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UBC Theses and Dissertations

The nature and role of oxidation state dependent conformational differences in cytochrome c Berghuis, Albert M.


The objective of the work described in this thesis was to study the nature and role of conformational differences between the oxidation states of cytochrome c. Using x-ray crystallographic techniques, the oxidized form of yeast iso-l-cytochrome c was solved and compared to the previously determined reduced state. The following differences between the oxidation states were identified. Three segments of polypeptide chain, located for the most part on the Met80 side of the protein, were shown to display an increase in mobility in the oxidized state. A conserved internal water molecule, Wat166, was observed to shift 1.7 A towards the heme iron atom and reorient its dipole moment in the oxidized state. As part of this movement several hydrogen bonds were broken including the interaction between Tyr67 OH and the Met80 SD heme ligand. Finally, differences between the two oxidation states were also observed for the conformation of the pyrrole A propionate and its associated hydrogen bond network, the distortion of the porphyrin ring plane, and the orientation of the imidazole plane of the His18 ligand. In order to assess the function of the observed conformational differences between the two oxidation states of cytochrome c, the three dimensional structures of five mutants (N52A, N521,Y67F, N52I-Y67F and 175M) were determined, of which three were completed in both oxidation states. Correlation of wild-type and variant protein structures with functional studies suggested that Wat166 was a central feature in oxidation state dependent differences, and three roles for this water molecule could be identified. First, the oxidation state dependent positioning and orientation of Wat166 appears to be particularly important for modulating the interaction between Tyr67 OH and Met80 SD. This hydrogen bond was shown to influence the electron withdrawing power of the Met80 ligand and therefore is a factor in controlling the midpoint reduction potential of cytochrome c. Secondly, the presence of Wat166 is necessary to maintain the spatial and hydrogen bonding relationships between residues in this region of the protein. Finally, Wat166 also appears to mediate the oxidation state dependent flexibility of selected polypeptide chain segments. The biological function of this phenomenon is still unclear, but our results suggest that it might play a role in interactions between cytochrome c and its redox partners. In conclusion, the work described in this thesis gives insight into the structure-function relationships in cytochrome c and provides a basis for future studies aimed at understanding the mechanism of electron transfer carried out by this protein.

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