Stray Cu(II) May Cause Oxidative Damage When Coordinated to the -TESHHK- Sequence Derived from the C-Terminal Tail of Histone H2A Marios Mylonas, † Gerasimos Malandrinos, † John Plakatouras, † Nick Hadjiliadis, † Kazimierz S. Kasprzak, ‡ Artur Kre ¸ z ˘ el, § and Wojciech Bal* ,§,| University of Ioannina, Department of Chemistry, Ioannina 45110, Greece, Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, Maryland 21702, Faculty of Chemistry, University of Wroclaw, 50-383 Wroclaw, Poland, and Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Received February 13, 2001 CH 3 CO-Thr-Glu-Ser-His-His-Lys-NH 2 , a hexapeptide representing the 120-125 sequence of histone H2A, coordinates Cu(II) ions efficiently. Monomeric complexes are formed. In the major complex at physiological pH, CuH -1 L, Cu(II) is coordinated equatorially through the imidazole nitrogen of the His-4 residue and the amide nitrogens of the Ser-3 and His-4 residues, and axially through the imidazole nitrogen of the His-5 residue. This complex reacts with H 2 O 2 and the resulting reactive oxygen intermediate efficiently oxidizes 2′-deoxyguanosine. The underlying mechanism involves the formation of Cu(III) and a metal-bound hydroxyl radical species. Introduction Histones are highly basic proteins that provide scaffold for DNA double helix in the cell nucleus. The resulting complex, nucleosome, is composed of a histone octamer (containing two copies of each of histones H2A, H2B, H3, and H4) and a 170 base pair DNA stretch. Studying the molecular mechanisms of nickel carcinogenesis, we pro- posed the existence of two Ni(II) binding sites in the octamer: one located in histone H3 in the center of nucleosome and another, on the periphery, within the C-terminal tail of histone H2A (2, 3). To obtain a detailed characterization of Ni(II) coordination properties of these sites we used their minimal peptide models. The se- quence of histone H3, -Cys-Ala-Ile-His- was studied first (2-4). The results of these studies were validated using the (H3-H4) 2 core tetramer (5). We subsequently studied Ni(II) coordination to the peptide Ac-Thr-Glu-Ser-His- His-Lys-NH 2 (-TESHHK-), 1 corresponding to positions 120-125 of H2A (6), and the hydrolytic and oxidative reactivity resulting from Ni(II) complex formation (6, 7). The results indicated that the stability of the Ni(II) complex with -TESHHK- at pH 7.4 might be high enough to ensure Ni(II) binding to nucleosome at certain patho- logical conditions (8). Histidine residues are also pre- ferred coordination sites for Cu(II), an essential metal ion (9). Preliminary experiments revealed that Cu(II) could also induce hydrolysis of -TESHHK- in a fashion similar to that of Ni(II) (7). It therefore seemed interest- ing to learn about Cu(II) binding to the same model peptide, as used in Ni(II) studies. Knowing the high redox reactivity of many, but not all, Cu(II) complexes, we tested such reactivity of Cu- TESHHK- with H 2 O 2 , using 2′-deoxyguanosine (dG) as a target/reporter molecule. In this system, unlike with DNA as a target, the yield of the oxidation product, 8-oxo- dG, is high and its quantification can be accomplished quickly and precisely by HPLC with a standard UV detector. Another reason for using dG rather than DNA, was the shortness of our model peptide which would not allow for reproduction of the protein-DNA interaction occurring in nucleosome. Experimental Procedures Materials. 2 Cu(NO3)2‚6 H2O, KNO3, HNO3, and NaOH volumetric solution (0.1 M) were obtained from E. Merck (Darmstadt, Germany). Sodium and potassium phosphates, N,N-dimethyl-p-nitrosoaniline (NDMA), Nitro Blue Tetrazolium (NBT) and Chelex 100 resin were purchased from Sigma Chemical Co. (St. Louis, MO). Ethanediol, acetone, ascorbic acid and methanol were obtained from POCH (Gliwice, Poland). Hydrogen peroxide aqueous solution (30%, analytical grade) was purchased from Chempur (Piekary Sl., Poland). The protected amino acids, Fmoc-His(Mtt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser- (tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Thr(tBu)-OH, and Fmoc- Ala-OH and the resin H-Linker-2-chlorotrityl were purchased from CBL Chemicals Ltd. (Patras, Greece). Peptide Synthesis. The peptide Ac-Thr-Glu-Ser-His-His- Lys-CONH2 (-TESHHK-, L) was synthesized in the solid state, using the H-Linker-2-chlorotrityl resin. The peptide was as- * To whom correspondence should be addressed. Phone: (48-71)- 3204-281. Fax: (48-71)328-2348. E-mail wbal@wchuwr.chem.uni.wroc.pl. † University of Ioannina. ‡ National Cancer Institute. § University of Wroclaw. | Polish Academy of Sciences. 1 Abbreviations: -TESHHK-, CH3CO-Thr-Glu-Ser-His-His-Lys-NH2; L, fully deprotonated form of -TESHHK-; Cu(II)-TESHHK-, any cupric complex of -TESHHK-; NDMA, N,N-dimethyl-p-nitrosoaniline; NBT, Nitro Blue Tetrazolium; CT, charge transfer; dG, 2′-deoxyguanosine; 8-oxo-dG, 7,8-dihydro-8-oxo-2′-deoxyguanosine; ESI-MS, electrospray ionization mass spectrometry. 2 Certain commercial equipment and materials are identified in this paper to adequately specify the experimental procedure, Such identi- fication does not imply recommendation or endorsement by the National Cancer Institute (Department of Health and Human Services, National Institutes of Health), nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. 1177 Chem. Res. Toxicol. 2001, 14, 1177-1183 10.1021/tx010031n CCC: $20.00 © 2001 American Chemical Society Published on Web 08/04/2001