Coordination Chemistry of Co(II)-Bleomycin: Its Investigation through NMR and Molecular Dynamics †,‡ Teresa E. Lehmann,* ,§ Maria Luisa Serrano, | and Lawrence Que, Jr. ⊥ Laboratorio de Ana ´ lisis Instrumental, Centro de Quı ´mica, Instituto Venezolano de InVestigaciones Cientı ´ficas (IVIC), Caracas 1090, Venezuela, Department of Chemistry and Center for Metals in Biocatalysis, UniVersity of Minnesota, Minneapolis, Minnesota 55455, and Laboratorio de Modelaje Molecular, Facultad de Farmacia, UniVersidad Central de Venezuela, Caracas 1041-A, Venezuela ReceiVed August 6, 1999; ReVised Manuscript ReceiVed NoVember 29, 1999 ABSTRACT: Previous studies on the coordination chemistry of Co-bleomycin have suggested the secondary amine in -aminoalanine, the N5 and N1 nitrogens in the pyrimidine and imidazole rings, respectively, and the amide nitrogen in -hydroxyhistidine as equatorial ligands to the cobalt ion. The primary amine in -aminoalanine and the carbamoyl group of the mannose have been proposed alternatively as possible axial ligands. The first coordination sphere of Co(II) in Co(II)BLM has been investigated in the present study through the use of NMR and molecular dynamics calculations. The data collected from the NMR experiments are in agreement with the equatorial ligands previously proposed, and also support the participation of the primary amine as an axial ligand. The paramagnetic shifts of the gulose and mannose protons could suggest the latter as a second axial ligand. This possibility was investigated by way of molecular dynamics, with distance restraints derived from the relaxation times measured through NMR. The molecular dynamics results indicate that the most favorable structure is six-coordinate, with the primary amine and either the carbamoyl oxygen or a solvent molecule occupying the axial sites. The analysis of the structures previously derived for HOO-Co(III)-bleomycin and HOO-Co(III)-pepleomycin led us to propose the six-coordinate structure with only endogenous ligands, as the one held in solution by the Co(II) derivative of bleomycin. Bleomycins (BLM) (Figure 1) are a family of glycopep- tide-derived antibiotics that have the ability to bind and degrade DNA in the presence of some metal ions (Fe 2+ , Fe 3+ , Mn 2+ , Co 3+ ), which is believed to be responsible for their antitumor activity (1-11). The presence of ferrous ion and molecular oxygen is proposed to be essential for DNA degradation in vivo (1, 10, 12). Knowledge of the structure of the biologically relevant Fe(II)BLM 1 is vital to further characterize the mechanism of action of the drug as well as to establish the drug-DNA spatial correlations that could explain the antibiotics specificity toward 5′ -GC- 3′ and 5′ -GT- 3′ DNA sequences (13). To this end, numerous structural studies on various metallo-BLMs (1), BLM analogues (14-20), and other antibiotics structurally related to BLM, such as pepleomycin (21) (PEP) and tallysomycin (22) (TLM), have been conducted. The results of these studies have led to very important findings about the coordination chemistry of the CO-Fe(II), Zn(II), Mn(II), Co(II), and Co(III) adducts of the drug (23-29). In previous work, we have structurally characterized the complex Fe- (II)BLM through NMR (30). Our NMR studies indicate that the primary and secondary amines in -aminoalanine, the pyrimidine, and the imidazole and the amide nitrogen in -hydroxyhistidine are coordinated to the iron in this metallobleomycin. The participation of the carbamoyl group in the mannose moiety as a ligand to the Fe center was ruled out for this complex, based on a structural correlation performed between the Fe(II)- and HOO-Co(III)BLM (31) derivatives. Even though the protons in both of the sugar residues exhibit paramagnetic behavior in Fe(II)BLM, the NMR data alone are not enough to determine if mannose is part of the first coordination sphere of the metal in this derivative. To further extend the comparison between the iron and cobalt complexes of bleomycin, both involved in DNA degradation, the coordination chemistry of the para- magnetic Co(II)BLM was investigated through NMR and molecular dynamics in the present study. The structural data derived from this investigation are important because: (1) the oxidation of Co(II)BLM by oxygen produces Co(III)- BLM and its peroxide form, HOO-Co(III)BLM, which is involved in DNA cleavage after photochemical activation † This work was supported by the Consejo de Desarrollo Cientı ´fico y Humanı ´stico (CDCH) Grant CDCH0610429698 and NIH Grants GM- 33162 and GM-51849. ‡ The PDB ID code for model I is 1DEY. * To whom correspondence should be addressed. Telephone: +58-(02)-504-1702. Fax: +58-(02)-504-1309. E-mail: tlehmann@ quimica.ivic.ve. § Instituto Venezolano de Investigaciones Cientı ´ficas (IVIC). | Universidad Central de Venezuela. ⊥ University of Minnesota. 1 Abbreviations: ALA, -aminoalanine; BIT, bithiazole; BLM, bleomycin; COSY, correlated spectroscopy; ESFF, extensible systematic force field; GUL, gulose; HIS, -hydroxyhistidine; HMQC, hetero- nuclear multiple quantum correlation; MAN, mannose; NOE, nuclear Overhauser effect; PEP, pepleomycin; PYR, pyrimidinylpropion- amide; THR, threonine; TOCSY, totally correlated spectroscopy; VAL, methylvalerate. 3886 Biochemistry 2000, 39, 3886-3898 10.1021/bi991841p CCC: $19.00 © 2000 American Chemical Society Published on Web 03/16/2000