ELECTROSPRAY MASS-SPECTROMETRY OF IRON BLEOMYCIN - DEMONSTRATION THAT ACTIVATED BLEOMYCIN IS A FERRIC PEROXIDE COMPLEX

The glycopeptide antibiotic bleomycin Aa (BLM) possesses potent antineoplastic activity, presumably due to its ability to bind iron and activate oxygen, forming a species, activated BLM, that is kinetically competent to cleave DNA. Activated BLM may be formed from Fe(II)BLM and O-2 followed by single electron reduction or directly from Fe(III)BLM and H2O2 (Burger, R.M.; Peisach, J.; Horwitz, S.B. J. Biol. Chem. 1981, 256, 11636-11644). We have used electrospray mass spectrometry to study the activation of oxygen by FeBLM. Upon reacting Fe(III)BLM with H2O2 Or Fe(II)BLM with O-2, we observe an intermediate that displays kinetics of formation and decay similar to those of activated BLM and a mass to charge ratio consistent with that of HOO-Fe(III)BLM. Formation of this species by reacting Fe(III)BLM with (H2O2)-O-18 and the observation of its increase in mass by 4 Da confirm that this species contains two oxygen atoms derived from hydrogen peroxide, These results strongly suggest that activated BLM is a ferric peroxide complex. Tandem mass spectrometry (MS/MS) of activated BLM was also performed, and the data indicate that the O-O bond is labile. The significance of these results to the activation of oxygen by FeBLM and other non-heme iron systems is discussed.