Spectroscopic studies of the interaction of ferrous bleomycin with DNA

Bleomycin is an antibiotic used in cancer chemotherapy for its ability to achieve both single-and double-strand cleavage of DNA through abstraction of the deoxyribose C4'-H. Magnetic circular dichroism (MCD) and X-ray absorption (XAS) spectroscopies have been used to study the interaction of the biologically relevant (FeBLM)-B-II complex with DNA. Calf thymus DNA was used as the substrate as well as short oligonucleotides, including one with a preferred 5'-G-pyrimidine-3' cleavage site [d(GGAAGCTTCC)(2)] and one without [d(GGAAATTTCC)(2)]. DNA binding to (FeBLM)-B-II significantly perturbs the Fe-II active site, resulting in a change in intensity ratio of the d --> d transitions and a decrease in excited-state orbital splitting (Delta(5)E(g)). Although this effect is somewhat dependent on length and composition of the oligonucleotide, it is not correlated to the presence of a 5'-G-pyrimidine-3' cleavage site. No effect is observed on the charge-transfer transitions, indicating that the H-bonding recognition between the pyrimidine and guanine base does not perturb Fe-pyrimidine backbonding. Azide binding studies indicate that (FeBLM)-B-II bound to either oligomer has the same affinity for N-3(-). Parallel studies of BLM structural derivatives indicate that Fe-II iso-PEPLM, in which the carbamoyl group is shifted on the mannose sugar, forms the same DNA-bound species as (FeBLM)-B-II. In contrast, (FeDP)-D-II-PEPLM, in which the beta-aminoalanine group is absent, forms a new species upon DNA binding. These data are consistent with a model in which the primary amine from the beta-aminoalanine is an Fe-II ligand and the mannose carbamoyl provides either a ligand to the Fe-II or significant second-sphere effects on the Fe-II site; intercalation of the bithiazole tail into the double helix likely brings the metal-bound complex close enough to the DNA to create steric interactions that remove the sugar groups from interaction with the Fe-II. The fact that the Fe-II active site is perturbed regardless of DNA sequence is consistent with the fact that cleavage is observed for both 5'-GC-3' and nonspecific oligomers and indicates that different reaction coordinates may be active, depending on orientation of the deoxyribose C4'-H.