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UBC Theses and Dissertations

Peroxidase activity of yeast iso-1-cytochrome c Villegas, Jose


Understanding the peroxidase activity of cytochrome c is essential for the development of potential applications of this useful property in diagnostics, chemical synthesis and bioremediation. Such knowledge may also be relevant for understanding the chemical basis of the physiological consequences of this reaction under conditions of oxidative stress induced by toxic species generated by various cellular processes. In the present work, the thermodynamic, spectroscopic and catalytic properties of two classes of cytochrome c variants have been studied in an effort to gain greater insight into the structural basis for the peroxidase activity of the cytochrome. The ultimate goal of this was to identify a strategy for the design or identification of new cytochrome c variants with significantly enhanced peroxidase activity. Development of one class of these variants was based on structural insight provided by X-ray crystallography, and the resulting variants were produced by site-directed mutagenesis. The other class of variants were obtained by a directed evolution method in which randomly-generated variants with elevated peroxidase activity were identified by the use of an activity screen. The results obtained suggest that the changes in the peroxidase activity observed for each variant are related not only to an overall destabilization of the protein structure but also to localized variations around the prosthetic group that function in combination to regulate the kinetic behavior of this protein. Such structural factors are discussed here in the context of the thermodynamic, spectroscopic and catalytic properties of each variant.

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