Q-band Electron Nuclear Double Resonance of Ferric Bleomycin and Activated Bleomycin Complexes with DNA:  Fe(III) Hyperfine Interaction with ³¹P and DNA-Induced Perturbation to Bleomycin Structure

Abstract

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 31 P of substrate DNA. Studies were focused on bleomycin complexes with a self-complementary duplex DNA 10-mer, d(GGAAGCTTCC) 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 31 P dipolar hyperfine couplings from which an Fe(III)-to-31 P distance was estimated at 7.4 ( 0.2 Å. High-resolution, angle-selected ENDOR of the Fe(III)-BLM 10-mer complex showed that the Fe(III)-to-31 P vector lay at 25 ( 5°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. Soc. 1995, 117, 7508-7512). Proton ENDOR features of the Fe(III)-BLM but not Act-BLM were perturbed by DNA substrate. In 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 Å from Fe(III) and to the frequencies of exchangeable, axially located protons. In contrast, the hyperfine couplings of covalently bonded, first-shell nitrogen and [ 17 O]peroxy ligands were unchanged.

Definition of the intermediates and mechanism of the anticancer drug bleomycin using nuclear resonance vibrational spectroscopy and related methods

“…We now consider the ðBLMÞFe III ðη 1 -OOHÞ model for ABLM, which had been suggested based on EPR (8,28,29), Mössbauer (30), EXAFS (9), electron nuclear double-resonance (ENDOR) spectroscopy (29,31), mass spectrometry (11), X-ray absorption spectroscopy (XAS) edge structure (9), and magnetic circular dichroism (MCD) spectroscopy (10). A ðBLMÞFe III ðη 1 -OOHÞ model, OOH-69-syn-α, was optimized based on the NMR structure of ðBLMÞCo III ─OOH (PDB ID 1MXK) (27).…”

Thermodynamics and kinetics of the cleavage of DNA catalyzed by bleomycin A5

“…The experimental apparent molar enthalpy changes for these reactions can be calculated using the integration type of Tian's equation and Eqn (16) from the calorimetric curves. The thermodynamic data for the binding, in which the values of K B , D b H 0 m and n are obtained by fitting the apparent molar enthalpy changes to Eqn (18), are summarized in Table 3. The v 2 value of Eqn (18) used to perform a nonlinear least-squares analysis for the binding of BLM-A 5 to DNA is 0.0268, indicating a good appropriateness of the model proposed.…”

“…It has been found that BLM-induced autoxidation of ferrous iron follows the Michaelis-Menten kinetics [9,10]. Although a significant number of experimental approaches have been used to elucidate the mechanism of DNA cleavage by BLM in the past two decades [2][3][4][5][6][11][12][13][14][15][16][17][18][19][20], thermodynamic information for the scission, which is necessary for a thorough understanding of the mechanism, is eagerly awaited. The purpose of this investigation is to provide detailed thermodynamic and kinetic data for BLMmediated DNA degradation to furnish insights into the anticancer mechanism of BLM.Microcalorimetry is an important tool for the study of both thermodynamic and kinetic properties of biological macromolecules by virtue of its general applicability, high accuracy and precision [21][22][23][24].…”

Nature of Activated Bleomycin

“…This stability and its diamagnetism have made the cobalt complex a useful analogue for activated bleomycin in NMR structural studies of bleomycin both alone [30, 67±70] and complexed to DNA [70±73]. Detailed NMR-derived structures are consistent with structural information obtained for Fe(II)ábleomycin by NMR [74] and activated bleomycin by ENDOR spectroscopy [75,76]. The structures deduced for the green complex and its synthetic congeners are therefore assumed to be good models for activated bleomycin.…”

“…If the former is the case, the hydrogen exchange must occur in seconds or less, because a change in activated bleomycin decay rate, monitored at 1 min intervals, occurs without a detectable lag when the solvent isotopic composition is rapidly changed. Exchangeable hydrogens have been found in the vicinity of the metal-peroxide complex, both by ENDOR [76] and by NMR [32] spectroscopy, but they are probably different atoms. The ENDOR proton signals are tentatively assigned to an axial metal ligand, probably b-aminoalanine, while the NMR protons are assigned to hydrogen bonds linking the peroxide with the threonine and methylvalerate moieties.…”