IRON COORDINATION OF ACTIVATED BLEOMYCIN PROBED BY Q-BAND AND X-BAND ENDOR - HYPERFINE COUPLING TO ACTIVATED O-17 OXYGEN, N-14 AND EXCHANGEABLE 1(H)

We provide initial electron nuclear double resonance (ENDOR) findings on the electronic structure of iron-containing bleomycin and its activ ated oxygen. Q-Band (34 GHz) ENDOR has resolved hyperfine couplings fr om the activated O-17 oxygen that originates in isotopically enriched O-17(2) gas. The largest O-17 hyperfine coupling, approximately 27 MHz , was best resolved at an intermediate g-value (g(y) 2.17), and a smal ler hyperfine coupling, approximately 10 MHz, was resolved at the extr emal g(z) (g(max)) and g(x) (g(min)) features as well as at g(y). The wider frequency range and anisotropy of O-17 ENDOR features from activ ated oxygen in bleomycin were in marked contrast to the narrow ENDOR f eature of the activated oxyferryl oxygen of horseradish peroxidase com pound I (Roberts, J. E.; Hoffman, B. M.; Rutter, R.; Hager, L. P. J. A m Chem. Soc. 1981, 103, 7654-7656). The implication of this contrast i s that the electronic structure of the activated oxygen differs marked ly in the two compounds. The agreement between Q-band ENDOR-resolved h yperfine couplings and hyperfine couplings estimated from X-band EPR l ine broadening at g(x) suggests that there is a large hyperfine coupli ng to only one oxygen. Hydroperoxide has been indicated as an iron lig and for activated bleomycin (Sam, J. FV.; Tang, X-J.; Peisach, J. J. A m. Chem. Sec. 1994, 116, 5250-5256), and strong hyperfine coupling to one oxygen is compatible with end-on ligation of the hydroperoxide. Ex changeable proton ENDOR patterns, well separated at Q-band from N-14 a nd O-17 features, had maximal couplings of the order 13 MHz near the g (z) axis, as might be expected for protons which are hydrogen bonded t o first shell amine nitrogen or oxygen axial ligands. Activated bleomy cin and ferric bleomycin (prepared by chelating ferric ion with bleomy cin) differed in details of their proton ENDOR. Nitrogen (N-14) ENDOR features and hyperfine couplings resembled those of low-spin ferric he me and imidazole nitrogens.