A model for the structure of the aquated form of cobalt bleomycin (H2O-CoBLM or CoBLM A2 brown), free and bound to d(CCAGGCCTGG)(2) (1) is reported based on molecular modeling using the constraints obtained from 2D NMR studies. CoBLM A2 brown has a chiral organization of its ligands, including the axial primary amine of beta-aminoalanine, identical to the hydroperoxide form of cobalt BLM (HOO-CoBLM or CoBLM A2 green). H2O-CoBLM forms a 1:1 complex with 1 with a K-d of 2 x 10(-6) M which is in slow exchange on the NMR time scale. The complex exhibits 44 intermolecular NOEs and 56 intramolecular NOEs within H2O-CoBLM itself. Molecular modeling reveals that H2O-CoBLM's mode of binding, basis for sequence specificity, and chemical specificity are almost identical to that previously reported for HOO-CoBLM (Wu, W.; Vanderwall, D. E.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem. Sec. 1996, 118, 1281-1294). The bithiazole tail is inserted from the minor groove 3' to C6 and the terminal thiazolium ring is well stacked between the bases G14 and G15, while the penultimate thiazolium ring is only partially stacked between the bases of C6 and C7. The basis for sequence specificity involves hydrogen-bonding interactions between the 4-amino group and N3 of the pyrimidine of BLM and the N3 and 2-amino group of G5 of DNA, forming an unusual base triple. Molecular modeling further reveals that the oxygen of the axial H2O ligand is 2.7 Angstrom from the 4' H of C6, the site of cleavage of iron BLM and light-activated cleavage by HOO-CoBLM. Preliminary studies of H2O-CoBLM with d(CCAGTACTGG) (4) and d(GGAAGCTTCC) (2) are also reported.