Characterization of cytochrome c structure and function by site-directed mutagenesis.
AuthorCaffrey, Michael Stephen.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractA dual Rhodobacter capsulatus/Rhodobacter sphaeroides genetic system has been used to study the structure and function of R. capsulatus cytochrome c₂. In the first part of this study, the processing, stability and in vivo, functionality of nine site-directed mutants have been examined. Mutations were designed to test various structural and functional properties of cytochrome c₂ such as redox potential (Y75C, Y75F and Y75S), surface charges (K12D, K14E, K32E and K14E/K32E), and protein conformation (P35A and W67Y). All R. capsulatus cytochrome c₂ mutants, except Y75C and Y75S, were overproduced in both R. capsulatus and R. sphaeroides suggesting that these mutations had no effects on heme attachment and protein stability. Furthermore, all R. capsulatus cytochrome c₂ mutants transcomplement for photosynthetic growth a cytochrome c₂ minus mutant of R. sphaeroides suggesting that these mutations function in vivo. Analysis of the spectroscopic, redox potential, kinetic and stability properties of mutants Y75C and Y75F suggested that R. capsulatus tyrosine 75 or its equivalent in other species plays an important role in formation of a hydrogen bonding network which results in maintaining redox potentials and stability of cytochromes c in general. It was found that the charge mutants exhibited small reductions in redox potentials that were consistent with the substitution of positively charged groups with negatively charged groups. Kinetic analyses of the charge mutant photooxidations by R. sphaeroides reaction centers suggested that the lysine groups surrounding the cytochrome c exposed heme edge are not critical to cytochrome c structure and function but play a role in optimal molecular orientation for electron transfer reactions. In addition, denaturation studies of the charge mutants indicated that lysine groups in the amino terminal alpha helix may be important to cytochrome c₂ stability. Analysis of the spectroscopic, redox potential, kinetic and stability properties of mutants P35A and W67Y suggested that proline 35 and tryptophan 67 of R. capsulatus cytochrome c₂ or their equivalents in other species are important to stability but not critical to the structure, redox potential, and function of cytochromes c in general.