70 species in solution a t the low chelate concentrations used here (0.007 to 0.009 M). A detailed analysis of the C P E results reported in this study must await further theoretical development of f-f optical activity in dissymmetric lanthanide complexes. Almost nothing is known about the mechanisms whereby lanthanide ion f-f transitions acquire optical activity in a chiral ligand environment nor about the influence of specific chelate-solvent interactions on optical activity in metal complexes in general. It seems clear that both TE and CPE are very sensitive probes for investigating chelate-solvent interactions and that they can provide a great deal of qualitative (and, in some cases, semiquantitative) structural information not readily obtainable by other techniques. However, the development of detailed and specific spectra-structure relationships must await further experimental and theoretical study. Acknowledgments. This work was supported by the National Science Foundation, the donors of the Petroleum Research Fund, administered by the American Chemical Society, and the Camille and Henry Dreyfus Foundation (through a Teacher-Scholar Awardto F.R.).
References and Notes (1) D. P. Craig, E. A. Power, and T. Thirunamachandran, Chem. Phys. Left., 27, 149 (1974).
(a) B. Bosnich, J. Am. Chem. Soc., 98, 6143 (1976); (b) B. Bosnich, "Fundamental Aspects and Recent Developments in Optical Rotatory Dispersion and Circular Dichroism," F. Ciardelli and P. Salvadori. Ed.. Heyden and Son Ltd., 1973, Chapter 3.8. L. D. Hayward and R. N. Totty, Can. J. Chem., 49, 623 (1971). S. Claesson and L. D. Hayward, Chem. Phys. Lett., 20,85(1973). P. E. SchiDDer, lnorq. Chim. Acta, 14, 161 (1975). P. E. Schipper, Cheh. Phys. Left., 30, 323 (1975). L. D. Hayward and S. Claesson, Chem. Scr., 9, 21 (1976). S.F. Mason, Chem. Phys. Left., 32, 201 (1975). (9) P. E. Schipper, Mol. Phys., 29, 1705 (1975). (10) P. E. Schipper, lnorg. Chim. Acta, 12, 199 (1975). (11) N. H. Andersen, B. J. Bottino, A. Moore, and J. R. Shaw, J. Am. Chem. Soc., 96, 603 (1974). (12) J. Diilon and K. Nakanishi, J. Am. Chem. Soc., 96,4057 (1974); 97, 5417 (1975). (13) See, for example, A. B. P. Sinha. Spectrosc. lnorg. Chem., 2, 255 (1971). (14) See. for example: (a) F. Halverson, J. S.Brinen, and J. R. Leto, J. Chem. Phys., 41, 157 (1964); (b) ibid., 41, 2752 (1964); (c) H. Samelson, C. Brecher, and A. Lempicke. J. Mol. Spectrosc., 19, 349 (1966). (15) G. A. Cotton, F. A. Hart, and G. P. Moss, J. Chem. Soc.. Dalton Trans., 221 (1975). (16) H. G. Brittain and F. S. Richardson, J. Chem. SOC., Dalton Trans., in press. (17) H. G. Brittain and F. S. Richardson, J. Am. Chem. Soc., 98, 5858 (1976). (18) R. G. Charles, lnorg. Synth., 9, 37 (1967). (19) C. K. Luk and F. S. Richardson, J. Am. Chem. Soc., 96, 2006 (1974). (20) C. K. Luk and F. S. Richardson, J. Am. Chem. Soc., 97, 6666 (1975). (21) For a discussion of the emission anisotropy factor, gem,see: I. Steinberg, "Concepts in Biochemical Fluorescence",Vol. I, R. F. Chen and H. Edelhock, Ed., Marcel Dekker, New York, N.Y., 1975, Chapter 3. (22) J. P. Riehl and F. S.Richardson, J. Chem. Phys., 65, 1011 (1976).
Proton Magnetic Resonance Study of Divalent Metal Ions Binding to Adenosine 5'-Triphosphate Joseph Granot*' and Daniel Fiat Contribution from the Department of Structural Chemistry, The Weizmann Institute of Science, Rehouot, Israel. Received April 1 , 1976
Abstract: The monometallic Co2+, Ni2+, Mg2+, and Ca2+ complexes with adenosine 5'-triphosphate have been investigated by proton magnetic resonance. The experimental pD values were adjusted so as to have the adenine ring either protonated or unprotonated, and the phosphate chain triple or fully ionized. The effect of ring protonation or the secondary phosphate hydrogen ionization on the metal-ring and metal-chain interactions has been studied. The formation of a bis M2+-ATP complex was established. The equilibria equations involving the formation of 1:l and 1.2 metal-ligand complexes and the nucleotide molecules self-association are discussed, and a method for calculating the formation constants from the N M R shifts is presented. A model for the bis complex, compatible with the experimental results, is proposed.
Nuclear magnetic resonance has been extensively used in the study of adenine nucleotides and their metal complexes.2 Concerning the complexes of adenosine 5'-triphosphate (1) and NH2
I
0-
0-
Iy
IB
6-P-0
II
0
-P
0-
-0-P-
II
0
II
Hj H
HI'
0 O'H
OH
1 divalent metal ions, the main results may be summarized as follows. The paramagnetic ions (Mn2+, Co2+,Ni2+) bind simultaneously to the three (a,p, y) phosphates and to the adenine The binding to the ring is not direct but via a water molecule which is coordinated to the metal ion and hydrogen bonded to N7 of the adenine ring.9,10In addition, the Journal of the American Chemical Society
metal complex contains another two water molecules in the inner sphere of co~rdination.~-"A more recent I3C N M R study of the Mn2+ complex of A T P indicated however a direct binding of the manganese ion to the N7 ring nitrogen.13 At high total A T P concentration it was found that 1:2 metal-ligand complexes are formed.'*,' The diamagnetic ions (Mg2+, Ca2+) were found not to interact with the adenine ring63l5 and their binding is to the /3 and y phosphated or to the /3 phosphate alone. A T P has two major ionization sites: the phosphate moiety and the adenine ring. The three primary phosphate hydrogens were estimated to have pK 5 2," and the final hydrogen was found to have pK 7.1s-23Hence the major part of A T P will be fully ionized (ATP4-) at p H 2 8. At a lower p H both ATP4- and ATP3- will be present, and a t p H 5 5 the major part will be ATP3-. 31PN M R s t ~ d i e s ~revealed ? ~ , ' ~ almost no difference in the metal-phosphate interaction in the p H range 5.5-8.5.In the present work we show that the same is true also regarding the metal-ring interaction. This metal-ring interaction, however, may be expected to depend on the ionization
/ 99:l / January 5, 1977
-
71
state of the ring. The pK associated with the ionization from the protonated ring of A T P was found to be -4.20-22 The problem was to determine the site of protonation/ionization in the adenine ring. Although some workers have claimed that the ionization is from the protonated Cb-amino g r o ~ p , ~ ~ , ~ ~ , ~ ~ - ~ ~ it is commonly agreed that the proton ionization site is the (4) N I H + g r o ~ p . ~ * We - ~ Inow present a study on the effect of the ring protonation on the metal-ring interaction in divalent metal KlK2 = K3K4 (5) complexes of ATP. The possibility of formation of 1:2 metalligand complexes has been taken into account (for the parawhere M represents a divalent metal ion, L represents ATP in magnetic ions) and formation constants for the various proany one of it's ionization states (Le., +DATP3-, ATP3-, or cesses have been calculated. ATP4-), and [MI and [L] denote free concentrations. The total concentrations LOand M Oare given by Experimental Section Lo = [L] 2[L2] [ML] 2[ML2] (6) Materials. ATP (disodium salt) and adenosine (A) of highest grade
+
were obtained from Sigma Chemical Co. and were used without further purification. The metal salts Co(N03)2-6H20 and MgC126H2O were obtained from Merck and Ni(N03)2*6H20and CaC122H2O from BDH Chemicals. The salts were dried overnight by slight heating in vacuo (to prevent decomposing). Stock solutions (2-3 M ) of the nickelous and cobaltous salts were prepared with D20 (99.7%) as solvent and standarized by EDTA complexometry with murexide as indicator. All other concentrations were determined gravimetrically. The ATP concentrations used were in the range 0.06-0.09 M, the Ni2+ and Co2+ concentrations varied from 1 X to 5 X IO-' M, and those of Mg2+ and C a 2 + from 8 X to 6 X lo-' M. In the higher C a 2 + concentrations a small volume of precipitate appeared in the APT solution and was dissolved after slight heating of the solution (to about 50 "C). The pH was adjusted, using concentrated acid or base solutions in D2O. NMR Spectra. Proton magnetic resonance measurements were recorded on a Bruker HFX-IO spectrometer operating at 90 MHz. A trace ofp-dioxane in the experimental solutions served as internal reference for shift measurements. All the measurements were performed at an ambient probe temperature of 27 O C , except for one case (see below) when a temperature study was made. The temperatures were stabilized by a Bruker B-ST 100/760 control unit and were calibrated by measuring the peak separation of an ethylene glycol sample. The experimental error in the field shift measurements was taken as about 5% of the line width values but not less than 1 Hz. An average of three or four measurements has been taken in order to improve the overall accuracy. Experimental pD. Considering the following equilibria between A T P and D + ( H + ) in D2O solutions: +DATP3- s ATP3ATP3- s ATP4-
+ D+
pK'
+ D+
pK"
-
-
Mo = [MI
+ [ML] + [ML2]
AHp = (1
(7)
+PM-')AH~
(8)
where AHp is the field shift between the signals of the free and bound ligands, AHa is the observed coalescent signal shift with respect to metal free solution, and P M is the mole ratio of bound and nonbound ligands. In our case, having two types of complexes, P M is given by
[MLI + 2 w - 2 1 Lo - [ML] - 2[ML2] Substituting eq 9 in 8 we get PM
=
(9)
It should be noted that in eq 10 an assumption has been made that the ligands bound either ig the 1:l or the 1:2 complexes undergo the same field shift. This assumption will be discussed later on. In principle, by solving simultaneously eq 1-4 one can get expressions for [ML] and [ML2] in terms of the formation constants. Then, by fitting eq 10 to the experimental results the formation constants may be calculated. Since eq 1-4 cannot be solved analytically, an approximate solution is required. Equations 1 and 2 give
4
7
[MLI = K I[MI [Ll [MLd = KiK2[M1[LI2 Substituting (1 1 ) and (12) in (7) gives
1OpD-pK'
M o = [M](1 + Ki[L]
-
+
Taking M to be a paramagnetic ion then under conditions of fast chemical exchange (in the NMR scale) between free and bound ligands, the following relation holds35
where +DATP3- denotes a protonated ring state, we have [ATP3-i = [+DATP3-]
+
(1 1) (12)
+ K1K2[LI2) E [MI * Q ( K I , K ~ , [ L ] )
(13) The concentrations of the various types present may be written now as
Accordingly, the experimental pD values, which are the pH meter readings +0.4,32 were chosen to be: pD 3 for the study of the +DATP3- species, pD 5-6 for the study of ATP3-, and pD 8 for the study of ATP4-.
-
Calculation of Formation Constants Taking a solution containing divalent metal ions and ATP, in appropriate pH (pD), so that the ATP-proton reactions can be neglected, the major processes that take place are the formation of 1: 1 and 1:2 metal-ligand complexes and dimerization of ATP molecules due to self-association.12-33-34 (The complexation and association of higher orders, Le., ATP, and M2+-ATP,, n > 2, are neglected.) The equilibria and the formation constants involved may be expressed as
Lo = [Ll
[MI = Mo/Q
(14)
[MLI = KiMo[LI/Q
(15)
WL21 = KiKMo[LI2/Q
(16)
[Lzl = K4tL12
(17)
+ 2K4P-I' + Mo(Ki[LI + 2KiKr[LI2)/Q
(18) (i) Linear Approximation Solution. In solutions where the paramagnetic ion concentrations are small relative to the ligands, Le., Mo [ML2].It is important to note that while a priori knowledge of AHp is needed in the nonlinear approximation, this may not be required in the linear approximation (eq 23). Moreover, the linear approximation may also be used to derive the values of AHp. In the case of A T P complexes with Co2+ and Ni2+, values of AHp could have been directly measured and hence the nonlinear apprximation could have been applied.
Results and Interpretation Co2+-ATP. The proton spectrum of ATP at pD 3.0 is shown in Figure 1. In the course of the experiment which consisted of titration of A T P with Co2+.ions,the shifts of Hg, H 2 , and H I ' , and also those of other ribose protons, which we denote as H,, H, (a double line), and H , , were observed. (Another ribose proton signal was obscured by the water signal.) Some of the shifts were upfield and others downfield, relative to their
January 5,1977
, 73
2 00
, 01 i
02
03
04
(MI
[Co]
Figure 5. Measured Hg field shifts (to low field) in the CoZ+-ATPsystem at pD 3 ([ATP10 = 0.081 M).
log
[col
Figure 4. Calculated concentrations of the various species present in the ATP (0.081 M ) solution as a function of added Co2+ion concentrations. The formation constants from Table 1 were used. Table 11. Bound State Field Shifts (SHp), Line Widths (AvzM), and Metal-Proton Distances ( R )for the CoATP ( 1 : l ) Complex at pD 3" SHp, HZ
H2 Hx HI' H, HI H.
170 f 5 -350 f 20 285 f 5 111 f 5 -60 f 20 -250 f 20
S V Z MH, Z
25 f 3 450 f 50 3053 24 f 3 24 f 3 100 f 5
- I5
R, A 5.0 3.1 4.8 5.1 5 .o 4.0
H,, H I , and H, denote ribose hydrogens. Negativeshiftsare to low field.
metal-free shifts, indicating the presence of both contact and pseudocontact hyperfine interactions contributing to the shifts (compare with the solely downfield shifts observed for the NiATP complex, cf. Table V below). Since the chemical exchange of ATP in the cobaltous ATP system is f a ~ tthe ~ ,above ~ method for calculation of the formation constants could be applied. Since the H2 and H I shifts could be observed throughout almost the whole measuring range, they were thus used for the calculation. First we applied the linear approximation solution assuming Ll = LO.It came out that eq 24 is the appropriate one for this case. A least-squares fit of the experimental results (Figure 2 ) for [Co2+]0>- A~W M ~hence , the relevant relation for the line-broadening analysis is Av2p = P M T 2 M - I . 3 6 Let A v ~ M ( I ) and A u ~ Mbe ( ~the ) line widths in the bound state in the 1:l and 1:2 complexes, respectively, then we have:
-
+
([ML] AVZM(I) 2[ML2] A U ~ U ( ~ ’ ) /(33) LO The Hz and HI’ line width data were analyzed with eq 33 to yield the values of A V Z M (and ’ ) A U Z M ((Table ’) VI). The water coordination in the nickelous-ATP 1:l and 1:2 complexes has been studied by fitting the line widths (Figure 12) with eq 32 (the water signal shifts were too small to enable significant study). The results are given in Table 111, indicating a more pronounced shortening of the electronic relaxation time in the nickelous relative to the cobaltous A T P complex. W e should point out that our results for the proton saturation shifts in the NiATP complex are lower than those obtained by Glassman et aL9 and Sternlicht et aL4 from temperature studies. It is our opinion however that temperature studies should be very carefully interpreted and that simple temperature-dependence assumption may be erroneous. To demonA ~ 2 p=
/ Divalent Metal Ions Binding to Adenosine 5‘-Triphosphate
76 -03
4451--1
- 437 I
1
9
4421 2g*
221 213 0.0
Figure 12. The paramagnetic line broadening of the water coordinated in the Ni2+-ATP system at pD 3 ( 0 )and pD 5.5 (0).
0.25
0.50
0.75
[M2+] (M)
Figure 14. Measured proton field shifts in the M2+-ATPsystem, M 2 + = Mg2+ ( 0 )or Ca2+ (0).The shifts (to low field) are given relative t o p dioxane ([ATP10 = 0.082 M).
Table VII. Proton Field Shifts in the Mg2+- and Ca2+-ATP ( 1 : l ) Complexes a t pD 3
2.5
2.7
2.9
3.1
3.3
3.5
(OK-‘)
10’1~
Figure 13. Temperature dependence of the H2 (A)and H 1’ (A)field shifts in 1 : I8 and in 1. I : 1 ( 0 .0 respectively) Ni2+-ATP systems ([ATP10 = 0.082 M, pD 3 ) .
strate this we studied the protons field shifts temperature dependence of l : 18 and l . l : l Ni2+:ATP solutions (Figure 13). (The paramagnetic shifts were measured relative to metal-free A T P shifts in the appropriate temperatures.) The temperature dependence of the shifts in the Ni2+ diluted solution may be interpreted4 as governed by a chemical exchange process: as the temperature is raised the exchange becomes more rapid and the shifts of the bulk signals increase. In the limit of fast exchange a 1 / T dependence is expected.46 Clearly, the decrease of the shift (Figure 13) is much faster than predicted, in a similar “anomalous” way as found in the CoATP comp l e ~Moreover, .~ in the study of the 1.1: 1 solution the bound signals were observed directly, and these are expected to shift with 1 / T dependence in the whole temperature range. The results contradict this expectation completely. Taking into account the possible degrees of freedom of the A T P molele^^^^^ and the complexation and self-stacking temperature dependence we conclude that the temperature study of A T P complexes reflects mainly conformational and complexational changes rather than exchange controlled temperature behavior. Additional study of the nickelous-ATP complex a t pD 8.5 yielded bound state parameters similar, within the error range, to those obtained a t pD 5.5 (Table V). Mg2+-ATP and Ca2+-ATP. Measurements of proton field shifts in the magnesium and calcium ATP complexes a t pD 5.5 and 8.5 revealed no significant change (within the experimental error) relative to the metal-free A T P solution, in accordance with previous s ~ d i e s .At ~ , pD ~ ~ 3 however significant shifts were observed (Figure 14). The data were analyzed assuming the formation mainly of 1:l complex in the metal’s concentrations range investigated and using the binding constants and l o 1 . ’for the MgATP and C a A T P complexes as determined by fluorescence study a t p H 2.8.49 The results are Journal of the American Chemical Society
/
99:l
/
M?+
Hz
Hx
HI‘
Mgz+ Ca2+
-4.2 f 0.2 -7.6 f 0.1
-2.7 f 0.2 -3.4 0.1
-1.4 f 0.2 -12.7 f 0.5
given in Table VII. The broadening of the lines in either complex was found to be negligible. Co2+-ATP-Adenosine. The complexation of adenosine (A) with Co2+ in the absence and the presence of A T P has been studied. The aim was to examine the possibility of the formation of a ternary Co2+ATP-A complex, i.e., to demonstrate the ability of the CoATP monocomplex to bind a second molecule via ring binding alone. The measurements were performed a t pD 2 . 5 . (Adenosine has a pK 3.6 for ring p r o t ~ n a t i o n . ~The ~ , ~effect ~ ) of the cobaltous ions (0.040 M) on the Hs, H2, and H I ’ resonance of A (0.038 M ) is shown in Figure 15b. The very small effect (H2 and H I ’ were almost unchanged and H8 only slightly broadened and downfield shifted) is expected, considering the low formation constant between A and divalent metal i o m 2 Moreover the solvent (HDO) signal was shifted downfield to an extent compatible with the formation of a regular hexacoordinated cobaltous aqueous complex. The adenosine binding is thus an outersphere coordination. When A T P (0.009 M ) was added to the Co2+-A solution, a considerable effect on the A resonance (with Hg undergoing the largest) was induced (Figure 15c). Clearly A which binds very weakly to the Co2+ ion binds more strongly to the CoATP complex. The dependence of the field shifts of the Hs, H2, and H I ’ resonances of A, as well as that of the solvent (HDO) on the concentration of A T P added to the 1:l Co2+:A solution, has been studied. The results are shown in Figure 16. As the ATP concentration was raised more A was bound and water molecules were expelled from the cobaltous coordination sphere. This resulted in shifting the water signal back upfield, and saturation was achieved a t about half the original shift, for 1:1 Co2+:ATP concentrations, indicating that three water molecules were taken out from the aqueous complex as the CoATP complex was formed. However, as the A T P concentration exceeded that of Co2+ (and A), the competition between the free A T P and the adenosine bound in the ternary complex resulted in the latter being removed from the complex, as is inferred from Figure 16. It should be noted, however, that an A T P concentration-dependent irregularity of the adenosine H8 and H2 shifts is observed. The H8 shift is being saturated much faster than that of H2 and “decays” much faster for [ATP] > [A]. This phenomenon is not yet
January 5, 1977
-
77
dA 1 -
560
480
400
320
240
160
Figure 15. Proton magnetic resonance spectra of the Hx, Hz, and H I ’ resonances of A (0.038 M), pD 2 . 5 , in (a) metal free solution, (b) with 0.040 M Co2+, (c) with 0.040 M Co2+and 0.009 M ATP. The shifts are measured downfield from a p-dioxane internal standard.
completely clear to us and a more thorough examination is needed. In order to estimate the influence of the ring-ring interacton on their resonance shifts in the Co2+-ATP-A complex the above study was repeated, but Mg2+ was used instead of Co2+. The results shown in Figure 16 reveal that negligible shifts are induced by one ring current on the second ring protons. We also consider this to be the case in the bis M2+-ATP complex.
Discussion (A) The Formation Constants. Comparing the formation constant obtained for the metal complexes of ATP3- and DATP3- (Table I), we find that the ring protonation does not significantly affect the metals binding to A T P as reflected in the formation constants. This implies that the main metal binding site is the phosphate chain, and indeed the secondary phosphate hydrogen ionization results in considerable changes in the binding constants.21It is interesting to note that in the Co*+-ATP system K2 5 K1, while in the Ni2+-ATP system Kz
References and Notes (1) D. P. Craig, E. A. Power, and T. Thirunamachandran, Chem. Phys. Left., 27, 149 (1974).
(a) B. Bosnich, J. Am. Chem. Soc., 98, 6143 (1976); (b) B. Bosnich, "Fundamental Aspects and Recent Developments in Optical Rotatory Dispersion and Circular Dichroism," F. Ciardelli and P. Salvadori. Ed.. Heyden and Son Ltd., 1973, Chapter 3.8. L. D. Hayward and R. N. Totty, Can. J. Chem., 49, 623 (1971). S. Claesson and L. D. Hayward, Chem. Phys. Lett., 20,85(1973). P. E. SchiDDer, lnorq. Chim. Acta, 14, 161 (1975). P. E. Schipper, Cheh. Phys. Left., 30, 323 (1975). L. D. Hayward and S. Claesson, Chem. Scr., 9, 21 (1976). S.F. Mason, Chem. Phys. Left., 32, 201 (1975). (9) P. E. Schipper, Mol. Phys., 29, 1705 (1975). (10) P. E. Schipper, lnorg. Chim. Acta, 12, 199 (1975). (11) N. H. Andersen, B. J. Bottino, A. Moore, and J. R. Shaw, J. Am. Chem. Soc., 96, 603 (1974). (12) J. Diilon and K. Nakanishi, J. Am. Chem. Soc., 96,4057 (1974); 97, 5417 (1975). (13) See, for example, A. B. P. Sinha. Spectrosc. lnorg. Chem., 2, 255 (1971). (14) See. for example: (a) F. Halverson, J. S.Brinen, and J. R. Leto, J. Chem. Phys., 41, 157 (1964); (b) ibid., 41, 2752 (1964); (c) H. Samelson, C. Brecher, and A. Lempicke. J. Mol. Spectrosc., 19, 349 (1966). (15) G. A. Cotton, F. A. Hart, and G. P. Moss, J. Chem. Soc.. Dalton Trans., 221 (1975). (16) H. G. Brittain and F. S. Richardson, J. Chem. SOC., Dalton Trans., in press. (17) H. G. Brittain and F. S. Richardson, J. Am. Chem. Soc., 98, 5858 (1976). (18) R. G. Charles, lnorg. Synth., 9, 37 (1967). (19) C. K. Luk and F. S. Richardson, J. Am. Chem. Soc., 96, 2006 (1974). (20) C. K. Luk and F. S. Richardson, J. Am. Chem. Soc., 97, 6666 (1975). (21) For a discussion of the emission anisotropy factor, gem,see: I. Steinberg, "Concepts in Biochemical Fluorescence",Vol. I, R. F. Chen and H. Edelhock, Ed., Marcel Dekker, New York, N.Y., 1975, Chapter 3. (22) J. P. Riehl and F. S.Richardson, J. Chem. Phys., 65, 1011 (1976).
Proton Magnetic Resonance Study of Divalent Metal Ions Binding to Adenosine 5'-Triphosphate Joseph Granot*' and Daniel Fiat Contribution from the Department of Structural Chemistry, The Weizmann Institute of Science, Rehouot, Israel. Received April 1 , 1976
Abstract: The monometallic Co2+, Ni2+, Mg2+, and Ca2+ complexes with adenosine 5'-triphosphate have been investigated by proton magnetic resonance. The experimental pD values were adjusted so as to have the adenine ring either protonated or unprotonated, and the phosphate chain triple or fully ionized. The effect of ring protonation or the secondary phosphate hydrogen ionization on the metal-ring and metal-chain interactions has been studied. The formation of a bis M2+-ATP complex was established. The equilibria equations involving the formation of 1:l and 1.2 metal-ligand complexes and the nucleotide molecules self-association are discussed, and a method for calculating the formation constants from the N M R shifts is presented. A model for the bis complex, compatible with the experimental results, is proposed.
Nuclear magnetic resonance has been extensively used in the study of adenine nucleotides and their metal complexes.2 Concerning the complexes of adenosine 5'-triphosphate (1) and NH2
I
0-
0-
Iy
IB
6-P-0
II
0
-P
0-
-0-P-
II
0
II
Hj H
HI'
0 O'H
OH
1 divalent metal ions, the main results may be summarized as follows. The paramagnetic ions (Mn2+, Co2+,Ni2+) bind simultaneously to the three (a,p, y) phosphates and to the adenine The binding to the ring is not direct but via a water molecule which is coordinated to the metal ion and hydrogen bonded to N7 of the adenine ring.9,10In addition, the Journal of the American Chemical Society
metal complex contains another two water molecules in the inner sphere of co~rdination.~-"A more recent I3C N M R study of the Mn2+ complex of A T P indicated however a direct binding of the manganese ion to the N7 ring nitrogen.13 At high total A T P concentration it was found that 1:2 metal-ligand complexes are formed.'*,' The diamagnetic ions (Mg2+, Ca2+) were found not to interact with the adenine ring63l5 and their binding is to the /3 and y phosphated or to the /3 phosphate alone. A T P has two major ionization sites: the phosphate moiety and the adenine ring. The three primary phosphate hydrogens were estimated to have pK 5 2," and the final hydrogen was found to have pK 7.1s-23Hence the major part of A T P will be fully ionized (ATP4-) at p H 2 8. At a lower p H both ATP4- and ATP3- will be present, and a t p H 5 5 the major part will be ATP3-. 31PN M R s t ~ d i e s ~revealed ? ~ , ' ~ almost no difference in the metal-phosphate interaction in the p H range 5.5-8.5.In the present work we show that the same is true also regarding the metal-ring interaction. This metal-ring interaction, however, may be expected to depend on the ionization
/ 99:l / January 5, 1977
-
71
state of the ring. The pK associated with the ionization from the protonated ring of A T P was found to be -4.20-22 The problem was to determine the site of protonation/ionization in the adenine ring. Although some workers have claimed that the ionization is from the protonated Cb-amino g r o ~ p , ~ ~ , ~ ~ , ~ ~ - ~ ~ it is commonly agreed that the proton ionization site is the (4) N I H + g r o ~ p . ~ * We - ~ Inow present a study on the effect of the ring protonation on the metal-ring interaction in divalent metal KlK2 = K3K4 (5) complexes of ATP. The possibility of formation of 1:2 metalligand complexes has been taken into account (for the parawhere M represents a divalent metal ion, L represents ATP in magnetic ions) and formation constants for the various proany one of it's ionization states (Le., +DATP3-, ATP3-, or cesses have been calculated. ATP4-), and [MI and [L] denote free concentrations. The total concentrations LOand M Oare given by Experimental Section Lo = [L] 2[L2] [ML] 2[ML2] (6) Materials. ATP (disodium salt) and adenosine (A) of highest grade
+
were obtained from Sigma Chemical Co. and were used without further purification. The metal salts Co(N03)2-6H20 and MgC126H2O were obtained from Merck and Ni(N03)2*6H20and CaC122H2O from BDH Chemicals. The salts were dried overnight by slight heating in vacuo (to prevent decomposing). Stock solutions (2-3 M ) of the nickelous and cobaltous salts were prepared with D20 (99.7%) as solvent and standarized by EDTA complexometry with murexide as indicator. All other concentrations were determined gravimetrically. The ATP concentrations used were in the range 0.06-0.09 M, the Ni2+ and Co2+ concentrations varied from 1 X to 5 X IO-' M, and those of Mg2+ and C a 2 + from 8 X to 6 X lo-' M. In the higher C a 2 + concentrations a small volume of precipitate appeared in the APT solution and was dissolved after slight heating of the solution (to about 50 "C). The pH was adjusted, using concentrated acid or base solutions in D2O. NMR Spectra. Proton magnetic resonance measurements were recorded on a Bruker HFX-IO spectrometer operating at 90 MHz. A trace ofp-dioxane in the experimental solutions served as internal reference for shift measurements. All the measurements were performed at an ambient probe temperature of 27 O C , except for one case (see below) when a temperature study was made. The temperatures were stabilized by a Bruker B-ST 100/760 control unit and were calibrated by measuring the peak separation of an ethylene glycol sample. The experimental error in the field shift measurements was taken as about 5% of the line width values but not less than 1 Hz. An average of three or four measurements has been taken in order to improve the overall accuracy. Experimental pD. Considering the following equilibria between A T P and D + ( H + ) in D2O solutions: +DATP3- s ATP3ATP3- s ATP4-
+ D+
pK'
+ D+
pK"
-
-
Mo = [MI
+ [ML] + [ML2]
AHp = (1
(7)
+PM-')AH~
(8)
where AHp is the field shift between the signals of the free and bound ligands, AHa is the observed coalescent signal shift with respect to metal free solution, and P M is the mole ratio of bound and nonbound ligands. In our case, having two types of complexes, P M is given by
[MLI + 2 w - 2 1 Lo - [ML] - 2[ML2] Substituting eq 9 in 8 we get PM
=
(9)
It should be noted that in eq 10 an assumption has been made that the ligands bound either ig the 1:l or the 1:2 complexes undergo the same field shift. This assumption will be discussed later on. In principle, by solving simultaneously eq 1-4 one can get expressions for [ML] and [ML2] in terms of the formation constants. Then, by fitting eq 10 to the experimental results the formation constants may be calculated. Since eq 1-4 cannot be solved analytically, an approximate solution is required. Equations 1 and 2 give
4
7
[MLI = K I[MI [Ll [MLd = KiK2[M1[LI2 Substituting (1 1 ) and (12) in (7) gives
1OpD-pK'
M o = [M](1 + Ki[L]
-
+
Taking M to be a paramagnetic ion then under conditions of fast chemical exchange (in the NMR scale) between free and bound ligands, the following relation holds35
where +DATP3- denotes a protonated ring state, we have [ATP3-i = [+DATP3-]
+
(1 1) (12)
+ K1K2[LI2) E [MI * Q ( K I , K ~ , [ L ] )
(13) The concentrations of the various types present may be written now as
Accordingly, the experimental pD values, which are the pH meter readings +0.4,32 were chosen to be: pD 3 for the study of the +DATP3- species, pD 5-6 for the study of ATP3-, and pD 8 for the study of ATP4-.
-
Calculation of Formation Constants Taking a solution containing divalent metal ions and ATP, in appropriate pH (pD), so that the ATP-proton reactions can be neglected, the major processes that take place are the formation of 1: 1 and 1:2 metal-ligand complexes and dimerization of ATP molecules due to self-association.12-33-34 (The complexation and association of higher orders, Le., ATP, and M2+-ATP,, n > 2, are neglected.) The equilibria and the formation constants involved may be expressed as
Lo = [Ll
[MI = Mo/Q
(14)
[MLI = KiMo[LI/Q
(15)
WL21 = KiKMo[LI2/Q
(16)
[Lzl = K4tL12
(17)
+ 2K4P-I' + Mo(Ki[LI + 2KiKr[LI2)/Q
(18) (i) Linear Approximation Solution. In solutions where the paramagnetic ion concentrations are small relative to the ligands, Le., Mo [ML2].It is important to note that while a priori knowledge of AHp is needed in the nonlinear approximation, this may not be required in the linear approximation (eq 23). Moreover, the linear approximation may also be used to derive the values of AHp. In the case of A T P complexes with Co2+ and Ni2+, values of AHp could have been directly measured and hence the nonlinear apprximation could have been applied.
Results and Interpretation Co2+-ATP. The proton spectrum of ATP at pD 3.0 is shown in Figure 1. In the course of the experiment which consisted of titration of A T P with Co2+.ions,the shifts of Hg, H 2 , and H I ' , and also those of other ribose protons, which we denote as H,, H, (a double line), and H , , were observed. (Another ribose proton signal was obscured by the water signal.) Some of the shifts were upfield and others downfield, relative to their
January 5,1977
, 73
2 00
, 01 i
02
03
04
(MI
[Co]
Figure 5. Measured Hg field shifts (to low field) in the CoZ+-ATPsystem at pD 3 ([ATP10 = 0.081 M).
log
[col
Figure 4. Calculated concentrations of the various species present in the ATP (0.081 M ) solution as a function of added Co2+ion concentrations. The formation constants from Table 1 were used. Table 11. Bound State Field Shifts (SHp), Line Widths (AvzM), and Metal-Proton Distances ( R )for the CoATP ( 1 : l ) Complex at pD 3" SHp, HZ
H2 Hx HI' H, HI H.
170 f 5 -350 f 20 285 f 5 111 f 5 -60 f 20 -250 f 20
S V Z MH, Z
25 f 3 450 f 50 3053 24 f 3 24 f 3 100 f 5
- I5
R, A 5.0 3.1 4.8 5.1 5 .o 4.0
H,, H I , and H, denote ribose hydrogens. Negativeshiftsare to low field.
metal-free shifts, indicating the presence of both contact and pseudocontact hyperfine interactions contributing to the shifts (compare with the solely downfield shifts observed for the NiATP complex, cf. Table V below). Since the chemical exchange of ATP in the cobaltous ATP system is f a ~ tthe ~ ,above ~ method for calculation of the formation constants could be applied. Since the H2 and H I shifts could be observed throughout almost the whole measuring range, they were thus used for the calculation. First we applied the linear approximation solution assuming Ll = LO.It came out that eq 24 is the appropriate one for this case. A least-squares fit of the experimental results (Figure 2 ) for [Co2+]0>- A~W M ~hence , the relevant relation for the line-broadening analysis is Av2p = P M T 2 M - I . 3 6 Let A v ~ M ( I ) and A u ~ Mbe ( ~the ) line widths in the bound state in the 1:l and 1:2 complexes, respectively, then we have:
-
+
([ML] AVZM(I) 2[ML2] A U ~ U ( ~ ’ ) /(33) LO The Hz and HI’ line width data were analyzed with eq 33 to yield the values of A V Z M (and ’ ) A U Z M ((Table ’) VI). The water coordination in the nickelous-ATP 1:l and 1:2 complexes has been studied by fitting the line widths (Figure 12) with eq 32 (the water signal shifts were too small to enable significant study). The results are given in Table 111, indicating a more pronounced shortening of the electronic relaxation time in the nickelous relative to the cobaltous A T P complex. W e should point out that our results for the proton saturation shifts in the NiATP complex are lower than those obtained by Glassman et aL9 and Sternlicht et aL4 from temperature studies. It is our opinion however that temperature studies should be very carefully interpreted and that simple temperature-dependence assumption may be erroneous. To demonA ~ 2 p=
/ Divalent Metal Ions Binding to Adenosine 5‘-Triphosphate
76 -03
4451--1
- 437 I
1
9
4421 2g*
221 213 0.0
Figure 12. The paramagnetic line broadening of the water coordinated in the Ni2+-ATP system at pD 3 ( 0 )and pD 5.5 (0).
0.25
0.50
0.75
[M2+] (M)
Figure 14. Measured proton field shifts in the M2+-ATPsystem, M 2 + = Mg2+ ( 0 )or Ca2+ (0).The shifts (to low field) are given relative t o p dioxane ([ATP10 = 0.082 M).
Table VII. Proton Field Shifts in the Mg2+- and Ca2+-ATP ( 1 : l ) Complexes a t pD 3
2.5
2.7
2.9
3.1
3.3
3.5
(OK-‘)
10’1~
Figure 13. Temperature dependence of the H2 (A)and H 1’ (A)field shifts in 1 : I8 and in 1. I : 1 ( 0 .0 respectively) Ni2+-ATP systems ([ATP10 = 0.082 M, pD 3 ) .
strate this we studied the protons field shifts temperature dependence of l : 18 and l . l : l Ni2+:ATP solutions (Figure 13). (The paramagnetic shifts were measured relative to metal-free A T P shifts in the appropriate temperatures.) The temperature dependence of the shifts in the Ni2+ diluted solution may be interpreted4 as governed by a chemical exchange process: as the temperature is raised the exchange becomes more rapid and the shifts of the bulk signals increase. In the limit of fast exchange a 1 / T dependence is expected.46 Clearly, the decrease of the shift (Figure 13) is much faster than predicted, in a similar “anomalous” way as found in the CoATP comp l e ~Moreover, .~ in the study of the 1.1: 1 solution the bound signals were observed directly, and these are expected to shift with 1 / T dependence in the whole temperature range. The results contradict this expectation completely. Taking into account the possible degrees of freedom of the A T P molele^^^^^ and the complexation and self-stacking temperature dependence we conclude that the temperature study of A T P complexes reflects mainly conformational and complexational changes rather than exchange controlled temperature behavior. Additional study of the nickelous-ATP complex a t pD 8.5 yielded bound state parameters similar, within the error range, to those obtained a t pD 5.5 (Table V). Mg2+-ATP and Ca2+-ATP. Measurements of proton field shifts in the magnesium and calcium ATP complexes a t pD 5.5 and 8.5 revealed no significant change (within the experimental error) relative to the metal-free A T P solution, in accordance with previous s ~ d i e s .At ~ , pD ~ ~ 3 however significant shifts were observed (Figure 14). The data were analyzed assuming the formation mainly of 1:l complex in the metal’s concentrations range investigated and using the binding constants and l o 1 . ’for the MgATP and C a A T P complexes as determined by fluorescence study a t p H 2.8.49 The results are Journal of the American Chemical Society
/
99:l
/
M?+
Hz
Hx
HI‘
Mgz+ Ca2+
-4.2 f 0.2 -7.6 f 0.1
-2.7 f 0.2 -3.4 0.1
-1.4 f 0.2 -12.7 f 0.5
given in Table VII. The broadening of the lines in either complex was found to be negligible. Co2+-ATP-Adenosine. The complexation of adenosine (A) with Co2+ in the absence and the presence of A T P has been studied. The aim was to examine the possibility of the formation of a ternary Co2+ATP-A complex, i.e., to demonstrate the ability of the CoATP monocomplex to bind a second molecule via ring binding alone. The measurements were performed a t pD 2 . 5 . (Adenosine has a pK 3.6 for ring p r o t ~ n a t i o n . ~The ~ , ~effect ~ ) of the cobaltous ions (0.040 M) on the Hs, H2, and H I ’ resonance of A (0.038 M ) is shown in Figure 15b. The very small effect (H2 and H I ’ were almost unchanged and H8 only slightly broadened and downfield shifted) is expected, considering the low formation constant between A and divalent metal i o m 2 Moreover the solvent (HDO) signal was shifted downfield to an extent compatible with the formation of a regular hexacoordinated cobaltous aqueous complex. The adenosine binding is thus an outersphere coordination. When A T P (0.009 M ) was added to the Co2+-A solution, a considerable effect on the A resonance (with Hg undergoing the largest) was induced (Figure 15c). Clearly A which binds very weakly to the Co2+ ion binds more strongly to the CoATP complex. The dependence of the field shifts of the Hs, H2, and H I ’ resonances of A, as well as that of the solvent (HDO) on the concentration of A T P added to the 1:l Co2+:A solution, has been studied. The results are shown in Figure 16. As the ATP concentration was raised more A was bound and water molecules were expelled from the cobaltous coordination sphere. This resulted in shifting the water signal back upfield, and saturation was achieved a t about half the original shift, for 1:1 Co2+:ATP concentrations, indicating that three water molecules were taken out from the aqueous complex as the CoATP complex was formed. However, as the A T P concentration exceeded that of Co2+ (and A), the competition between the free A T P and the adenosine bound in the ternary complex resulted in the latter being removed from the complex, as is inferred from Figure 16. It should be noted, however, that an A T P concentration-dependent irregularity of the adenosine H8 and H2 shifts is observed. The H8 shift is being saturated much faster than that of H2 and “decays” much faster for [ATP] > [A]. This phenomenon is not yet
January 5, 1977
-
77
dA 1 -
560
480
400
320
240
160
Figure 15. Proton magnetic resonance spectra of the Hx, Hz, and H I ’ resonances of A (0.038 M), pD 2 . 5 , in (a) metal free solution, (b) with 0.040 M Co2+, (c) with 0.040 M Co2+and 0.009 M ATP. The shifts are measured downfield from a p-dioxane internal standard.
completely clear to us and a more thorough examination is needed. In order to estimate the influence of the ring-ring interacton on their resonance shifts in the Co2+-ATP-A complex the above study was repeated, but Mg2+ was used instead of Co2+. The results shown in Figure 16 reveal that negligible shifts are induced by one ring current on the second ring protons. We also consider this to be the case in the bis M2+-ATP complex.
Discussion (A) The Formation Constants. Comparing the formation constant obtained for the metal complexes of ATP3- and DATP3- (Table I), we find that the ring protonation does not significantly affect the metals binding to A T P as reflected in the formation constants. This implies that the main metal binding site is the phosphate chain, and indeed the secondary phosphate hydrogen ionization results in considerable changes in the binding constants.21It is interesting to note that in the Co*+-ATP system K2 5 K1, while in the Ni2+-ATP system Kz
