118
J. Phys. Chem. 1980, 84, 116-118
Noncovalent Interactions and Paramagnetic Relaxation Probes. Investigation of the Correlation Timest Elena Gaggelll, Claudlo Rossi, and Enzo Tlezzl* InsfEtute of General Chemistry, University of Siena, Siena, Ita& (Received June 29, 1979)
Publication costs assisted by the Institute of General Chemistry
An EPR and NMR combined analysis has been carried out in the study of noncovalent interactions involving metal ions. Manganous ion was chosen as the paramagnetic relaxation probe. The 5’-ATP-Mn(II)-(apo-BCA) and the 5’-ATP-Mn(II)-Trpternary systems have been investigated under different experimental conditions. The EPR line shape was analyzed in terms of a relaxation model based on a distribution of ZFS sites. Typical inverted spectra have been found whenever stacking interaction occurred. Paramagnetic contributions of Tt;l and T2;’ to 13Cand IH relaxation rates were investigated by taking into account a distribution of correlation times and the competition between T, and 7, connected with the formation of ternary species.
Introduction Stacking adducts between amino acid aromatic moieties and purine or pyrimidine bridged by metal ions are involved in many biological processes. Several papersM pointed out that stacking adduct formation is a driving force in cellular biochemistry, underlining the fundamental role of metal ions as far as both enzyme activations and specific interactions between proteins and nucleic acids are concerned. In this report we take into account the 5’-ATP-Mn(11)-Trp (tryptophan) complex where the metal ion favors stacking of indole and purine moieties;lI2data on the 5’ATP-Mn(I1)-BCA (bovine carbonic anhydrase) are also reported. Manganous ion was chosen as paramagnetic probe because of its suitability to give information both on structural and dynamic feature~.~-’OMoreover the use of a relaxation probe allows a combined EPR and NMR analysis based on (i) EPR line shape study of the metal ion; (ii) 13CNMR relaxation rates of the ligand nuclei; and (iii) lH NMR relaxation rates of the ligand protons; the experiments concerning the last two points may be perM) of the metal formed at very low concentrations ( N ion due to the detectable effects of the paramagnetic contribution to nuclear spin relaxation. Recently we presented’l EPR experimental evidence of ternary complex formation in the case of the 5’-ATPMn(I1)-Trp system. The aim of this paper is to interpret the EPR spectra in terms of both the electron spin relaxation analysis and the dynamics in solution. On the other hand, the EPR line shape analysis allows a deeper insight into the Solomon-Bloembergen-Morgan (SBM) equations in terms of the correlation times modulating the paramagnetic interactions, namely, the presence of the stacking interaction and the formation of a ternary species lead to different values of the structural and dynamic parameters in the SBM expressions. In order to understand this varition several experiments have been performed by changing concentrations, temperature, and frequency. Moreover particular attention has been devoted to the through-water interaction between the paramagnetic metal ion and the ligand nuclei, since the exchange of molecules +Dedicatedto Sam Weissman. 0022-3654/80/2084-0116$01.OO/O
in- and outside the metal solvation shell may lead to mechanisms involving solvent-separated species (outer sphere).
Experimental Section 5’-Adenosine triphosphate disodium salt and tryptophan were Merck reagent grade. Mn(C104)2.6H20(Alpha Inorganic) was used in order to minimize anionic complexation. NMR solutions were prepared by dissolving weighted amounts of ligand in 99.75% D20 (Merck). Apo-BCA was kindly supplied by Professor I. Bertini, University of Florence. pH values were measured by a Metrohm Model E-388 pH meter. EPR spectra were recorded with a Bruker ER 200tt spectrometer operating at X-band (9200 MHz) and with a Varian V-4651 spectrometer operating at Q-band (34 350 MHz). Temperature variations were obtained with a Bruker B-ST 100/700 temperature control unit (accuracy fl K). NMR spectra were obtained by using a Bruker WH-90 spectrometer operating at 90 and 22.63 MHz for lH and 13C,respectively. Transverse relaxation times were measured from half-height line widths assuming Lorentzian line shape. Longitudinal relaxation times were calculated from partially relaxed spectra by using the pulse sequence (180-~-90-t),. T1values were averaged over at least three measurements, and the error was calculated as f 5 % , The paramagnetic contributions T1;l and T2C1 were obtained from
All 13C NMR spectra were performed by using a broad band modulated proton decoupling frequency.
Results and Discussion Since the electron spin relaxation of manganous ion in solution is dominated by the modulation of the zero field splitting (ZFS) term,s the EPR line shape analysis yields information on structural and dynamic features. In fact the ZFS is the major structure-sensitive term, whereas dynamic information is provided by correlation times. Whenever stacking occurs between two different ligands and the metal ion stabilizes the adduct, the asymmetry of the ternary species is reflected in the ZFS term. In this case a loss in the EPR intensity is expected, due to very short electron spin relaxation times and to the existence of slow exchange conditiondl in the EPR time scale. 0 1980 American Chemical Society
The Journal of Physical Chemistty, Vol. 84, No. 1, 1980
Paramagnetic F;elaxation Probes
117
hi,
Q-
band
+J.-L
Flgure 1. Q-band (w = 2.16 X 10‘‘ rad/s) EPR spectrum of the 5’-ATP-Mn(II)-apo-BCA complex at pH 7 and T = 300 K: [Mn’”] = 2.5 X M; [B‘-ATP] = 2.5 X lo-* M; [apo-BCA] = 2.5 X
M.
The Q-band EPR spectrum of the 5’-ATP-Mn(II)-apoBCA system in Figure 1is a typical example of “inversion”, that is, the line width variation of the hyperfine components causes the inner, instead of the outer, components to become This corresponds to the w o 2 ~ , 2:> 1 condition. Moreover the X-band spectrum displays a broad cunresolved line with a strong frequency dependence. The observed intensity loss, which is particularly relevant whenever ternary species are involved,ll is consistent with electron spin relaxation times much shorter than those of the firee ion; thus it is possible to suggest a distribution of T,, which is the electron spin correlation time in the SBM equations for nuclear relaxation, ranging from 5 X to Ei X s. Both EPR intensity loss and “inversion” o f the spectra point to the presence of a stacking interaction. Moreover EPR spectra of the binary Mn(I1)-npo-BCAsystem do not give rise to similar features. Nevertheless, if, on one hand, it is possible to state that a ternary speciies is present, on the other hand, due to the complexity of the BCA molecule, it is only possible to suggest the presence of a stacking interaction between BCA and 5‘-ATP in the ternary complex. Figure 2 relers to the predominant presence of the ternary species. In fact in these experimental conditions an increase in the 5’-ATP concentration from 4 X to 7.2 X M leads to a dramatic change of the EPR line shape, accompanied by an intensity decrease;ll whereas in the parent blinary system, 5’-ATP-Mn(II), over a larger range of concentration ratios, these effects are not present. It is noteworthy that Trp does not contribute remarkably to line shape variations even at the highest Trp:metal ratios. In this3 case line shape analysis points out the suitability of tlhe EPR technique to provide evidence for stacking adducts bridged by the metal ion: the noncovalent interaction yields shorter T, values (Figure 2B). Another feature, which is usually omitted, is the event of outer-sphere coordination, that is, a through-water interaction of the biomolecule with the metal ion. Electron spin relaxation offers LL dual time scale from this point of view; in fact the exchange between different complexes is in the slow exchange limit in the EPR time scale (giving rise to the intensity loss), whereas exchange between free ions and outer-sphere coordinated ions is in the fast exchange lirnit,l0 so that the electron spin relaxation shortening is partially due to outer-sphere species which usually display T ) N 5 X 10-lo-1 X s. Thus all EPFl data point outs-l2 a randomly dispersed distribution of ZFS terms and correlation times. Table I show,sthe paramagnetic contributions to both
R
Ik Flgure 2. X-band (w = 5.8 X 10‘’ rad/@ EPR spectra of the 5’ATP-Mn(I1)-Trp complex at pH 7 and T = 300 K: [Mn2+] = 8 X 1 O3 M; [Mn]:[Trp]:[ATP] (A) 1:8.5:0.5; (B) 1:8.5:0.0.
TABLE I: Paramagnetic Contributions to l 3C Relaxation Rates at pH 7 and T = 300 Ka Mn(11)-ATP-Trp 6.3 Ti, s T -1 s-l 0.2 TZP- 1 ’ s-l 1.3
0.32 4.9 4.9
5.7 0.2
1.8
0.18 3.5 17.1
6.34 1.0 5.4
0.13 5.0 28.6
6.54 2.8 33.4
Mn(I1)-ATPb Tl,
Tip‘:r, sTzp-
6.33 1.1 4.1
0.17 G0.03
5.9 0.59 10.7
[Mn2+]= 5 X lo-’ M; [5’-ATP] = 5 X lo-’ M; [Trp] = From Y. F. Lam, G. P. P. Kuntz, and G. M. Kotowycz, J. Am. Chem. SOC.,96, 834 (1974). a
5
x
the spin-lattice and transverse nuclear relaxation rates for 13Cnuclei of the 5’-ATP molecule in the ternary system compared with the same data in the binary one. The TIP to Tzpratio is usually greater for 13Cthan for protons, so that in this case the contribution to Tl may be considered almost purely dipolar allowing some considerations on tlhe complex structure, whereas a scalar contribution is present for Tz. As pointed out earlier, when dealing with a dis-
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The Journal of Pbysical Chemistry, Vol. 84, No. I , 1980
Gaggelli, Rossi, and Tiezzl
b “2
TABLE 11: Paramagnetic Contributions to H Relaxation Rates at pH 7 a n d T = 300 Ka
H(8)
Mn(II)-ATP-Trr,
T1P- 1 s-l T2P- 1 s - l T,”-’, T.=-Is-l s-l
14.5‘
9
7
R- r i boryl -5'- t r iphoipha t e
ZP
64.7 69.5
9
/
a I
I
14.0 97.5
s-l
-1
1P
7
[Mn2+]= 5
‘
1.4 3.3 3.5 4.2
-
Mn(I1)-ATP 1.5
Tlp-l,s-l T
[Mn2+l/ H(1’) [ATP] . .
H(2)
X
a term A7,,/[1 + expre~sion:’~
2.9
M ; [Trp] = 5 x w,~T,?]
0.5 7.5 2.3 15.1
10-4 10-4 10-3 10-3
0.9 4.3
10-4 10-3
M.
must be considered in the TI
I
\
0
O r p /
’0
61
\
1
/ O ‘OOp\O
-N
Figure 3. (A) Standard numbering of the 5‘-ATP molecule. (B) Tentative and simplified structure of the 5’-ATP-Mn(IIt.Trp complex (from ref 1).
Since the experimental EPR spectra display inhomogeneous features, the EPR line shape is described by an equation of the type8 Y’(w) = CPkYk(H)where Yk(H) (F:F)f,(q,r,nw),that is a sum of 12-degenerate lines with different widths. This results in a distribution of T, values which, particularly in the case of protons, makes the calculation of distances impractical. Acknowledgment. We are grateful to Professor E. Ferroni for the facilities offered at the Institute of Physical Chemistry, University of Florence. Thanks are also due to Professor C. Giori and Mr. B. Valenti, Institute of Physics, University of Parma, for the use of the EPR $-band spectrometer, and to Mr. F. Brogi and Dr. G. Sabatini for their technical assistance. Q:
tribution of correlation times, distances cannot be estimated. Moreover ternary complex formation gives rise to slower rotation so that an increase in T,, the rotational correlation time, is expected together with a decrease in T ~ as , experimentally verified in the EPR experiments. This finding assumes major relevance in light of the novel derivation of the SBM equations, including ligand-field effects.14 From data in Table I it is apparent that the stacking interaction, favored by the metal ion, occurs; in fact a comparative analysis with the values of the binary system gives evidence of a noticeable variation for C(2) and of different Tl,/Tz ratios for C(5), C(8), and C(4) as a consequence of the Aifferent structure of the ternary system: namely, the distance C(2)-metal must be reduced by the stacking between the aromatic moieties. Figure 3 shows the standard numbering of the atoms in the 5’-ATP molecule (Figure 3A) and a tentative1 and simplified structure of the 5’-ATP-Mn(I1)-Trpcomplex (Figure 3B). The proton paramagnetic relaxation rates are shown in Table I1 at two different [metal]/[ATP] ratios. The comparison between the binary and ternary systems allows the same evidence as derived from data at the [metal] / [ATP] = loT3ratio; nevertheless, proton data do not allow conclusive remarks since the interplay of scalar and dipolar contributions to both T1;l and T2p-lis apparent N T2c1 for H(8)). As previously outlined in this case (T1g1 7,may compete with 7, in determining the dipolar term whenever a noncovalent interaction is present. Moreover,
References and Notes (1) H. Sigel and C. F. Naumann, J. Am. Cbem. Soc., 98, 730 (1976). (2) C. F. Naumann and H. Sigel, Febs. Left., 47, 122 (1974). (3) Y. Fukuda, P. R. Mltchell, and H. Sigel, Helv. Cbim. Acta, 61, 638 (1978). (4) J. L. Dimicoli and C. Helene, Biochemistry, 13, 714 (1974). (5) C. F. Naumann and H. Sigel, J . Am. Cbem. Soc., 96, 2750 (1974). (6) C. F. Naumann, B. Prijs, and H. Sigel, fur. J . Blochem., 41, 209 (1974). (7) R. A. Dwek, “Nuclear Magnetlc Resonance in Biochemistry”, Clarendon Press, Oxford, 1973. (8) L. Burlamacchi, G. Martini, M. F. Ottaviani, and M. Romanelll, Adv. Mol. Relaxation Processes, 12, 145 (1978). (9) R. Basosi, N. Niccolai, E. Tlezzl, and G. Valensin, J. Am. Chem. Soc., 100, 8047 (1978). (10) L. Burlamacchi, G. Martini, and E. Tiezzi, J. Phys. Cbem., 74, 1809 (1970). (11) R. Basosi, E. Gaggelli, and E. Tiezzi, J. Chem. Res., (S) 278 (1977). (12) L. Burlamacchi, G. Martini, and E. Tiezzi, Chem. Phys. Left., 23, 294 (1973). (13) W. G. Espersen and R. B. Martin, J. Phys. Cbem., 80, 161 (1976). (14) S. H. Koenlg, J. Magn. Reson., 31, l(1978).
