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Title:Semisynthetic cytochrome c site-67 substitutions
Author(s):Frauenhoff, Mary Mills
Doctoral Committee Chair(s):Kenneth S. Suslick
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
Chemistry, Inorganic
Abstract:Highly conserved tyrosine-67 of mitochondrial cytochrome $c$ is thought to be involved in important hydrogen bonding interactions in the hydrophobic heme pocket of the protein. In order to investigate the hydrogen bonding role of this residue, two site-67 analogs were prepared by semisynthetic methods: (Hse-65, Phe-67)cytochrome $c$ and (Hse-65, p-F-Phe-67)cytochrome $c$. The analog proteins were reconstituted from a native heme-containing peptide, residues 1 to 65, prepared by CNBr degradation of horse heart cytochrome $c$, and a synthetic peptide, residues 66-104, prepared by solid-phase peptide synthesis. The analogs were characterized and compared to (Hse-65)cytochrome $c$ and the native protein. Both analogs have well developed 695-nm visible absorption bands and are active in a cytochrome $c$ oxidase assay. The redox potentials of (Hse-65, p-F-Phe-67)cytochrome $c$ and (Hse-65, Phe-67)cytochrome $c$ were lower than the native protein by 45 and 50 mV respectively. Both analogs had similar binding constants for imidazole, increased approximately 10-fold over the native protein and (Hse-65)cytochrome $c$. In a cyanide binding study, however, (Hse-65, p-F-Phe-67)cytochrome $c$ had a 3- to 5-fold lower binding constant than the other proteins. The pK$\sb{\rm a}$ values for the alkaline transition for (Hse-65, p-F-Phe-67)cytochrome $c$ was 9.7, and for (Hse-65, Phe-67)cytochrome $c$ was $\geq$10.3, compared to 9.4 for the native protein. The redox potential result may be explained on the basis of a more open heme pocket in the two site-67 analog proteins. Results from ligand binding and alkaline transition studies may be explained best as stabilization or destabilization of the forms of the analogs in which the Met-80 to heme iron bond has been displaced with another ligand, rather than as stabilization of the forms in which the Met-80 to heme iron bond is intact.
Issue Date:1990
Rights Information:Copyright 1990 Frauenhoff, Mary Mills
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9026184
OCLC Identifier:(UMI)AAI9026184

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