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Title:Long-range electron transfer in cobalt-labeled cytochrome c
Author(s):Conrad, David William
Doctoral Committee Chair(s):Faulkner, Larry R.; Scott, R.A.
Department / Program:Chemistry, Analytical
Chemistry, Inorganic
Discipline:Chemistry, Analytical
Chemistry, Inorganic
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Analytical
Chemistry, Inorganic
Abstract:In order to investigate the relationship between intramolecular electron transfer kinetics and redox site separation distance, a semisynthetic two-site donor/acceptor system, in which the distance between redox sites was fixed and known, was prepared. The approach taken involved attachment of the macrocyclic cage complex (Co(diAMsar)) $\sp{3+}$ to various sites on the surface of horse heart cytochrome c. In this way, a second redox site, in addition to the heme, was introduced. Labelling of the protein at several surface sites, allowed several donor/acceptor distances to be evaluated within the same protein system.
The attachment procedure utilized the water-soluble carbodiimide, 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), to promote the condensation of carboxylates provided by the protein with pendant amine groups furnished by the cobalt complex, resulting in a stable amide-bond linkage. Purification of the reaction products allowed isolation of seven different singly modified derivatives. Tryptic peptide mapping experiments, coupled with the results of amino acid analysis, peptide sequencing, and FAB-MS (MS/MS), established the unique modification site in each of these cobalt-labeled proteins.
The long-lived excited state of Ru(bpy)$\sb3\sp{2+}$ was used as both a powerful photooxidant and photoreductant in flash photolysis experiments designed to measure the intramolecular electron transfer rate constants (k$\sb{\rm et}$) from Co(II) $\to$ Fe(III) in the thermodynamically unstable mixed-valence state. Although the results of molecular modelling studies established that derivatives spanned a range of heme edge-to-cobalt distances of 10 to 20 A, each displayed similar values for k$\sb{\rm et}$ (1-3 s$\sp{-1}$). Possible explanations for this apparent lack of a distance dependence are discussed.
The modified protein derivatives were also covalently attached to the surface of an edgeplane pyrolytic graphite electrode in an effort to electrochemically measure k$\sb{\rm et}$ using a second independent technique. Although this approach was unsuccessful in yielding a rate constant, due to the presence of large background currents, the procedure did allow for the development of a novel directional attachment procedure. Through proper control of such variables as reaction pH, or electrode potential, the protein was able to be attached in specific molecular orientations with respect to the electrode surface.
Issue Date:1990
Rights Information:Copyright 1990 Conrad, David William
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9021665
OCLC Identifier:(UMI)AAI9021665

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