CATALYTIC RELEASE OF DEOXYRIBONUCLEIC-ACID BASES BY OXIDATION AND REDUCTION OF AN IRON-BLEOMYCIN COMPLEX

The kinetics and stoichiometry of several reactions involving bleomycin, iron, DNA, oxygen, and sulfhydryls were examined in order to assess their possible role in degradation of DNA by bleomycin. Oxidation of Fe(II) in the presence of bleomycin resulted in an iron(III)-bleomycin complex, having an optical absorption spectrum with a broad shoulder at 320-400 nm, which was stable for several hours. If Fe(II) was allowed to oxidize before bleomycin addition, the complex did not form. The complex was reduced by dithiothreitol 5 times faster than unchelated Fe(III), and reduction of the complex was inhibited by high concentrations of DNA. However, stopped-flow studies showed that, when sufficient DNA was present to bind most of the iron(II)-bleomycin, its rate of oxidation by molecular oxygen was 60 times faster than that of unbound iron(II)-bleomycin. Under the same conditions, oxidation of each mole of DNA-bound iron(II)-bleomycin released 0.18 mol of thymine. Treatment of pyrimidine-labeled Escherichia coli DNA with bleomycin and high concentrations of Fe(II) and 2-mercaptoethanol resulted in the release of up to 2.4 mol of pyrimidines (of which 60% were thymine) per mol of bleomycin. This result implies that each Fe·bleomycin complex went through several cycles of oxidation and reduction and that bleomycin usually was not inactivated in the base-release reaction. In supercoiled pDR3709 DNA, one base was released per single-strand break (measured in alkali), eliminating the possibility of multiple base release during a single bleomycin-DNA interaction. Thus, the iron·bleomycin complex acts as a catalyst which, after being reduced by sulfhydryls, binds to DNA in a way which facilitates both the oxidation of the chelated Fe(II) and the degradation of the DNA backbone by the products of this oxidation. © 1979, American Chemical Society. All rights reserved.