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Kenneth S. Suslick Frauenhoff, Mary Mills 2011-05-07T13:00:53Z 2011-05-07T13:00:53Z 10000-01-01 1990 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. Made available in DSpace on 2011-05-07T13:00:53Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9026184.pdf: 6628344 bytes, checksum: 517e6c421bc54279dc3df340f402cbf5 (MD5) Previous issue date: 1990 Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:49:02Z Item is restricted indefinitely. Restriction data tranferred 2014-07-01T11:22:16-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission ETDs are only available to UIUC Users without author permission U of I Only AAI9026184 (UMI)AAI9026184 http://hdl.handle.net/2142/21183 eng Copyright 1990 Frauenhoff, Mary Mills Chemistry, Biochemistry Chemistry, Inorganic Semisynthetic cytochrome c site-67 substitutions text Chemistry Chemistry University of Illinois at Urbana-Champaign Dissertation Ph.D.