Q-band electron nuclear double resonance (ENDOR) has measured anisotropic, distance-dependent dipolar hyperfine couplings from iron in ferric bleomycin [Fe(III)-BLM] and in activated bleomycin [Act-BLM] to P-31 of substrate DNA. Studies were focused on bleomycin complexes with a self-complementary duplex DNA 10-mer, d(GGAA<(GC)under bar>TTCC)(2), containing a 5'-G-C-3' sequence that is selective for bleomycin cleavage (Mao, Q.; Fulmer, P.; Li, W.; DeRose, E. G.; Petering, D. H. J. Biol. Chem. 1996, 271, 6185-6191). Bleomycin complexes with high molecular weight calf thymus DNA were also used. Fe(III)-BLM and Act-BLM complexes with the 10-mer and the calf thymus DNA showed anisotropic P-31 dipolar hyperfine couplings from which an Fe(III)-to-P-31 distance was estimated at 7.4 +/- 0.2 Angstrom. High-resolution, angle-selected ENDOR of the Fe(III)-BLM 10-mer complex showed that the Fe(III)-to-P-31 vector lay at 25 +/- 5 degrees to the maximal g value direction, where the latter direction pointed near the exchangeable protons on axial BLM ligands and is associated with the maximal hyperfine couplings of these protons (Veselov, A.; Sun, H.; Sienkiewicz, A.; Taylor, H.; Burger, R. M.; Scholes, C. P. J. Am. Chem. Sec. 1995, 117, 7508-7512). Proton ENDOR features of the Fe(III)-BLM but not Act-BLM were perturbed by DNA substrate. Zn the presence of the 10-mer and the calf thymus DNA, proton ENDOR revealed distinct perturbation to the frequencies of three sets of nonexchangeable protons assigned to the BLM macrocycle and estimated to be 2.9-3.5 Angstrom from Fe(III) and to the frequencies of exchangeable, axially located protons. In contrast, the hyperfine couplings of covalently bonded, first-shell nitrogen and [O-17]peroxy ligands were unchanged.