Intermediates in the ferrous oxidase cycle of bleomycin

We have previously shown that the bleomycin-induced autooxidation of ferrous iron follows Michaelis--Menten kinetics which are characteristic of enzymatic reactions [Caspary, W. J., Lanzo, D. A., Niziak, C., Friedman, R., & Bachur, N. R. (1979) Mol. Pharmacol. 16, 256]. In this paper, we identify the iron complexes formed during this reaction. The first is a ferrous iron--bleomycin complex which can be considered the catalyst substrate complex. The product of this reaction is a ferric iron--bleomycin complex which is found in a low-spin and a high-spin form. The relative concentrations of these two forms are a function of pH. Glutathione, a biologically relevant reducing agent, binds to the ferric iron--bleomycin complex, reduces it, and may serve as a model for the reduction of the ferric iron--bleomycin complex to the ferrous state during the catalytic cycle. Oxygen uptake induced by bleomycin and ferrous iron is not inhibited by superoxide dismutase (SOD) or catalase. In the absence of bleomycin, catalase strongly inhibits oxygen uptake. This suggests the presence of a relatively stable intermediate in which the superoxide radical is not readily accessible to superoxide dismutase. At pH 9.3, we are able to observe a transient species by electron spin resonance (ESR). When potassium superoxide is added to the ferric iron--bleomycin complex, the same ESR spectrum is produced. We suggest that a transient species composed of a ferric iron, the superoxide ion, and bleomycin is formed. The precise nature of the binding cannot be determined from the data presented.