The long-term goal of this research is to understand the role of mitochondrially-encoded proteins in structure and function of the respiratory chain. This is an area with relevance to public health as there is a growing awareness that a number of human disorders are associated with defects in the mitochondrial respiratory chain. The more immediate focus is upon the molecular enzymology of electron transfer and proton uptake/release in the bc1 complex. The proposed research involves a mutational analysis of the Qi or ubiquinone reductase center of the protonmotive cytochrome beta protein, a mitochondrial gene product. A unique set of mutations in the mouse cytochrome beta gene have been isolated and shown to affect inhibitor binding and catalytic function at the Qi center. These mutations will be analyzed further to determine the effect of the amino acid substitutions upon the quinone redox chemistry at this site including the formation of the stable Qi semiquinone anion. As a complementary approach, site-directed mutagenesis of the Paracoccus cytochrome beta gene will be undertaken. The initial studies will involve the mutation of evolutionarily conserved acidic and basic amino acid residues which may be involved in controlling Qi semiquinone anion stability and function, and particularly the interaction between this redox intermediate and the cytochrome betaH heme group.
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