The work proposed in this competing renewal application is to continue studies on transition metalloproteins and complexes whose metal centers can be probed by various physical techniques. Particular attention will be given to bleomycin (BLM), a glycopeptide antibiotic that cleaves DNA and requires a transition metal for this activity. We will determine that metal ligand in Fe (III)-BLM and activated BLM, elucidating the structure of the coordination site from magnetic field and frequency dependent electron spin echo modulation (ESEEM) studies. We will determine how the structure is change when the drug binds to DNA. We will study the interaction of metallo bleomycins with oligo- and polynucleotides as a means of relating the proposed mechanism of DNA cleavage to the specificity of binding of the drug. With Fe(III)-, activated- and 02Co(II)-BLM, ESEEM studies will be used to determine the distance of the paramagnetic probe to deuterons specifically labelled on polynucleotide sugar. Resonance Raman studies are proposed to elucidate the structure of bound oxygen in an activated belomycin derivative that does not cleave DNA. As fundamental differences have already been seen in the mechanism of Fe-bleomycin action with purified DNA as compare to cell nuclei, experiments are proposed to study the DNA cleavage activity in nuclei and to relate this to the in vitro mechanism. We will assess the requirement for copper in the antibiotic action of BLM in cells where metallothionein level are elevated and copper is sequestered. We will continue the development of electron spin echo envelope modulation (ESEEM) spectroscopy with particular attention to the study of 170, 23NA and 39K interactions with Mn(II)-ATP in kinase, both to quantify the number of interacting nuclei and to determine their distances from the paramagnetic center. Our continuing ESEEM studies with substituted imidazole- 14N interactions with Cu(II), heme, and Fe(III)- tetraphenylporphyrin, models for copper oxidase and mitochondrial cytochrome b, will assess the relative contributions of steric as compared to electronic effects on electron-nuclear coupling.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM040168-32
Application #
2180199
Study Section
Special Emphasis Panel (NSS)
Project Start
1988-05-01
Project End
1998-04-30
Budget Start
1994-05-01
Budget End
1995-04-30
Support Year
32
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Pharmacology
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
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Vergara, Alessandro; Franzese, Marisa; Merlino, Antonello et al. (2007) Structural characterization of ferric hemoglobins from three antarctic fish species of the suborder notothenioidei. Biophys J 93:2822-9
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Peisach, Jack (2003) An appreciation of William H. Orme-Johnson III. J Inorg Biochem 93:6-10
Burns, Colin S; Aronoff-Spencer, Eliah; Legname, Giuseppe et al. (2003) Copper coordination in the full-length, recombinant prion protein. Biochemistry 42:6794-803
Legler, Patricia M; Lee, H Caroline; Peisach, Jack et al. (2002) Kinetic and magnetic resonance studies of the role of metal ions in the mechanism of Escherichia coli GDP-mannose mannosyl hydrolase, an unusual nudix enzyme. Biochemistry 41:4655-68

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