The Interaction of Metal Ions with Polynucleotides and Related

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Downloaded by CALIFORNIA INST OF TECHNOLOGY on March 30, 2017 | http://pubs.acs.org Publication Date: January 1, 1967 | doi: 10.1021/ba-1967-0062.ch025

The Interaction of Metal Ions with Polynucleotides and Related Compounds VIII. The Selectivity of Metal Ions for Coordination Sites on Biological Macromolecules GUNTHER L. EICHHORN Gerontology Branch, National Institute of Child Health and Human Development, National Institutes of Health, PHS, U . S. Department of Health, Education, and Welfare, Bethesda, M d . and the Baltimore City Hospitals, Baltimore, M d .

The nucleic acids offer a diversity of electron donor sites among which a coordinating metal ion may choose. There is opportunity for coordination to phosphate groups as well as to oxygen and nitrogen atoms on the heterocyclic com ponents of the nucleotides. Some metals (e.g., zinc (II), nickel (II), lanthanum (III)) select phosphate. Others, such as copper (II), silver (I), and mercury (II), prefer the heterocyclic electron donors. The phosphate binders stabilize the hydrogen-bonded conformation of the nucleic acids at low temperatures; at high temperatures they cleave the phosphodiester bonds. The metals binding to heterocyclic donors bring about the destruction of the hydro gen bonds because they react with hydrogen-bonded sites. Thus, metal binding to nucleic acids aids in understanding selective coordination and many facets of coordinated ligand reactions.

Among the applications of Werner's coordination theory that have gained momentum i n recent years has been the study of coordination com pounds of biological importance. The participation of metal ions i n enzymatic reactions is so widely understood that the activation of enzymes by metal ions is one of the properties of enzymes that is almost universally studied when new enzymes are isolated. Such activation involves the 378 Kauffman; Werner Centennial Advances in Chemistry; American Chemical Society: Washington, DC, 1967.


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formation of a metal complex of the enzyme protein. M a n y studies have been carried out recently on the nature of this metal-protein interaction. We have been particularly interested in the reaction of metal ions with another group of biological macromolecules—the nucleic acids. Interest in such reactions was initiated in the hope of attaining two objectives of biochemical importance: (1) the determination of nucleotide sequence (the genetic code) by selective reactions of metal ions with the various nucleotide bases and (2) the elucidation of the biological role of metal ions i n the transmission of hereditary information. Although our primary interest has been the use of coordination chem istry to solve biological problems, I believe that these studies have aided i n elucidating some problems of coordination chemistry. The coordination chemist has long been interested in the selectivity of the various metal ions for different electron donors. The attention of inorganic chemists i n terested in this phenomenon has been generally directed to a comparison of stability constants of metal complexes that are mostly similar but differ in some one structural feature. The study of biological macromolecules has led to an understanding of the behavior of metal ions with long poly mers that provide a large variety of electron donors among which a metal ion can choose. It is interesting that different metal ions make different choices and that the nature of the metaFs choice can have a profound effect upon the structure of the macromolecule. In the present paper we shall be concerned mainly with the results of previously published experiments on the reaction of metal ions with nucleic acids. This symposium presents a good opportunity to bring to gether the results scattered i n the literature and to make appropriate generalizations. The experiments themselves can only be alluded to, but the references cited should provide the reader with ample opportunity to verify the conclusions. Structure

of Nucleic Acids

In order to understand the nature of the interaction of metal ions with macromolecular ligands, it is necessary to understand the structure of the ligands themselves. There are two types of naturally occurring nucleic a c i d s — R N A and D N A . Each of these consists of a sugar ribose backbone to which the four bases shown in Figure 1 are attached (in R N A ) . (Ribose attached to 3 or 9 positions produces a nucleoside, and ribose phosphate a nucleotide. When the ribose has no 2 ' - O H group, deoxynucleoside and deoxynucleotide are produced.) The relationship of these bases to the sugar phosphate is indicated in Figure 2. A s shown, the bases are attached in various sequences. The top ribose shows the numbering used. (Adenine and guanine are bound by loss of the 9-proton; cytosine and uracil by that of the 3-proton.) The biological function of one type of R N A

Kauffman; Werner Centennial Advances in Chemistry; American Chemical Society: Washington, DC, 1967.


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WERNER

H

H

2

0

Cytosine

Uracil

NH

CENTENNIAL

Figure 1. Structures of nucleotide bases

molecule is to transmit the genetic message from D N A in the nucleus to the cytoplasm where protein synthesis occurs. D N A resembles R N A i n that i t contains adenine, cytosine, and guanine, but instead of uracil it has a 5-methyl derivative called thymine. The sugar in D N A is deoxyribose—i.e., the 2'-hydroxy group of the ribose present in R N A has been removed. The native D N A structure consists of two strands of "polydeoxyribonucleotide," each containing the four bases, intertwined as shown in Figure 3, with hydrogen bonding between the guanines on either chain and the cytosines on the other, and the adenines on either chain and the thymines on the other. It is immediately apparent that there are two very different kinds of sites for metal coordination available on an R N A and D N A molecule—i.e., (1) the phosphate groups on the sugar phosphate backbone and (2) the oxygen and nitrogen donor atoms on the "bases." We shall first discuss the consequences of binding to phosphate. Metal Ions Binding to Phosphate The first such consequence is to stabilize the ordered form of the nucleic acid molecule. In D N A this means that phosphate-binding metals will stabilize the double helix. It was discovered quite some time ago that, if D N A is dissolved in distilled water, the double helical structure is



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"denatured"—i.e., it is dissociated into two randomly coiled, single strands, as determined primarily from absorbance measurements (25, 27). The reason for this dissociation lies in the fact that the surface of the D N A molecule is laden with negative charges on the phosphates that repel each other, thus rendering the ordered structure unstable (5, 18, 27, 29). The neutralization of this charge by metal ions therefore stabilizes the molecule. High concentrations of alkali or alkaline earth metal salts will serve in this stabilization process as will much lower concentrations of divalent transi tion metals such as M n (II), Co (II), N i (II), and Zn (II). Thus, metal ions are required to preserve the structural integrity of the D N A molecule. Peculiarly these same metal ions that stabilize the ordered structure of the D N A molecule can be used for the depolymerization of

0

HO

0 CH OPO-

5'

2

0 0P0 .0

guanine

HO

0 0P0 ,0

HO

0

adenine

HO

CH

0 0P0 2

0 0P0

Figure 2.

Portion of RNA structure


382

CENTENNIAL


WERNER

B Figure 3. Structure of DNA: A. Hydrogenbonded base pairing of adenine and thymidine; B. Portion of double helix structure (30)



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R N A and synthetic polyribonucleotides that, like R N A , contain ribose as the sugar component but, unlike R N A , contain only one of the four bases in a polymeric chain (e.g., polyadenylic acid, or poly A ) . This depolymerization was discovered by Dimroth et al. (6, 7, 8, 9) and Bamann, Trapmann, and Fischler (1) and has been investigated ex tensively recently in various laboratories (21, 23), including our own (2,12). It consists of the scission of the phosphodiester linkages between the ribose and phosphate i n the backbone of the molecules. The reaction does not occur with D N A (1, 12), thus implicating the participation of the 2 ' - O H group of ribose and leading to a structural intermediate involving complex formation as follows:

0CH

BASE

\& 2

OH

0^0

(T 0-La x

0

The mechanism of this reaction resembles that of many coordinated ligand reactions in that the positive charge on the metal draws electrons (11, 16) from the phosphate and further labilizes the weakest link i n the chain, which happens in this instance to be the 5'-linkage. Among the divalent ions of the first transition series, rates of depoly merization were studied with M n (II), Co (II), N i (II), C u (II), and Zn (II). This is illustrated in Figure 4, where the ordinate is a measure of the extent of depolymerization (2). It is of interest to note that the reaction with zinc is approximately 10 times as rapid as with any of the other metals. Rates were also determined with trivalent rare earths [La (III), Ce (III), L u (III)], and these were similar to the rate with zinc (II). It should be pointed out that these depolymerization reactions are carried out at elevated temperatures. A t low temperatures R N A , like D N A , is stabilized by metal ions through the charge neutralization effect (18, 29). We have thus seen that coordination of metal ions with the phosphate group produces two strikingly different results with nucleic acid. A t low temperatures the conformation of the macromolecules is stabilized by a charge neutralization mechanism, and at high temperatures R N A and the polyribonucleotides are depolymerized. Metal Ions Binding to "Bases'

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Some of the most interesting reactions of the nucleic acids with metals are those i n which the electron donors are the heterocyclic "bases." I n -


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tensive studies have been carried out with copper (II), especially the reac tion of C u (II) with D N A . These studies have shown that C u (II) binds weakly to three of the four D N A bases (adenine, cytosine, and guanine), but apparently not thymine (15, 17). N M R studies to this effect are shown in Figure 5, where the compounds are all 0 . 1 M solutions in D 0 . For each set of spectra in this figure the top curve is the metal-free solution and the copper concentration is indicated for the others. The consequence of this type of binding on the structure of D N A has led to some very i n teresting findings.


2

15

20HrsO

5

10

15

20Hrs0

20

40

60

80

100 Min

TIME HEATING AT 64° C

Figure 4.

Depolymerization

of polyadenylic acid by divalent ions

When native double-stranded D N A molecules are heated with copper (II) at low ionic strength, some of the copper ions displace the hydrogen bonds that hold the two strands together (10, 14, 20, 28); the resulting structure is one in which single strands of D N A are weakly held together at some points by copper ions binding the bases. When the elec trolyte concentration of such a solution is then increased, the copper ions are displaced from their coordination sites, and the hydrogen bonds of the double-helical structure are reformed. (The evidence for the initial scission of the hydrogen bond b y copper (II) is derived mainly from " m e l t i n g " curves, which show that the double-helical structure of D N A "melts o u t " into single strands of D N A at a much reduced temperature in the presence of copper (II). The most essential evidence that copper ions take the place of hydrogen bonds is provided by the fact that the D N A regenerated



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Figure 5,


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Proton NMR spectra of the copper complexes of deoxynucleosides of (A) adenine, (B) cytosine, (C) guanine, and (D) thymine

by strong electrolyte is completely double-helical, as determined by several criteria—e.g., absorbance, sedimentation rate, optical rotation, and density gradient sedimentation. The reformation of the double helix after its destruction by copper ions can be explained only if the copper (II) holds the complimentary base pairs of the single strands i n register while the D N A is in the denatured state.) The reaction of D N A with copper (II) therefore takes place i n a delicate equilibrium, low ionic strength favoring displacement of hydrogen bonds by copper (II), and high ionic strength favoring displacement of copper (II) by hydrogen bonds. A few other metal ions [e.g., C d (II)] behave in a similar fashion, but copper (II) is virtually unique among metal ions i n its pronounced tendency to function in this reversible reaction. The reason for the ability of copper (II) to demonstrate such behavior must lie, at least in part, in the relative stability of the complexes of copper (II) with the "bases" and of the double-stranded structure of native D N A . If the copper complexes were weaker, they would probably not form at all. If they were stronger, it would be impos sible to replace copper (II) by hydrogen bonds through the addition of electrolyte. The fact that the "reversible" reaction with copper occurs must be explained by the circumstance that the copper complexes of the "bases" exhibit just the right stability range for the process to occur. It is in this manner, in fact, that it is possible to explain why some other metal ions that might be expected on the basis of their ordinary coor dination behavior to react in a manner similar to copper (II) do not do so. For example, M n (II), Co (II), N i (II), and Z n (II) do not displace the


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hydrogen bonds from double-stranded D N A (10). One could invoke complicated steric or electronic explanations for the difference between C u (II) and the other first transition elements, but the simplest explana tion appears to be that the copper complex, though weak, is just strong enough to compete with the hydrogen-bonded double helical structure of DNA. The nickel (II) complex simply doesn't make the grade. Another type of interaction between metal ions and the bases of D N A is represented by the mercury (II) and silver (I) complexes. These sub stances, first discovered by K a t z (22), and having received considerable attention recently by a number of investigators (3, 13, 26), especially Davidson and co-workers (4, 19, 24, 31, 32), resemble the copper (II) complexes in that silver and mercury ions also displace the hydrogen bonds of the double helix of D N A . They differ from the copper complex in that the reactions of these metals with D N A are not reversed by addition of electrolyte; in fact, the complexing reactions are carried out in the presence of strong electrolyte. The mercury (II) and silver (I) can be displaced from D N A binding by strong complexing agents such as E D T A . Conclusions The effects of different metal ions in the nucleic acids can be sum marized by considering the action of nickel (II), copper (II), and mercury (II). Nickel (II) reacts only with the phosphate groups; it stabilizes the ordered structure of D N A or R N A at low temperature by neutralizing the mutually repulsive negative charges on phosphate. A t higher temperature it depolymerizes R N A and R N A - l i k e structures by the scission of the ribose-phosphate bonds. Copper (II) ions react with the phosphate in a similar manner, and will also cleave the phosphodiester linkage of R N A . In addition, copper (II) ions react weakly with the heterocyclic "bases" and, for this reason, will bring about the splitting of the double helix in weak electrolyte and its reformation in strong electrolyte. Copper ions can therefore split an R N A molecule perpendicular to its axis and a D N A molecule along its axis. Mercury (II) does not react with the phosphates on the nucleic acids. It does react very strongly with the "bases" and therefore replaces the hydrogen bonds of D N A in a manner not reversible by strong electrolyte. To illustrate the versatility of the metal complexes of polynucleotides, it has recently been shown that zinc (II) can bind to the phosphate groups of the macromolecules to which "bases" silver (I) has already been attached. Evidence for this simultaneous binding is provided by the fact that the characteristic effect of silver (I) on the spectrum of the polynucleo-


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tides is observed at the same time that depolymerization by zinc (II) takes place.


Literature Cited (1) Bamann, E., Trapman, H., Fischler, F., Biochem. Z. 328, 89 (1954). (2) Butzow, J. J., Eichhorn, G. L., Biopolymers 3, 97 (1965). (3) Daune, M., Dekker, C. A., Schachman, H. K., Biopolymers 4, 51 (1966). (4) Davidson, N., Jensen, R. H., Biopolymers 4, 17 (1966). (5) Dove, W. F., Davidson, N., J. Mol. Biol. 5, 467 (1962). (6) Dimroth, K., Jaenicke, L., Heinzel, D., Ann. Chem. 566, 206 (1950). (7) Dimroth, K., Jaenicke, L., Vollbrechtshausen, Z. Physiol. Chem. 289, 71 (1952). (8) Dimroth, K., Witzel, H., Ann. Chem. 620, 109 (1959). (9) Dimroth, K., Witzel, H., Hülsen, W., Mirbach, H., Ann. Chem. 620, 94 (1959). (10) Eichhorn, G. L., Nature 194, 474 (1962). (11) Eichhorn, G. L., Bailar, J. J., Jr., J. Am. Chem. Soc. 75, 2905 (1953). (12) Eichhorn, G. L., Butzow, J. J., Biopolymers 3, 79 (1965). (13) Eichhorn, G. L., Clark, P., J. Am. Chem. Soc. 85, 4020 (1963). (14) Eichhorn, G. L., Clark, P., Proc. Natl. Acad. Sci., U. S. 53, 586 (1965). (15) Eichhorn, G. L., Clark, P., Becker, E. D., Biochem. 5, 245 (1965). (16) Eichhorn, G. L., Trachtenberg, I. M., J. Am. Chem. Soc. 76, 5183 (1954). (17) Fiskin, A. M., Beer, M., Biochem. 4, 1289 (1965). (18) Fuwa, K., Wacker, W. E. C., Druyon, R., Bartholomay, A. F., Vallee, B. L., Proc. Natl. Acad. Sci., U. S. 46, 1298 (1960). (19) Gillen, K., Jensen, R., Davidson, N., J. Am. Chem. Soc. 86, 2792 (1964). (20) Hiai, S., J. Mol. Biol. 11, 672 (1965). (21) Huff, J. W., Sastry, R. S., Gordon, M. P., Wacker, W. E. C., Biochem. 3, 501 (1964).

(22) Katz, S., J. Am. Chem. Soc. 74, 2238 (1952).

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Nandi, U. S., Wang, J. C., Davidson, N., Biochem. 4, 1687 (1965). Shack, J., Jenkins, R. J., Thompsett, J. M., J. Biol. Chem. 203, 373 (1953). Singer, B., Fraenkel-Conrat, H., Biochem. 1, 852 (1962). Thomas, R., Trans. Faraday Soc. 50, 304 (1954). Venner, H., Zimmer, Ch., Biopolymers 4, 321 (1966). Wacker, W. E. C., Vallee, B. L., J. Biol. Chem. 234, 3257 (1959). Watson, J. D., Crick, F. H. C., Nature 171, 737 (1953). Yamane, T., Davidson, N., J. Am. Chem. Soc. 83, 2599 (1961). Yamane, T., Davidson, N., Biochim. Biophys. Acta 55, 609 (1962).

RECEIVED June 30, 1966.


The Interaction of Metal Ions with Polynucleotides and Related

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