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Two Metal Ions Coordinated to a Purine Residue Tolerate Each Other Well.

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Angewandte
Chemie
Stability Constants
Two Metal Ions Coordinated to a Purine Residue
Tolerate Each Other Well**
Bernd Knobloch, Roland K. O. Sigel,*
Bernhard Lippert, and Helmut Sigel
In biological systems two (or more) metal ions are often
located relatively close to one another.[1] For example, in the
active-site cavity of Cu,Zn superoxide dismutase the two
metal ions are linked by the imidazolate of a histidine residue,
and in certain iron clusters the metal centers may be bridged
by sulfide ions or cysteine residues. Not only proteins, but also
RNA molecules require monovalent and divalent metal ions
(usually Mg2+) for folding and activity.[2a–f] Several examples
are known in which up to five Mg2+ ions are located close to
one another,[2g–m] either to stabilize local structures and
tertiary contacts[2g–j] or to participate directly in catalysis.[2k–m]
The binding of Mg2+ (sometimes outersphere through water
molecules)[2c,i] often occurs at N7 and/or C(6)O of a guanine
residue.[2h,i,l] However, recently it was also proposed that the
deprotonated N1 position participates directly in catalysis by
the hairpin ribozyme.[2n] In proteins and RNA molecules
metal ions are held in place through binding to further
ligation sites located within the molecules. What happens in
the absence of such additional supporting binding sites? From
studies with kinetically inert metal ions, such as PtII, it is
known that two metal ions can bind simultaneously to N1 and
N7 sites of adenine[3a,b] and guanine.[3c,d] Except for the results
of one study with Pd2+,[4] no equilibrium data are available on
the simultaneous binding of two biologically relevant or
kinetically labile metal ions to a nucleobase in solution.
However, such information would be of high relevance with
regard to understanding the structure and function of
ribozymes.
To quantify the effect that two metal ions coordinated to
the same purine moiety exert on one another with respect to
[*] Dr. B. Knobloch, Prof. Dr. R. K. O. Sigel
Institute of Inorganic Chemistry
Universit6t Z8rich
Winterthurerstrasse 190, 8057 Z8rich (Switzerland)
Fax: (+ 41) 1-635-6802
E-mail: roland.sigel@aci.unizh.ch
Prof. Dr. B. Lippert
Department of Chemistry
Universit6t Dortmund
Otto-Hahn-Strasse 6, 44227 Dortmund (Germany)
Dr. B. Knobloch, Prof. Dr. H. Sigel
Department of Chemistry, Inorganic Chemistry
Universit6t Basel
Spitalstrasse 51, 4056 Basel (Switzerland)
[**] This study was supported by the Swiss National Science Foundation
under grant numbers PP002-68733/1 (R.K.O.S., FErderungsprofessur) and 20-66647.01 (H.S.), the Deutsche Forschungsgemeinschaft (B.L.), and the Fonds der Chemischen Industrie (B.L.), as well
as by the Swiss Federal Office for Education and Science within the
COST D20 programme (H.S.).
Angew. Chem. Int. Ed. 2004, 43, 3793 –3795
their binding stability, we studied the
metal-ion-binding properties of a
guanine residue with a metal ion
already coordinated at N7. The cationic complex [(dien)Pt(9EtG-N7)]2+
(Scheme 1), which consists of 9-ethylguanine (9EtG) with a kinetically inert
(dien)Pt2+ unit at N7, is suitable for this
purpose, as after deprotonation of the
H(N1) site, the coordination of an
additional metal ion can occur. Therefore, by potentiometric pH titrations
(25 8C; I = 0.1m, NaNO3)[5a,b] we determined first the acidity constant
KH
ðdienÞPtð9EtG-N7Þ for the equilibrium (1a):
Scheme 1. What is the
effect of metalation at
N7 on the binding of
M2+ to N1 in the cationic complex [(dien)Pt(9EtG-N7HN1)]+?
(dien = diethylenetriamine = 1,4,7-triazaheptane; 9-EtG = 9ethylguanine)
½ðdienÞPtð9EtG-N7Þ2þ Ð ½ðdienÞPtð9EtG-N7HN1Þþ þHþ
KH
ðdienÞPtð9EtG-N7Þ ¼
½½ðdienÞPtð9EtG-N7HN1Þþ ½Hþ ½½ðdienÞPtð9EtG-N7Þ2þ ð1aÞ
ð1bÞ
We obtained the result pKH
ðdienÞPtð9EtG-N7Þ = 8.17 0.03,
which agrees perfectly with the value 8.14 0.06 previously
determined by NMR spectroscopy.[5c] Comparison with the
[5a]
value for free 9EtG (pKH
shows that
9EtG = 9.57 0.05)
2+
coordination of a (dien)Pt unit at N7 of the guanine residue
increases the acidity of the proton at N1 by DpKa/Pt,H = 1.40 0.06.
The coordination properties of the deprotonated N1 site
of [(dien)Pt(9EtG-N7HN1)]+ were investigated with Cu2+
and Mg2+ according to the equilibrium (2a), in which
2+
PtN7·G·N1 represents the complex [(dien)Pt(9EtGN7HN1)]+ and (PtN7·G·N1M)3+ the bimetallic species
formed between [(dien)Pt(9EtG-N7HN1)]+ and M2+:
2þ
PtN7 G N1 þM2þ Ð ðPtN7 G N1MÞ3þ
KM
PtN7
G
N1M ¼
½ðPtN7 G N1MÞ3þ ½ PtN7 G N1 ½M2þ 2þ
ð2aÞ
ð2bÞ
Cu2+ was used because of its high affinity for nitrogen
ligands[6] and Mg2+ because of its biologically important role
in ribozymes.[2] The following stability constants as defined by
Equation (2b) were determined: lg KCu
PtN7
G
N1Cu = 3.45 0.25
and lg KMg
PtN7
G
N1Mg = 0.65 0.3. These results become meaningful if compared with the affinity of these metal ions for the
N1 position of deprotonated 9EtG, that is, lg KCu
Cuð9EtGHN1Þ =
4.45 0.25 for [Cu(9EtGHN1)]+[7a] and lg KMg
Mgð9EtGHN1Þ =
1.0 0.2 for [Mg(9EtGHN1)]+:[7b] The stability differences
due to (dien)Pt2+ coordination at N7 are Dlg KPt,Cu = (4.45 0.25)(3.45 0.25) = 1.0 0.35 (3s) and Dlg KPt,Mg = (1.0 0.2)(0.65 0.3) = 0.35 0.35 (3s) for the Cu2+ and Mg2+
systems, respectively. Clearly there is a repulsive interaction
between the two divalent metal ions coordinated at N1 and
N7, but surprisingly this repulsion is relatively small. As
expected, the repulsive effect upon the coordination of Mg2+
is smaller, as this metal ion commonly interacts with N sites of
this type in an outersphere manner.[7c–e] Thus repulsion is
reduced in accord with theoretical calculations.[8] However,
DOI: 10.1002/anie.200453987
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3793
Communications
the apparently low stability constant of the Mg2+ complex
should not be underestimated. In an RNA structure the water
molecules of the relatively stable hydration sphere of a Mg2+
ion are strongly linked through hydrogen bonds to a number
of other sites, including nucleobases,[2g–j,m] thus compensating
for the intrinsic low stability of the Mg2+ coordination.
How do the metal–metal and metal–proton interactions
compare to the proton–proton interaction between the N7
and N1 positions? The micro acidity constant[9a] pkH
N7N1
H
N7N1
H =
7.22 0.01 describes the deprotonation of the H(N1) site of
an N7-protonated H(9EtG)+ to give the zwitterionic tautomer (+HN7·9EtGHN1) . Therefore, the proton–proton
interaction amounts to DpKa/H,H = (9.57 0.05)(7.22 0.01) = 2.35 0.05 (3s). This repulsion is considerably larger
than the Pt2+–proton (DpKa/Pt,H = 1.40 0.06) and the Pt2+–
M2+ interactions (Dlg KPt,Cu = 1.0 0.35 (3s) and Dlg KPt,Mg =
0.35 0.35 (3s); see above). Thus, it follows that the clustering
of metal ions in RNA, especially of Mg2+, can occur
surprisingly easily, and we therefore expect that more
metal-ion clusters will be identified in RNA structures. Such
clusters are able to stabilize certain folds in the tertiary
structure or to serve as tools to promote catalysis. Indeed, it
has been proposed that several metal ions cooperate with one
another in ribozymes such as RNase P.[2f]
The results presented show that for divalent metal ions the
metal–metal repulsive interaction is smaller than the metal–
proton repulsion,[9b] which is in turn much smaller than the
proton–proton interaction. These findings are in agreement
with observations[4a] based on equilibrium studies of Pd2+–
purine systems by 1H NMR spectroscopic shift experiments.[4b] The effects of charges located at N7 and N1 of
purine residues are reciprocal and equal. That is, the effect of
a charge at N1 on that at N7 is equal to the effect of a charge
at N7 on that at N1; this equivalence has been shown for M2+–
H+ interactions.[9b]
The acidity constant pKH
ðdienÞPtð9EtG-N7Þ = 8.17 is close to the
physiological pH range. For guanosine and 2’-deoxyguanosine
residues pKa values are even lower by about 0.3 pK units.[5a,b]
In addition to the increase in acidity of H(N1) through
coordination of a metal ion at N7, the coordination of a
second metal ion at N1 further facilitates the simultaneous
deprotonation of H(N1) because of competition between the
metal ion and the proton for the binding site. Therefore, at a
physiological pH value of about 7.5, purine residues in
ribozymes may well coordinate two metal ions simultaneously
at N7 and N1.
Experimental Section
9-Ethylguanine was purchased from Sigma Chemical Co., St. Louis,
MO (USA). The complex [(dien)Pt(9EtG-N7)](ClO4)2·2 H2O was
prepared via the intermediate [(dien)PtI]I.[3c, 10a] For the other
reagents, see reference [5a,b]. The potentiometric pH titrations in
aqueous solution were carried out and evaluated as described
previously.[5b] For the determination of the acidity constant
pKH
ðdienÞPtð9EtG-N7Þ [Eq. (1b)], aqueous HNO3 (0.2 mm, 25 mL, 25 8C;
I = 0.1m, NaNO3) was titrated both in the presence and in the absence
of [(dien)Pt(9EtG-N7)]2+ (0.29 to 0.41 mm) with NaOH (0.03 m,
0.8 mL) under N2. The stability constants KM
PtN7
G
N1M [Eq. (2b)] of the
Mg2+ and Cu2+ complexes were determined under the same
3794
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
conditions,[10b] but with NaNO3 partly or fully replaced by M(NO3)2.
The ratios [(dien)Pt(9EtG-N7)]2+/M2+ were 1:90 for Mg2+ and 1:58,
1:45, or 1:36 for Cu2+. The error limits of the measured stability
constants are relatively large because the buffer depression (which is
the difference between the pKa value of the ligand and the apparent
acidity constant, pKa’, measured in the presence of M2+)[10b] in the
Mg2+ system is small and the experiments with the Cu2+ system were
hampered by the formation of hydroxo complexes. The value for
KH
ðdienÞPtð9EtG-N7Þ [Eq. (1b)] was calculated as the average of five
independent pairs of titrations; the stability constants [Eq. (2b)] for
the Cu2+ and Mg2+ systems are the average of three and four
independent pairs of titrations, respectively. The error limits indicated
correspond to three times the standard error of the mean value (3s)
or the sum of the probable systematic errors, whichever value is
larger.
Received: February 10, 2004 [Z53987]
.
Keywords: magnesium · metal–metal interactions · ribozymes ·
RNA · stability constants
[1] Handbook on Metalloproteins (Eds.: I. Bertini, A. Sigel, H.
Sigel), Dekker, New York, 2001, pp. 1 – 1182.
[2] a) R. K. O. Sigel, A. M. Pyle, Met. Ions Biol. Syst. 2003, 40, 477 –
512; b) A. M. Pyle, J. Biol. Inorg. Chem. 2002, 7, 679 – 690;
c) S. E. Butcher, Curr. Opin. Struct. Biol. 2001, 11, 315 – 320;
d) R. K. O. Sigel, D. G. Sashital, D. L. Abramovitz, A. G.
Palmer III, S. E. Butcher, A. M. Pyle, Nat. Struct. Mol. Biol.
2004, 11, 187 – 192; e) R. K. O. Sigel, A. Vaidya, A. M. Pyle, Nat.
Struct. Biol. 2000, 7, 1111 – 1116; f) M. BrInvall, L. A. Kirsebom,
Proc. Natl. Acad. Sci. USA 2001, 98, 12 943 – 12 947; g) C. C.
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B. C. N. M. Jones, R. Cosstick, T. R. Cech, Nature 1997, 388,
805 – 808; l) S.-O. Shan, A. Yoshida, S. Sun, J. A. Piccirilli, D.
Herschlag, Proc. Natl. Acad. Sci. USA 1999, 96, 12 299 – 12 304;
m) S.-O. Shan, A. V. Kravchuk, J. A. Piccirilli, D. Herschlag,
Biochemistry 2001, 40, 5161 – 5171; n) P. C. Bevilacqua, Biochemistry 2003, 42, 2259 – 2265.
[3] a) R. K. O. Sigel, S. M. Thompson, E. Freisinger, B. Lippert,
Chem. Eur. J. 2001, 7, 1968 – 1980; b) R. K. O. Sigel, S. M.
Thompson, E. Freisinger, B. Lippert, Chem. Commun. 1999, 19 –
20; c) G. Frommer, H. Schoellhorn, U. Thewalt, B. Lippert,
Inorg. Chem. 1990, 29, 1417 – 1422; d) B. Longato, G. Bandoli, G.
Trovo, E. Marasciulo, G. Valle, Inorg. Chem. 1995, 34, 1745 –
1750.
[4] a) R. B. Martin, Met. Ions Biol. Syst. 1996, 32, 61 – 89, see
page 84; b) K. H. Scheller, V. Scheller-Krattiger, R. B. Martin, J.
Am. Chem. Soc. 1981, 103, 6833 – 6839.
[5] a) B. Song, J. Zhao, R. Griesser, C. Meiser, H. Sigel, B. Lippert,
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[6] a) R. B. Martin, Inorg. Chim. Acta 2002, 339, 27 – 33; b) H. Sigel,
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H
[7] a) As the acidity constants pKH
9EtG = 9.57 0.05 and pK9MeG =
9.56 0.02 (9 MeG = 9-methylguanine) are identical, the averaged value of the stability constants of the corresponding Cu2+
complexes[5a] was used for comparison; b) the stability constant
of the Mg2+ complex of (dGuoHN1) (N1-deprotonated 2’[5b]
as the aciddeoxyguanosine) is lg KMg
MgðdGuoHN1Þ = 0.94 0.14;
[5b]
ity constant pKH
=
9.25
0.02
is
only
slightly
smaller than
dGuo
[5a]
and the stability of Mg2+
that of 9EtG (pKH
9EtG = 9.57 0.05)
www.angewandte.org
Angew. Chem. Int. Ed. 2004, 43, 3793 –3795
Angewandte
Chemie
complexes formed with N donor ligands is practically independent of the basicity of the N atom involved,[7c–e] the stability
constant for the [Mg(9EtGHN1)]+ complex is probably very
close to lg KMg
Mgð9EtGHN1Þ = 1.0 0.2 and well within the error
limits indicated; c) L. E. Kapinos, H. Sigel, Inorg. Chim. Acta
2002, 337, 131 – 142; d) L. E. Kapinos, B. Song, H. Sigel, Chem.
Eur. J. 1999, 5, 1794 – 1802; e) L. E. Kapinos, B. Song, H. Sigel,
Inorg. Chim. Acta 1998, 280, 50 – 56.
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Hobza, J. Phys. Chem. B 2000, 104, 7535 – 7544; b) J. E. Šponer,
J. Leszczynski, F. GlahL, B. Lippert, J. Šponer, Inorg. Chem.
2001, 40, 3269 – 3278.
[9] a) G. Kampf, L. E. Kapinos, R. Griesser, B. Lippert, H. Sigel, J.
Chem. Soc. Perkin Trans. 2 2002, 1320 – 1327; b) R. Griesser, G.
Kampf, L. E. Kapinos, S. Komeda, B. Lippert, J. Reedijk, H.
Sigel, Inorg. Chem. 2003, 42, 32 – 41.
[10] a) G. W. Watt, W. A. Cude, Inorg. Chem. 1968, 7, 335 – 338;
b) R. K. O. Sigel, B. Song, H. Sigel, J. Am. Chem. Soc. 1997, 119,
744 – 755.
Angew. Chem. Int. Ed. 2004, 43, 3793 –3795
www.angewandte.org
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3795
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