Structure Function Studies of Cytochromes

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9707680 McLendon The aims of this research are to examine structure function relationships in the protein cytochrome C1. Genetic and molecular biological procedures will be used to systematically alter cytochrome c1 (Cc1) in the yeast Saccharomyces cerevisiae. Cc1 mutants will be generated by three procedures: conventional oligonucleotide-directed mutagenesis, transformation of yeast directly with synthetic oligonucleotides, and PCR-generated random mutagenesis. The Cc1 mutants will be tested for functionality by growth on nonfermentable media (Nfs+ or Nfs-) , Cc1 levels by spectral analysis, and preservation of the over-all structure of Nfs- mutants by dominate-negative genetic analysis. The altered forms will be used to investigate structure-function relationships of Cc1 and the interactions between Cc1 and cytochrome c (Cc), both in vivo and in vitro. The sites of interaction between Cc1 and Cc, as well as between the cytochromes and Qcr6p, will be investigated with genetic approaches using suppressor mutations. Reactivity in vitro will be studied by steady state and single turnover methods, including highly exergonic (G( reactions using photoinduced electron transfer from Cc. The objective of this research is to examine structure function relationships in the protein cytochrome c1 (Cc1). This protein plays a critical role in mitochodrial electron transport, and thereby helps control energy production within cells. This research will probe factors which influence the redox potentials of Cc1, and thereby affect both thermodynamic and kinetic reactivity of this system. The work will employ genetic, molecular biological and biophysical procedures. ***