Electronic structure of activated bleomycin: Oxygen intermediates in heme versus non-heme iron

Bleomycin (BLM) is a glycopeptide antibiotic produced by the fungus Strepto myces verticillus which is used clinically for anticancer therapy. It is mo st active as an iron complex. A detailed spectroscopic and theoretical stud y of the ferric form of the drug, Fe(III)BLM, and its activated form, ABLM, is reported. ABLM, which has been shown to be a ferric hydroperoxide compl ex, is the last detectable intermediate in the reaction cycle of BLM that l eads to a DNA radical and subsequent cleavage. Both forms of the drug are l ow-spin Fe(III) complexes and were studied with electron paramagnetic reson ance (EPR), magnetic circular dichroism (MCD), and absorption (ABS) spectro scopy. In addition, resonance Raman (rR) and circular dichroism (CD) spectr a are reported for Fe(III)BLM. The g matrix was analyzed in detail, and Gri ffith's model for row-spin d(5) complexes was experimentally evaluated. A l igand field analysis of the d-d region of the optical spectra resulted in a complete determination of the ligand field parameters for both forms of th e drug. Analysis of the optical and rR data led to assignment of the main A BS band at 386 nm in both Fe(III)BLM and ABLM as deprotonated amide-to-iron ligand-to-metal charge-transfer (LMCT) transitions. Revised vibrational as signments are proposed on the basis of B3LYP frequency calculations on mode ls of Fe(III)BLM. On the basis of energetically optimized geometric models for Fe(III)BLM and ABLM the electronic structures of both forms were analyz ed using density functional theory (DFT), ab initio Hartree-Fock, and INDO/ S methods. Conjugation of the deprotonated amide and pyrimidine functionali ties is not proposed to be a major factor in the BLM electronic structure. In agreement with experimental data the calculations show that the ligand f ields of both Fe(III)BLM and ABLM are dominated by the deprotonated amide t hat also determines the orientation of the t(2g) hole, the orientation of t he g matrix and Mossbauer quadrupole tensor. The main LMCT band was shown t o be an amide pi -LMCT band. The calculations were extended to study the re activity of the drug toward DNA. The possibilities of (a) heterolytic cleav age of the O-O bond in ABLM to give Fe(V)BLM = O and H2O, (b) homolytic cle avage to give Fe(IV)BLM = O and OH., and (c) direct attack of the hydropero xide of ABLM on DNA to give Fe(IV)BLM = O, water, and a DNA radical were ev aluated. Importantly, heterolytic cleavage is strongly suggested to be ener getically unfavorable because it leads to a product which is best described as [Fe(IV) = O BLM'] with a high-energy hole localized on the deprotonated amide. Initial homolytic cleavage is also discarded on the basis of the sp ecificity of the reaction; this leads to the new working hypothesis that AB LM chemistry proceeds by direct attack of the substrate C-H bond by the low -spin ferric hydroperoxide complex ABLM. The electronic structure contribut ions to such a reaction are discussed, and the relationship of ABLM and cyt ochrome P450 is addressed.