Inorg. Chem. 1982,21, 1846-1854
1846
Contribution from the Laboratoire de Chimie de Coordination du CNRS and the Laboratoire de Pharmacologie et Toxicologie Fondamentales du CNRS, Universitt Paul Sabatier, 3 1400 Toulouse, France, and the Dtpartement de Chimie, UniversitB de Sherbrooke, Sherbrooke, QuBbec, Canada J1K 2R1
[5-Leucine]- and [5-Methionine&nkephalin-Copper(11) Complexation under Physiological Conditions JEAN-EDOUARD GAIRIN,IavbHONOR& MAZARGUIL,lCPATRICK SHARROCK,Idand RAYMOND HARAN*l8 Received June 2, I981
Paramagnetic perturbations in I3C and 'H NMR spectra of [Leus]- and [Met5]enkephalinsare caused by copper(I1) ions and interpreted in terms of the major and minor species formed as a function of pH. The shifts in the electronic absorption spectra and the bonding parameters determined by EPR spectroscopy point to the formation of strong, covalently bonded species as the pH increases. The major species found under physiological conditions is described as involving the tyrosine amino group, two glycine peptide linkages, and the carboxylate function of the C-terminal residue. NMR broadening results are attributed to minor species involving axially bonded functional groups in rapid exchange. The significance of enkephalin-metal complexation lies in its possible use in determining the structures of the biologically active conformations.
Introduction Since the discovery in brain tissues of the two pentapeptides Tyr-Gly-Gly-Phe-Leu and Try-Gly-Gly-Phe-Met by Hugues et al. in 1975,2 interest in the properties of these natural enkephalins (Figure l), and in related peptides, has increased dramatically. Research into the pharmacological properties of enkephalins has shown them to be potent analgesics3 that operate at the same receptors as natural opiates and their antagonist^.^ Structure-function relationships have been investigated both theoretically and e~perimentally.~ The solution conformational states of the enkephalins have been intensively studied by nuclear magnetic resonance.6-'0 Initially, contradictory results were obtained, which were then resolved by admitting several sets of stable conformations featuring various types of 0 bends and different rotamer populations. The lability of the side-chain conformations of Tyr', Phe4, and Met5 residues remains a debated question. Recent relaxation rate measurements focused on the structural information which can be derived from relaxation data dominated by dipolar mechanisms6 in the presence of various solvents' and to possible biological binding components.'' Lanthanum and gadolinium have been used to relate solvent polarity with the fraction of folded conformations and the separation of the charged end groups.'2 , 'K Na+, Ca2+, and Mn2+ have also been used to study alterations in the circular dichroism spectra,13 but metal ion complexation has (1) (a) Laboratoire de Chimie de Coordination du CNRS, Universit6 Paul Sabatier. (b) Holder of a 1980 FranctQuebec Scientific Exchange Scholarship. (c) Laboratoire de Pharmacologie et Toxicologie Fonda(2) (3) (4) (5)
(6)
mentale du CNRS, Universitt Paul Sabatier. (d) Universitt de Sberbrooke. Hughes, J.; Smith, T. W.; Kosterlitz, H. W.; Fothergill, L. A.; Morgan, B. A.; Morris, R. H. Nature (London) 1975, 258 577. Belluzzi, J. D.; Grant, N.; Garsky, V.; Sarantakas, D.; Wise, C. D.; Stein, L. Nature (London) 1976, 260, 624. Bradbury, A. D.; Smyth, D. G.; Snell, C. R.; Birdsall, N. J. M.; Hulm, E. L. Nature (London) 1976, 260, 793. (a) Loew, G. H.; Burt, S . K. Proc. Natl. Acad. Sei. U.S.A. 1978. 75, 7. (b) Isogai, Y.;Nemethy, G. and Scheraga, H. A. Zbid. 1977,74,414. (c) Balodb, Yu;Nikiforvich, G. V.; Grinsteine, I. V.: Vegner, R. E. Chipens, G. I. FEBS Leu. 1978, 86, 939. Nimlai, N.; Garsky, V.; Gibbons, W. A. J. Am. Chem. SOC.1980,102,
1517. (7) Kobayashi, J.; Nagai, U.; Higashijima, T.; Mijazawa, T. Biochem. Biophys. Acta 1979, 577. 195. (8) Roques, B. P.; Garbay-Jaureguiberry, C.; Oberlin, T.; Anteunis, M.; Lala, A. K. Nature (London) 1976, 262, 778. (9) Bleich, H. E.; Cutnell, C. D.; Day, A. R.; Freer, R. J.; Clasel, J. A.; McKelvy, J. F. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 2589. (10) Khaled, M.A.; Long, M. M.; Thompson, W. D.; Bradley, R. J.; Brown, G. B.; Urry, D. W. Biochem. Biophys. Res. Commun. 1977, 76, 224. ( 1 1) Jarrell, H. C.; Deslauriers, R.; McGregor, W. H.; Smith, I. C. P. Biochemistrv 1980. 19. 385. (12) Higashijima, T.';Kobayashi, J.; Nagai, U. and Miyazawa, T. Eur. J . Biochem. 1979, 97, 43.
not yet been reported for the enkephalins. Though no involvement of enkephalins in interactions with metal ions in vivo is known up to date, complex formation with metal ions seems to promise interesting re~u1ts.l~Several studies with model peptides have shown them to be strong complexing agents,lS and one can in effect consider bioorganic compounds as competing for the various trace elements available. Even though much is known about the formation of copper complexes with the amino acids, this information cannot be easily transposed to the case of peptides, where detailed coordination studies have to be carried out in each case. The dispersity of the theoretical results and the divergence of interpretations of the experimental results concerning the solution structure of enkephalins calls for the application of additional techniques. Since N M R line-broadening data have been used and questioned repeatedly,16 we have used 'H and "C N M R together with EPR and visible spectroscopy results. We now wish to report on the pH-dependent interactions of copper(I1) with natural enkephalins in aqueous solution simulating physiological serum media.
Experimental Section Synthesis. [Leus]enkephalin and [Me5]enkephalinwere both synthesized by solution methods. The 'H and 13Cspectra are similar to published ones, with no spurious absorptions. Instnunentation. Fresh D20solutions, 0.9 X M in NaCl and 0.08-0.10 M in enkephalins, were used for NMR. DCl and NaOH solutions were used to adjust the pH which was measured with Tacusel 268 N and Orion Research pH meters and a Markson microelectrode. pH values are not corrected for deuterium isotope effects. Solutions with variable copper concentrations were made up by micropipetting from a concentrated D20 solution of the anhydrous copper chloride. Spectra were recorded at ambient probe temperature (300 K), and the shifts S(Ci), 6(Hi) are expressed in ppm relative to external Me& Bruker WH90 and WH 400 NMR spectrometers operating in the Fourier transform mode were used for I3C and 'H spectra, respectively. The number of scans varied from 1OOOO to 25 OOO for 13C and from 40 to 100 for 'H spectra. Continuous-wave H 2 0 spectra were obtained on a Varian A-60 instrument, with a coaxial M sample tube containing acetone as a line-width standard. A Cu2+solution containing a tenfold excess of ligand was repeatedly scanned at 50 Hz width, resulting in an accuracy of better than 0.1 Hz. (13) Hollosi, M.; Dobolyi, Z.; Bajusz, S.FEBS Letf. 1980, 110, 136. (14) Biological activity studies in vitro will be reported in a forthcoming
paper.
(15) Margerum, D. W.: Wong, L. F.; Bossu, F. P.; Chellappa, K. L.;
Czarnecki, J. J.; Kirksey, S.T., Jr.; Nubecker, T. A. 'Bioinorganic Chemistry"; American Chemical Society: Washington, D.C., 1977; Adv. Chem. Ser. No. 162, p 281. (16) Kuroda, Y.;Aiba, H. J. Am. Chem. SOC.1979, 101, 6837.
0020-1669/82/1321-1846$01.25/00 1982 American Chemical Society
Enkephalin-Copper(I1) Complexation
Inorganic Chemistry, Vol. 21, No. 5, 1982 1847
OH
'~HZ
YN- C,H- c'II 0
H I N-$H&-N
H
I
It 0
H 'CH2 -c,H&-
I
II
0
N
I - ;H-c~-N II
H 'CHz 1
- ~1H - C5O O H
0
Figure 1. Primary structure of natural enkephalins. In [Mets]R is CH(CH3)2. enkephah, R is CH2SCH3;in [Le~~lenkephalin,
150
loo
.
50
3
LOO
Figure 2.
500
700
600
Absorption spectra of copper(I1) [Le~~lenkephalin com-
plexes. Solutions of 2 X lo-' M Cuz+in a 1-cm length cell and a Cary 14 spectrophotometerwere used for visible absorption studies. An E-9Varian EPR spectrometer was used with quartz sample tubes of various sizes, and the low-temperature spectra were recorded as previously described." Theory Interpretation of the magnetic resonance results in the presence of Cu2+ion is not straightforward and requires some theoretical background. N M R line widths are determined by the spin-lattice relaxation time l/T1 and the spin-spin relaxation time 1/T2 = K ( A V ~ where / ~ ) A V , /is ~ the width of the absorption at half-height. The presence of a paramagnetic metal ion can greatly change the parameters describing the N M R absorption, both by direct dipole-dipole interaction between the nuclear spin and the electron spin and by a contact interaction which transfers electron spin density from the metal ion to the nucleus being o b ~ e r v e d . ~ Since ~ J ~ copper(I1) ions have long electronic relaxation times ( Tl, = 2 X s) and the complexes fast rotational rates,20 the contribution of the electronic spin to the nuclear spin relaxation is given by eq 1 where r is the distance between the two spin centers, 7,the
correlation time for the dipolar interaction, p the magnetic moment of the paramagnetic ion, and A the hyperfine coupling constant. Results Visible Absorption Spectra. Formation of the different copper(I1) enkephalin complexes with increasing pH can be ~
~~
(17) Sharrock, P. J . Magn. Reson. 1979, 33, 465. (18) Solomon, I. Phys. Reo. 1955, 99, 559. (19) Wllthrich, K. 'NMR in Biological Research. Peptides & Proteins"; Elsevier: New York, 1976; p 226. (20) Dwek, R. A.; Williams, R. J. P.; Xavier, A. V. Met. Ions Bioi. Syst. 1974, 4, Chapter 3.
. 6
.
.
.
9
.
.
Figure 3. pH dependences of the wavelength maxima and the molar absorbance of copper(I1) [Leu5]enkephalin(full points) and copper(I1) [Mets]enkephalin (open symbols). Below pH 4.5 A,, is not well defined in the 700-nm region and the e values are calculated for a A,, = 700 nm.
followed by visible spectroscopy in the 350-700-nm range. Copper(I1) [Leus]enkephalin was titrated from pH 3.5 to 12.5 and copper(I1) [Met5]enkephalin from pH 5 to 1 1 . Absorption spectra were taken at various points during the titration. At low p H ( 4[u(F,)].
Introduction Recently we have studied the structures of a variety of seven-coordinate molybdenum(I1) and tungsten(I1) isocyanide complexes containing unidentate ligand^.^ Criteria were developed for classifying the geometries and analyzing the factors that dictate the choice of stereochemistry for the ML72+/ML6X+families, where L = RNC and X = halide or pseudohalide. With the synthesis of the [Mo(diphosphine)(iso~yanide)~]~+cations,6 exploration of related complexes having the stoichiometry [M(BL)(UL),] (BL = bidentate ligand; UL = unidentate ligand) became possible. In such molecules the constraints of the chelating ligand will (1) Part 16 of a continuing series on higher coordinate cyanide and isocyanide complexes. For part 15 see ref 2. (2) Giandomenico, C. M.; Lam, C. T.; Lippard, S.J. J . Am. Chem. Soc. 1982, 104, 1263. (3) Ligand abbreviations: dppm = bis(dipheny1phosphino)methane; dppe = 1,2-bis(diphenylphosphino)ethane; dppp = 1,3-bis(diphenylphosphin0)propane; diars = o-phenylenebis(dimethy1arsine); rac-diars = rac-o-phenylenebis(methylpheny1arsine); meso-diars = meso-ophenylenebis(methylpheny1arsine). (4) (a) Columbia University. (b) Purdue University. (5) Smlda, D. J.; Dewan, J. C.; Lippard, S.J. Inorg. Chem. 1981,20, 3851 and references cited therein. ( 6 ) Wood, T. E.; Deaton, J. C.; Corning, J.; Wild, R. E.; Walton, R. A. Inorg. Chem. 1980, 19, 2614.
influence the structure, and there are highly regarded' theoretical model^*^^ for analyzing the resulting stereochemistry. Here we report the structures of the pentagonal-bipyramidal complexes [MO(~~~~)(CNCH,)~](PF~)~ (1)3 and [Mo(dppe)(CNCH3)s](PF6)2(2)3having four- and five-membered chelate rings, respectively. Prior to this study, the only structurally characterized [M(BL)(UL),] complexes were [Nb(02)Fs]3- and [Ta(02)Fs]3-.10.'1 Experimental Procedure and Results Collection and Reduction of X-ray Data. (Bis(dipheny1phosphino)methane)pentakis(methyl isocyanide)molybdenum(II) HexafluorophaPphate,[Mo(dpPm)("3)51(pF6)2 (1). The synthesis and crystallization of 1 have been described previously.6 The orange-red crystal used in the diffraction study had approximate dimensions 0.20 X 0.17 X 0.53 mm and was sealed in a capillary to minimize decomposition. Study on the diffractometer suggested that the crystal belonged to the monoclinic crystal system. Its quality was Hoffmann, R.; Beier, B. F.; Muetterties, E. L.; Rossi, A. R. Inorg. Chem. 1977, 16, 511. Kepert, D.L. Prog. Inorg. Chem. 1979, 25, 41. Dewan, J. C.; Henrick, K,; Kepert, D. L.; Trigwell, K. R.; White, A. H.; Wild, S. B. J . Chem. SOC.,Dalton Trans. 1975, 546. Ruzic-Toros, Z.; Kojic-Prodic,B.; Gabela, F.; Sljukic, M. Acta Crystallogr., Sect. E 1977, E33, 692. Ruzic-Tom, Z.; Kojic-Prodic,B.; Sljukic, M. Acta Crystallogr., Sect. E 1976.832, 1096.
0020-1669/82/ 1321-1854$01.25/0 0 1982 American Chemical Society
