A 1H nuclear relaxation study of the Mn2+ . bleomycin complex. Proximity of the metal to the DNA-binding site.

The antineoplastic action of bleomycin is thought to involve the aerobic degradation of DNA by the Fe2+ . bleomycin complex. Different parts of the bleomycin molecule have been implicated in metal binding and DNA binding. To probe the structure of a metal-containing bleomycin, we studied the effects of the high spin Mn2+ ion in the Mn2+ . bleomycin complex on the longitudinal nuclear relaxation rates of various protons in the molecule. Complexation of Mn2+ to bleomycin was also studied by EPR, and a Scatchard plot of the EPR data revealed a single tight divalent cation-binding site per molecule. From the magnitudes of the paramagnetic effects of Mn2+ on the nuclear relaxation rates of several assigned resonances, we calculate the relative distances of the corresponding protons from the metal. Using a pyrimidine methyl to metal distance of 6.5 A, consistent with the metal coordination of this aromatic group of bleomycin established on the basis of other studies, we find from our data that the bithiazole and COOH-terminal portions of the molecule are located spatially very close to the metal. These groups have previously been implicated in DNA binding. Our metal to bithiazole proton distances (approximately 5.4 A) are consistent with bithiazole as a metal ligand, although possible involvement of interactions other than direct coordination in maintaining close proximity cannot be excluded. Our distance data also argue against the imidazole ring of beta-hydroxyhistidine as a ligand. The short distance between the metal- and DNA-binding sites indicated by our studies would help ensure that the reactive reduced oxygen radicals produced at the metal site during Fe2+ oxidation in the aerobic Fe2+ . bleomycin complex reach the substrate DNA before the destruction of these radicals can occur in other ways.