.1 4.5 7.0
~km. . . . . (E, M - i cm- i)
766 604 760 582 758 534
(13.35) (sh) (13.68) (7.3) (5.77) (11.06)
Aspartic acida-benzylester
3.3
764 (12.39) 611 (sh)
Succinic acid
3.3
767 615 793 617 809 583 834 576
4.6 5.1 5.7 a: sh, shoulder
(18.79) (sh) (18.39) (8.67) (17.78) (13.13) (18.79) (sh)
CD km..... 808 561 784 561 791 556
28
R. P. Ferrari et al.
HOOC-CH= \ /CH
O
c-o<-.-ll o ----->V2 ." H20
......
~.... O H 2 OH=
OOC\ O
O C iF • - - O . . " ii "
>OHa
O O i C-~O "" 11 - " - H 2 N
CH2 /
.
HC
'!N~ ...... ',---'):OH, H2C-- C-,, O O II
FIGURE 4.
k~H, "/"-
......
H2C--C-O % O
~\
-'~o-;c " ' O
Ill
Structures of VO(IV)-Asp complex species 1, lI. III.
(Table 1). Confirmation of the structure of species II and lII could come also from the agreement of their A o experimental values with those of VO(GIy), VO(Gly) 2 [6, 17] and the theoretical estimates A o for model complexes VO(N~H2 O 0 2 ) and VO(N{NH2)20;), respectively [8]. Additionally, by taking into account that aspartic acid can act as tridentate ligand [11] and since the A i = 159.53 ~ 1 0 4 cm ~ value of the species III is lower than the A i! = 165 × 10 ~ cm " value for the VO{GIy) 2 equivalent species, one can conclude that lI and II1 also have an axial coordination of metal-ion to ~t-COO . Finally these structures are in agreement with the formation constant measurements recently reported [121. Another tridentate ligand, 2-mercapto succinic acid, exhibits behavior similar to that of aspartic acid: -SH is present here instead of the -NH2 group, and gives species type II and IIl with chelate rings (-COO /-S ) instead of ( - C O 0 /-NH2). The A o = 81 x 10 4 cm ~ for bis-chelate 2-mercapto succmate species Vl [141 is much lower than A 0 = 87.18 × 10 '* cm ~ for bis-chelate aspartate species III because the ligand fiekt strength due to the equatorial cluster ( 0 : $ 2 ) is higher than that of (O2 N 2 ). When the c~-COOH is blocked, as in D-aspartic acid ce-benzylester, it seems from EPR data reported in Table 1, that an alternative monocoordination of ~-COO to oxovanadium(IV) is not law)red at pH < 4. Monocoordinated species are generally not very stable and in these conditions /3-COOH is mostly protonated. Increasing pH to 4.6 and 7.0, the variations in the spin Hamiltonian parameters are indicative of interaction o f oxovanadium(IV) with the ligand. In particular, from their typical features we can argue the presence of the species VO(Asp-~bz) IV as chelate (ce-NH2/fl-COO ), and VO(Asp-c~bz)2V as bis-chelate ((~-NH~ 'i~-COO ) ( F i g , 5). The formation of a six-atom chelate ring structure is also confirmed by the noncoincidence o f its EPR parameters with those of a six-membered chelate ring (-COO / - C O O ) for VO(IV)-malonate species Vll [131 (Fig. 6). The electronic and CD data of 1:2 complexes are collected in Table 2, The data for 1 : 1 complexes are not reliable for their low stability with such a small amount of ligand. The same happens for 1: I and 1:2 oxovanadium(lV)--aspartic acid c,-benzylester complexes.
EPR OF VO(IV)-D-ASPARTIC ACID ct-BENZYLESTER
o
O
o
j&o----li\,, ...... . , N - . d H=C
~ _
/
~v--~. .
c-o--((
"~-J"
29
))
6H=
O~ ; H - N H , ......OH= =...... O-C O
V
IV
FIGURE 5. Structures of VO(IV)-Asp-c~bz complex species IV and V. Two d-d bands are observed in the visible spectra of oxovanadium(IV)-D-aspartic acid 1:2 complexes. On the basis of their Car geometry the corresponding transitions can be observed: 2B2(xy)-*2E(xz,yz )
and
2B2(xy)--*2Bn(x2-y2 )
in order of increasing energy. With increasing pH, both the electronic and CD bands in the visible region shift to higher energy. These changes are in agreement with the successive appearance of species VO(Asp) II and VO(Asp) 2 III. The CD spectra display a rather invariant pattern of bands with pH. The position of the CD bands roughly matches the absorption bands but their intensity is markedly dependent on pH. At acidic pH, where the monodentate species I is present, the optical activity is extremely low. It is possible that the CD spectrum observed depends on the small amount of chelate species II present. Species II maximizes near pH = 4.5 and corresponds to the most intense CD spectrum. The observed optical activity then decreases at higher pH but is due to the faster oxidation of the VO 2+ species in these conditions and the relatively long time required to record the CD spectra. The electronic absorption maxima of VO-succinate (Table 2) support and confirm the EPR results according to which monocoordinated species (type I) are formed at low pH and hydrolytic oxovanadium(IV) species dominate when pH is above 5. Conversely, the electronic spectra of VO(IV)-2-mercaptosuccinate support the EPR analysis of chelate species VI (type IV) formation [14]. CONCLUSIONS The detailed and quantitative EPR analysis, conducted at room temperature and 77 K, on aqueous solutions of VO(IV)-aspartic acid and VO(IV)-a-blocked aspartic acid allowed us to clearly distinguish: (a) the formation, close to physiological pH range, of predominant species VO(Asp) II, VO(Asp) 2 III, VO(Asp-abz) IV, and VO(Aspctbz) 2 V with an approximate C4v geometry; Co) the type of equatorial ligands and
O
O
-OOC.. CHa
O
........ -=s/
H2C--C%~O
O
O
O
......... o-'c
~O
OH2 O VII
Vl
FIGURE 6. Structures of VO(IV)-2-mercapto succinic acid (VI) and VO(IV)-malonic acid (VII) complex species.
30
R. P. Ferrari et aL
the size of chelate ring (nitrogen/oxygen donors groups); (c) the additional apical interaction of /3-COO- o f aspartic acid with the metal-ion which enhances the stability of complexes II and III; (d) the possibility to differentiate selectively a-COO-/~-COOmetal-ion interaction; (e) the formation of chelate species VI, VO(IV)-2-mercaptosuccinate and VII, VO(IV)-malonate, support the formation of V, VO(IV)-aspartate species as a six-atom chelate ring with (N20 ~ ) donor system on the equatorial plane. In conclusion, we can tentatively state that B-COO- group of aspartic acid residues, present in some proteins, can act as a binding site tbr vanadium (V) and (IV), particularly in proximity of amino acidic residues with nitrogen/sulphur donor groups [181. This work was supported by Ministero Italiano Pubblica lstruzione and by Centro di Calcolo CSI Piemonte.
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