Bleomycin (BLM), a glycopeptide antibiotic chemotherapy agent, is capable of single- and double-strand DNA damage. Activated bleomycin (ABLM), a low-spin Fe
III-OOH complex, is the last intermediatedetected prior to DNA cleavage following hydrogen-atom abstraction from the C-4' of a deoxyribose sugarmoiety. The mechanism of this C-H bond cleavage reaction and the nature of the active oxidizing speciesare still open issues. We have used kinetic measurements in combination with density functional calculationsto study the reactivity of ABLM and the mechanism of the initial attack on DNA. Circular dichroismspectroscopy was used to directly monitor the kinetics of the ABLM reaction. These experiments yield adeuterium isotope effect,
kH/
kD ![](/images/entities/ap.gif)
3 for ABLM decay, indicating the involvement of a hydrogen atom in therate-determining step. H-atom donors with relatively weak X-H bonds accelerate the reaction rate,establishing that ABLM is capable of hydrogen-atom abstraction. Density functional calculations were usedto evaluate the two-dimensional potential energy surface for the direct hydrogen-atom abstraction reactionof the deoxyribose 4'-H by ABLM. The calculations confirm that ABLM is thermodynamically and kineticallycompetent for H-atom abstraction. The activation and reaction energies for this pathway are favored overboth homolytic and heterolytic O-O bond cleavage. Direct H-atom abstraction by ABLM would generatea reactive Fe
IV=O species, which would be capable of a second DNA strand cleavage, as observed
invivo. This study provides experimental and theoretical evidence for direct H-atom abstraction by ABLMand proposes an attractive mechanism for the role of ABLM in double-strand cleavage.