The Synthesis and Characterization of Inorganic Redox Reagent-Modified Cytochromes C

Citation

Yocom, Kathryn Mary (1982) The Synthesis and Characterization of Inorganic Redox Reagent-Modified Cytochromes C. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/5tfv-gx02. https://resolver.caltech.edu/CaltechTHESIS:10082012-092613083

Abstract

A stable complex is formed between pentaammineruthenium(III) and the imidazole moiety of histidine-33 in cytochrome c. This complex is the major mono-substituted product of the reaction between aquopentaammineruthenium(II) and horse heart cytochrome c at pH 7. It is isolated and purified by ion exchange chromatography on CM-cellulose. High-pressure liquid chromatography of the tryptic hydrosylate of the modified cytochrome c is shown to be an effective method for the identification of the pentaammineruthenium binding site. The spectrum of the modified peptide mimics that of the pentaamminehistidineruthenium(III) model complex. Spectro-electrochemical and optical absorption measurements show that the integrity of the native structure in the vicinity of the heme c group is maintained in the ruthenium-modified protein. The reduction potentials of the two redox sites of the modified protein, derived from cyclic voltammetric measurements at a gold electrode in the presence of 4,4'-bipyridyl, are: heme c (Fe^3+/2+), 0.26 V; Ru(NH₃)₅(His-33)^3+/2+, 0.15 V (vs. NHE). A-15 Å separation between the two redox sites in this system is estimated from molecular models of cytochrome c. It is suggested that the specificity, stability, and redox properties exhibited by aquopentaammineruthenium(II) render it an ideal protein modification reagent for the production of "synthetic" multisite metalloproteins.

The criterion of product stability is not met by the aquopentacyanoferrate(II) ion. The products isolated from the reaction of this reagent with cytochrome c are believed to be a mixture of histidine and methionine substitution products, and ionic association complexes. The relatively rapid dissociation of the pentacyanoferrate(II) moiety from the protein severely limits the extent to which the products can be characterized.

The synthesis of a cobalt(III)-cytochrome c complex is described. Tetrachloroplatinate(II) reacts specifically with methionine-65. Pyridine-4-carboxylatopentaamminecobalt(III) is subsequently reacted with the platinum center. Unfortunately, the cobalt(II) form of the derivative is substitution labile, and the redox properties of the cobalt complex are highly unfavorable.

Intermolecular reductions of horse heart cytochrome c, Pseudomonas aeruginosa cytochrome c(551), Pseudomonas aeruginosa azurin, and Rhus verniaifera stellacyanin by hexaammineruthenium(II) are reported. Rate constants and activation parameters are presented. The results are discussed in terms of electron transfer distances for metalloprotein redox reactions.

Thesis Files