Copper(I)-bleomycin [Cu(I).BLM] was characterized in detail by 13C and 1H NMR. Unequivocal chemical shift assignments for Cu(I)-BLM and Cu(I)-BLM.CO were made by two-dimensional 1H-13C correlated spectroscopy and by utilizing the observation that Cu(I)-BLM was in rapid equilibrium with Cu(I) and metal-free bleomycin, such that individual resonances in the spectra of BLM and Cu(I)-BLM could be correlated. The binding of Cu(I) by bleomycin involves the β-aminoalaninamide and pyrimidinyl moieties, and possibly the imidazole, but not Na of β-hydroxyhistidine. Although no DNA strand scission by Cu(II)-BLM could be demonstrated in the absence of dithiothreitol, in the presence of this reducing agent substantial degradation of [3H]DNA was observed, as was strand scission of cccDNA. DNA degradation by Cu(I)-BLM was shown not to depend on contaminating Fe(II) and not to result in the formation of thymine propenal; the probable reason(s) for the lack of observed DNA degradation in earlier studies employing Cu(II)-BLM and dithiothreitol was (were) also identified. DNA strand scission was also noted under anaerobic conditions when Cu(II)-BLM and iodosobenzene were employed. If it is assumed that the mechanism of DNA degradation in this case is the same as that under aerobic conditions (i.e., with Cu(I).BLM + 02 in the presence of dithiothreitol), then Cu-BLM must be capable of functioning as a monooxygenase in its degradation of DNA.
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