May GSH and L-His contribute to intracellular binding of zinc? Thermodynamic and solution structural study of a ternary complex Artur Kr ˜ e˙ zel, a Jacek Wójcik, b Maciej Maciejczyk b and Wojciech Bal* b a Faculty of Chemistry, University of Wroc ´ law, Joliot-Curie 14, 50-383 Wroc ´ law, Poland. E-mail: arti@wchuwr.chem.uni.wroc.pl; Fax: +48-71-3282348; Tel: +48 71 3757264 b Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawi´ nskiego 5a, 02-106 Warszawa, Poland. E-mail: wbal@ibb.waw.pl; Fax: +48-22-6584636; Tel: +48-22-6597072 x2353 Received (in Cambridge, UK) 16th January 2003, Accepted 6th February 2003 First published as an Advance Article on the web 21st February 2003 GSH and L-His are abundant biomolecules and likely biological ligands for Zn(II) under certain conditions. Potentiometric titrations provide evidence of formation of ternary Zn(II) complexes with GSH and L-His or D-His with slight stereoselectivity in favour of L-His (ca. 1 log unit of stability constant). The solution structure of the ZnH(GSH)(L-His)(H 2 O) complex at pH 6.8, determined by NMR, includes tridentate L-His, monodentate (sulfur) GSH, and weak interligand interactions. Calculations of com- petitivity of this complex for Zn(II) binding at pH 7.4 indicate that it is likely to be formed in vivo under conditions of GSH depletion. Otherwise, GSH alone emerges as a likely Zn(II) carrier. Reduced glutathione (GSH) is one of the most abundant and ubiquitous molecules of life, at 1–20 mM intracellularly, with strong compartmentalisation and various functions in cellular metabolism and defenses, including detoxication of heavy metals. 1 Zn(II) is involved, among others, in DNA transcription (enzymes, zinc fingers) and intracellular signaling. O’Halloran et al. demonstrated the absence of free Zn(II) in E. coli. 2 On the other hand, estimates for free Zn(II) in the cytoplasm of eukaryotic cells range from 10 212 M to 10 29 M, depending on cell type and state, and up to 10 23 M in specific secretory vesicles. 3,4 Zn(II) is an emerging signalling ion, and thus its metallothionein (MT)-bound pool ought to be easily mobili- sable. Glutathione is capable of releasing Zn(II) from MT in a redox reaction involving its oxidised form (GSSG). 5 A lack of consensus regarding interactions between GSH and Zn(II) ions in vitro, 6 and the absence of specific information on possible interactions in vivo, made it difficult to predict further steps in Zn(II) release. Also transport of Zn(II) outside and into the eukaryotic cell is not understood very well. Histidine, which is present ubiquitously in the body at ca. 10 24 M, has been implicated as a possible Zn(II) shuttle in some tissues. 7 In order to provide a chemical basis for assessment of the possible participation of glutathione in Zn(II) transport, we have examined the acid–base chemistry and Zn(II) coordination of GSH, GSSG and many of their analogues, using potentiometry and NMR. 8,9 In the course of these studies we noted that GSH readily forms ternary complexes with Zn(II) and amino acids and peptides. Here we present the results for such complexes, involving histidine. Protonation constants of GSH, L-His and D-His, as well as stability constants for their binary and ternary complexes with Zn(II), were obtained from potentiometric titrations and con- firmed by one-dimensional 1 H-NMR spectra at 300 and 500 MHz. These constants are provided in Table 1. The values for major complexes of L-His and D-His, ZnA and ZnA 2 , agree well with those determined previously. 10 The same can be stated for GSH complexes, where our model is practically identical with the previous one. 11 The only difference is our ZnH 22 L 2 62 species instead of ZnH 21 L 22 , postulated previously, and the differences in values of constants can be ascribed to differences in ionic strengths of determinations. Ternary Zn(II) complexes with GSH and L-His or D-His are novel. The analysis of 1D NMR spectra indicated that Zn(II) in these complexes is coordinated to all three donors of histidine (imidazole and amine nitrogens and carboxylate oxygen) and to the thiol sulfur of GSH. The deprotonation yielding ZnLA 22 from ZnHLA 2 is, as indicated by the spectra, that of the uncoordinated amine. Its pK a is the same in both diastereomers (8.2 ± 0.1), and lowered compared to free GSH by 1.5 log units, apparently due to the increased overall charge in the complex and altered electrostatics in bonded GSH compared to free GSH (neutralisation of the thiolate by Zn(II) and spatial shielding of the amine from the Gly carboxylate). 8 There is a significant difference in stabilities of diastereomers, ca. 1 order of magnitude in favour of the L-His containing species, which translates into DDG of 2.8 kJ mol 21 . Fig. 1 presents a simplified species distribution diagram, calculated for the L-His system at the concentrations of the NMR experiments. The abundance of the ZnHLA 2 complex at weakly acidic to weakly basic pH allowed for the determination of its structure by 2D NMR at 500 MHz and molecular mechanics. 13 Fig. 2 presents the resultant lowest-energy structure and the overlap of ten low- energy conformers, allowed by NOE connectivities. The complex formation is primarily due to high enthalpies of Zn(II) bonding by thiol sulfur and histidine donors. Its structure is additionally stabilised by a local network of weak C–H…C, C– H…N and N–H…C type hydrogen bonds (Fig. 2, dashed lines), which is anchored on the b-methylene protons of His and Cys residues. 14 The differences in the involvement of individual protons of each pair in H-bonding are reflected in the spectacular differences of half-widths of their 1 H NMR signals, shown in Fig. 3. Stability constants allow us to estimate the competitiveness of the system studied towards zinc proteins. 15 The competitivity Table 1 Protonation and stability constants, I = 0.1 M, T = 25 °C 12 log b ijkl a log b ijkl a GSH b L-His HL 22 9.655(2) HA 9.129(1) H 2 L 2 18.391(2) H 2 A + 15.165(2) H 3 L 21.903(3) H 3 A 2+ 16.85(5) H 4 L + 24.029(7) D-His Zn(GSH) HA 9.129(2) ZnHL 14.74(2) H 2 A + 15.142(3) ZnL 2 8.31(2) H 3 A 2+ 16.83(6) ZnH 2 L 2 22 29.50(4) ZnHL 2 32 22.533(5) Zn(L-His) ZnL 2 42 13.617(5) ZnA + 6.567(5) ZnH 21 L 2 52 3.817(6) ZnA 2 12.025(5) ZnH 22 L 2 62 26.485(6) ZnH 21 A 22 1.18(2) ZnH 22 A 2 22 29.90(2) Zn(L-His)(GSH) ZnHLA 2 21.46(5) Zn(D-His) ZnLA 22 13.26(8) ZnA + 6.61(1) ZnA 2 12.09(1) Zn(D-His)(GSH) ZnH 21 A 22 1.15(4) ZnHLA 2 20.37(10) ZnH 22 A 2 22 29.83(3) ZnLA 22 12.24(5) a log b ijkl = log([M i H j L k A l ]/[M] i [H] j [L] k [A] l ). b Ref. 8. This journal is © The Royal Society of Chemistry 2003 704 CHEM. COMMUN. , 2003, 704–705 DOI: 10.1039/b300632h