HEME PROTEIN STRUCTURE AND FUNCTION

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DESCRIPTION: (Verbatim from the Applicant's Abstract) One focus of this study is to further understand the role of specific domains of the activated form of the anticancer antibiotic bleomycin (BLM) in site recognition of DNA cleavage. Present understanding of site recognition is based on a model whereby a ligand of Fe in the drug, amino pyrimidine, H-bonds with guanine in DNA. However, tallysomycin (TLM), a BLM congener containing the same metal ligands and additionally an L-talose away from the metal binding site, has different site specificity, whereas removing the bithiazole moiety as in pseudotetrapeptide A (PTP) abolishes site specificity. This suggests that the metal binding site alone does not govern site recognition. A series of experiments are therefore proposed that aim at understanding site recognition in FeTLM and PTP with DNA. It is hypothesized that the difference in structure of TLM as compared to BLM is manifest in a direct effect on the binding of drug to DNA and an indirect effect on the electron distribution in the metal binding site and the oxygenous ligands of Fe, thereby altering activity. This study will include an NMR investigation of HOO-Co(III)TLM both in the presence and absence of DNA to elucidate the 3-D structure, an ESEEM study of O2-Co(II) and Fe(III)TLM with specifically deuterated oligonucleotides to determine the distance from the metal to the targeted C4,H of deoxyribose as well as (1) the mechanism of formation and the structure of the activated complexes of TLM and PTP using mass spec and (2) the rate and stoichiometry of products formed in the reaction in single and multiple turnover experiments with DNA to relate activity to kinetic competence and reaction mechanism. A second focus is to carry out EPR, ESEEM, and HYSCORE (2D ESEEM) studies of the deoxy Co subunits of Fe-Co hybrid hemoglobins (Hbs) that are in the R, T, and the newly proposed T' (monoligated and diligated tetramers in which two subunits in the same dimer are ligated) quaternary state. These studies will examine electron-nuclear coupling to the metal and its nitrogen ligands, as well as relative orientation of the axial His imidazole, in order to test the hypotheses that there exists a quaternary state structurally distinct from the T and R state in the Monod-Wyman-Changeux two-state model, and that quaternary structural change is accompanied by structural change in the metal center of the alpha-subunits only.