3209 J. Kraut and J. J. Reed, Acta Crystallogr., 15, 747 (1962). I. L. Karle and K. Britts. Acta Crystal/ogr., 20, 118 (1966). J. Pletcher, M. Sax, S. Sengupta. J. Chu, and C. S. Yoo, Acta CrystaC logr., Sect. 8,28, 2928 (1972). C. H. Carlisle and D. S. Cook, Acta Cfystallogr., Sect. B, 25, 1359 (1969). A. F. Cameron, K. P. Forrest, and G. Ferguson, J. Chem. SOC.A, 1286 (1971). T. V. Long, 11, A. W. Herlinger. E. F. Epstein. and i. Bernal, horg. Chem.,
9, 459 (1970). (32) J. T. Veal and D. J. Hodgson. Inorg. Chem., 11, 597 (1972). (33) W. C. Hamilton and J. A. Ibers, "Hydrogen Bonding in Solids", W. A. Benjamin, New York, N.Y., 1968. (34) I. D. Brown, Department of Physics, McMaster University, Hamilton, Ontario, private communication, 1974. (35) L. Pauling. "The Nature of the Chemical Bond", 3rd ed, Cornell University Press, lthaca, N.Y.. 1960, p 260. (36) F. Jordan, J. Am. Chem. SOC.,g6,3623 (1974).
Nucleic Base-Metal Ion Interactions. Acidity of the N( 1) or N(3) Proton in Binary and Ternary Complexes of Mn2+, Ni2+, and Zn2+ with the 5'-Triphosphates of Inosine, Guanosine, Uridine, and Thymidine Helmut Sigel Contribution from the Institute of Inorganic Chemistry, University of Basel. CH-4056 Basel, Switzerland. Received August 20, 1974
Abstract: By uv difference spectra and potentiometric titrations the acidity constants, K H ~ r pof, the nucleic base residues, i.e., the proton at N( 1) in ITP4- and GTP4-, or at N(3) in U T P - and TTP-, and of their complexes with Mn2+, Ni2+, and Zn2+ ( K H ~ ( ~ have ~ p )been ) determined; the data for the Cu2+ and Mg2+ complexes were taken from earlier work. The influence of the metal ions was characterized by A ~ K A = pK"~p p K H ~ ( ~ which ~ p ) ,is in the order of 0.2 for MgZ+,0.3 for Mn2+, 0.9 for NiZ+,1.9 for Cu2+,and 1.2 for Zn2+. As the difference in ~ K for A the proton of N(l) or N(3) for the nucleotides and the corresponding nucleosides is about 0.4 log units, the following is concluded. (i) There is a considerable interaction between the metal ion and the nucleic base residue in the nucleotide complexes of Ni2+, Cu2+, and Zn2+. (ii) On the structure of the complexes with Mg2+ and MnZ+nothing can be said, as the small shift in acidity may simply be due to the partial neutralization of the negatively charged phosphate chain by the metal ion. In the mixed-ligand 2,2'-bipyridyl nucleo~) about 0.6, thus indicating similar structures. Based on tide complexes with Ni*+, Cu2+, and Zn2+ A P K A ( B ~is~always known structures, a folded form is suggested which allows an intramolecular charge-transfer interaction between the purine or pyrimidine and pyridyl moieties. The tendency of the binary complexes to form hydroxo species, M(L)(OH)"-. increases for a given nucleotide, with Mn2+ < Ni2+ < Zn2+ < Cu2+, while it decreases for a given metal ion, with L = CTP4ATP4- > (UTP-lH)S- (TTP-1H)5- > (ITP-lH)S- (GTP-IH)S-, hence, indicating in the latter series a n increasing degree of saturation of the coordination sphere of the metal ion.
-
-
-
-
Virtually all enzymes requiring nucleoside phosphates as substrates need in addition a divalent metal ion.' T o understand the role of these metal ions, it is necessary to learn more about the structures of their b i n a r ~ ~and - ~ ternary (mixed-ligand)6-8 nucleotide complexes. So far the 3d metal ion-adenine nucleotide complexes are best studied; several of the corresponding A T P complexes exist in the ring bound Le., the metal ion coordinates both to the phosphate chain and to N(7) of the adenine moiety. Considerably less is known on the interaction between metal ions and the base moieties of other nucleotides. Even though the stability of divalent earth alkali and 3d metal ion nucleotide complexes is governed by the coordination tendency of the phosphate chain^,^.^.'^-'^ the nucleic bases determine a t least in part the specificity because in most enzyme reactions the nucleotides are not freely interchangeable. Hence, there is a clear relationship between structure and reactivity. This is also evident from the Cu2+ promoted dephosphorylation of nucleoside 5'-di- and -triphosphates, where the rate depends on the extent of the metal ion-nucleic base i n t e r a ~ t i o n , ' ~which - ' ~ in turn is dependent on the nucleic base. Before the understanding of such structure-reactivity relationships will improve, it is necessary to study metal ionnucleic base interactions more in detail. I t is the aim of this study to shed some light on the structures of the complexes
formed by Mn2+, Ni2+, and Zn2+ with ITP, GTP, UTP, and TTP. The I U P A C numbering system is indicated on Chart I; for convenience N ( 1) of the purine and N(3) of the pyrimidine nucleotides are both labeled N(A). In contrast to A T P and CTP. the nucleotides of Chart I
R =ribosyl 5'4rIphosphate ITP : R2 = - H
UTP:Rg:-H
GTP: R 2 = - N H 2
TTP: R5'-CH3
-
Abbr: N(N N(t) of ITP or GTP,
and
N(3) of UTP or TTP
have a proton at N(A). In case the base moieties of the nucleotides participate in complex formation one expects that this proton ionizes a t a lower p H than in the free 1igands.l' Due to the recent work of Clarke and Taubels on guanosine-ruthenium complexes, it is now possible to reach conclusive results also for "labile" metal ions, like Ni2+ and Zn2+. Certainly, if the base moieties are prevented from a Sigel
/ Nucleotide Complexes of M n 2 + , N i 2 + , and Zn2+
3210 direct coordination by the formation of ternary complexes, e.g., with 2,2'-bipyridyl, the acidification of the proton a t N(A) should be considerably smaller; this is found indeed.
I
i
Experimental Section Materials. All materials were reagent grade and used without further purification. The sodium salts of the nucleoside 5'-triphosphates ("Type I" or "Sigma grade") were purchased from Sigma Chemical Co., St. Louis, Mo. The perchlorates of Mn", Ni", Zn" (all purum) and Na' (purissimum), and 2,2'-bipyridyl (purissimum) were from Fluka AG, Buchs, Switzerland. The exact metal ion concentration of the perchlorate stock solutions was determined with EDTA. Apparatus. The potentiometric titrations were carried out with a Metrohm Potentiograph E 336 and Metrohm EA 121 UX glass electrodes. The absorption spectra were recorded with a Beckman spectrophotometer DB, connected with a W + W Electronic Hispeed Recorder 202. The p H measurements were performed with a Metrohm potentiometer E 353 B with microglass electrodes E A 147. Potentiometric Measurements. Stock solutions of N T P - (cf. ref 19) were always freshly prepared by rapidly titrating with N a O H the dissolved di- or trisodium salts to the equivalence point. Also, reaction solutions containing metal ions were immediately titrated after mixing to prevent dephosphorylation.'5.'6 The acidity constant for the deprotonation of N ( A ) was determined from automatic titrations of aqueous solutions containing 2 X M HClO4 and NaC104 ( I = 0.1) in the presence and absence of NTP4- (8 X M ) under N2 with 0.1 M N a O H (25O). The difference of such a pair of titrations was evaluated and the constants were calculated from the p H range which corresponded to a degree of neutralization between 0.1 and 0.9. Of T T P only limited amounts were available; therefore, the titrations were carried out in solutions containing 3.2 X M TTP4- by using 4 X IO-* M N a O H . The acidity constants for the deprotonation of N ( A ) in the binary complexes were determined in solutions that contained in addition the metal ion in a ratio of 1:l with regard to NTP4-. The binary complexes are formed under these conditions to a high degree. The same is true for the ternary complexes containing 2,2'bipyridyl, where the acidity constants were determined from solutions containing NTP4-, metal ion, and Bipy in a ratio of 1: 1:1. For the binary systems the neutralization degree exceeded a value of 1, indicating the formation of hydroxo complexes. The corresponding constants were calculated by taking into account (where necessary) the overlapping buffer regions.20 ATP4- or CTP4- and M2+ M ) in 1:l solutions were also titrated and the constants for the hydroxo complexes calculated. Constant values were only obtained within the range of the degree of neutralization 0.1--0.6 (cf. also ref 15). These values for the A T P complexes agree reasonably with those determined by Brintzinger.21Certainly, these constants were obtained only from titrations at a single concentration and, therefore, do not exclude ,~~ the possible formation of dimers, e+, [ Z ~ ( A T P ) ( O H ) ] I ~ -but they completely satisfy the titration curves within the given limits. Spectrophotometric Measurements. The stock solutions of NTP4- were prepared as described. The deprotonation of N ( A ) in the nucleotides and in their binary complexes results in a spectral change (cf. ref 17) that may be used to determine the acidity constants. To obtain a clear-cut situation, uv difference spectra (cf. ref 17) had to be recorded ( I = 0.1, NaC104; 25'). They were taken by M placing in the reference beam one quartz cuvette with 4 X NTP4- (pH 6.2-6.8) in aqueous 0.1 M NaC104 and a second one M ) also in 0.1 M with the metal ion perchlorate (4 X NaC104; the sample beam contained one quartz cuvette with the mixed system in 0.1 M NaC104 (with values of p H between 5 and 10) and one with the solvent (aqueous 0.1 M NaC104). As the absorption of the reference solutions was high, the spectrophotometer was used on "manual" with the slit at 2.2 mm. With I T P and G T P 2-mm cuvettes were used and with L'TP and T T P 1-cm cuvettes. The desired p H in the mixed NTP-metal ion system was adjusted under N2 by dotting with a glass stick and 2 N N a O H (or 2 N HC104); the change in volume was negligibly small. The acidity constants were determined according to the proce-
Journal of the American Chemical Society
97:11
1.5
'-K~"(,rp,=-"52.10-5 -10
ApK~(~ipy).~~ For the binary metal ion-nucleotide systems the degree
1 M a y 28, 1975
321 1 Table I. Acidity Constants, Determined from Potentiometric Titrations, for the Deprotonation of N(A) in Nucleoside 5'-Triphosphates,Q,b in Their Binary Mn2+, Ni*, or ZnZf Complexes, and in Their Corresponding Ternary Complexes23Formed with 2,2'-Bipyridyl (I = 0.1, NaC10,; 25")b
M
NTP
PK~M(NTP)
kinZ+
ITP GTP UTP TTP I TP GTP UTP TTP I TP GTP UTP TTP
8.93 i 0.02 9.36 k 0.01 9.45 i 0.02 9.67 i 0.03 8.39 i 0.03 8.64 i 0.03 9.10 * 0.03 9.08 i 0.09 8.31 i 0.04 8.39 t 0.05 8.71 i 0.04 8.35 t 0.05
NiZ+
Zn2+
APKA
pKHM(Bipy)(NTP)
ApKA(Bipr)
0.33 0.43 0.25 0.22 0.87 1.15 0.60 0.81 0.95 1.40 0.99 1.54
8.77 i 0.05 9.16 i 0.04 9.24 i 0.03 9.42 i 0.05 8.87 i 0.02 9.20 ? 0.03 9.13 i 0.02 9.06 t 0.05
0.59 0.63 0.46 0.47 0.39 0.59 0.57 0.83
'7pK"~p = 9.26 i 0.01 for ITP, 9.79 t 0.02 for GTP, 9.70 i 0.01 for UTP, and 9.89 * 0.03 for TTP. b The results are the average of the constants calculated from three independent titrations. The single exceptions are the data for the TTP systems; these were titrated only once due t o scarcity of this nucleotide. The range of error is three times the standard deviation. Table 11. Tendency for the Formation of Hydroxo Complexes (I = 0.1, NaC10,; 25')Q
9.58 9.41 9.7 9.9 10.6 10.57
10.87 t 0.13 10.7 11.1 11.2 11.24 i 0.15 11.3
CTP4ATP4(UTP-1 H) '(TTP-1 H)5(ITP-1H ) ~ (GTP- 1H) -.
i
0.15
+ 0.08
* 0.05
8.79 i 0.05 8.87 i 0.04 9.24 i 0.11 9.2 9.4 9.48 f 0.07
7.6b 7.9,b 8.17C 8.4b 8.2b 9.2b 9.3b
QTherange of error is three times the standard deviation; where no number is given the corresponding range is approximately k0.2 log units (cf. footnote b of Table I). b From ref 17. CFrom ref 15. Table 111. Acidity Constants, Determined from Spectrophotometric Measurements, for the Deprotonation of N(A) in Nucleoside 5'-Triphosphates and in Their Binary MnZ+, Ni2+, or Zn" Complexes (I = 0.1, NaC10,; 25")a NTP ITP GTP UTP TTP
9.0b 9.5b 9.6b 10.lb
8.8 9.3 9.3 9.6
0.2 0.2 0.3 0.5
8.2 8.6 9.1 9.3
0.8 0.9 0.5 0.8
8.2 8.3 8.8 8.7
0.8 1.2 0.8 1.4
aThe results are the average constants obtained from at least two (usually three) series of spectral measurements. The range of error corresponding to three times the standard deviation is approximately k0.2 log units, with the exception of Zn(GTP) and the TTP systems; in these latter cases only a single determination could be carried out and hence the error may be rather large. b From ref 17.
of neutralization exceeded unity in the potentiometric titrations (cf. Experimental Section). This was attributed to the formation of hydroxo complexes according to eq 6 . For
Discussion As we have seen, the ionization of the proton a t N ( A ) is facilitated in all the binary metal ion-nucleotide complexes. The characteristic quantities, i.e., the values of APKA, are M(NTP-1H)(H,0),3- === M(NTP-lH)(OH)'- + H' (6) summarized in Table IV, together with the data of Cu2+ K H M m T p - l q H , o ) = [M(NTP-1H)(OH)4-I[H']/[M(NTP-1H)3-] and those obtained by Sari and B e l a i ~ hfor ~ ~the Mg2+ complexes. The proton a t N ( A ) in the binary nucleotide comparison the formation of hydroxo complexes containing CTP"- or ATP4- was also studied. The results are summacomplexes is increasingly acidic within the series Mg2+ < rized in Table 11. Mn2+ < Ni2+ < Zn2+ < Cu2+. The ionization of the proton a t N ( A ) is coupled with a On the Structure of the Ni2+, Cu2+, and Zn2+ Nucleosignificant spectral change in all these n ~ c l e o t i d e s , as ' ~ well tides. For a further evaluation of the results it is helpful to as in their binary metal ion complexes. Hence, it was possicompare the data of these nucleotide complexes with those ble to determine by an independent method, i.e., by recordof nucleoside ones. In the latter complexes no phosphate ing uv difference spectra, the acidity constants for the binagroups are present and therefore the only binding sites are ry complexes according to eq 2. The results are given in those of the base. From the data given by Fiskin and Beer25 Table 111, and their comparison with the data of Tables I for the guanosine-Cu2+ system one may ~ a l c u l a t e 'the ~ and I1 demonstrates that the (so far) tentative assignments shift of the acidity constant for the proton of N ( l ) under for the acidity constants obtained from the potentiometric the influence of Cu2+ coordinated to N(7) and obtains: titrations are correct; Le., the first acidity constant (Table APKA = p K H ~ u o- p K H ~ ( ~ u=o 9.24 ) - 7.05 = 2.2. ObI) is always due to the deprotonation of N ( A ) in the binary viously, the increased acidity of the proton at N ( 1 ) is only nucleotide complexes while the second one (Table 11) must stronger by a factor of -2 (0.3 log units), compared with then refer to the formation of hydroxo complexes. The the corresponding Cu2+ nucleotides (Table IV). Hence, in values of A ~ K A determined by potentiometry and spectrothese latter complexes a Cu2+-nucleic base interaction defiphotometry agree satisfactorily. nitely exists. The somewhat smaller increase of the acidity Sigel 1 Nucleotide Complexes of Mn2+,N i 2 + ,and Zn2+
3212 Table IV. Values of APKA = P K H ~ -~ pp K H ~ ( ~ for ~ p ) Several M2+ Nucleotide Systems
ITP GTP UTP TTP
0.2a 0.2a 0.2a
0.33 0.43 0.25 0.22
0.87 1.15 0.60 0.81
1.7b 1.9b 1.7b 2.1b
0.95 1.40 0.99 1.54
~~
aValues from Sari and B e l a i ~ hb. ~From ~ ref 17.
in the nucleotide complexes is certainly due to the additional coordination of Cu2+ to the fourfold negative phosphate chain. Of further substantial help are the data of Clarke and Taube18 on pentaammineruthenium-guanosine complexes, where Ru(I1) or Ru(II1) is firmly coordinated to N(7): A P K A / R ~ ( ~=I )0.8 and A P K A / R ~ ( I I= I ) 2.2. These results allow to conclude that values of A ~ K A as small as -0.8 still evidence a considerable metal ion-base interaction. For further reasonings on the structure of these complexes it should be noted that Sternlicht et a1.26 proposed in 1968 that under the conditions [M2+]/[ATP4-]
