The ubiquinol:cytochrome c oxidoreductase (bc1 complex) is an essential component of cellular energy transduction, and is involved in the formation of a membrane potential and a proton gradient necessary to produce ATP. The long term goal of this project is to understand, in detailed molecular terms, how the cyt bc1 complex functions during electron transfer and vectorial proton translocation. This widespread complex is essential for living cells, and its improper function leads to severe neurological and muscular diseases. Our experimental system derives from the procaryote Rhodobacter capsulatus which is a model organism for mitochondrial and chloroplast energy transduction. Previously, the primary structure of the cyt bc1 complex was established using molecular genetic techniques. The location of one of its active domains, the quinol oxidation (Qo) site, is now emerging from the analysis of inhibitor resistant (InhR) mutants. This project will continue molecular genetic and biochemical analyses of the cyt bc1 complex to correlate its functional sites [quinol oxidation (Qz,o,p) and quinone reduction (Qc,i,n)] with different regions of its structural subunits (FeS protein, cyt b, cyt c1). For this purpose, (1) Role of the specific amino acid residues of cyt b located in the vicinity of the Qo domain will be analyzed by saturation mutagenesis; (2) Intrasubunit and intersubunit interactions at the Qo domain will be analyzed by second site suppressors of non functional cyt b mutants; (3) Contribution of the universally conserved residues of the FeS protein to the Qo site will be assessed by mutagenesis; (4) Location of the Qi domain of a bacterial bc1 complex will be defined by analysis of InhR mutants of Rhodospirillum rubrum, a naturally sensitive species and by construction of Qi mutants of R. capsulatus; and (5) Structural and functional implications of the addition of a residue between the axial ligands of the cyt bH and bL hemes will be tested. These mutants will be characterized by genetic, physiological and biochemical analyses. Insights gained in this bacterial system are generally applicable to the structurally more complex and yet functionally similar mitochondrial and chloroplast oxidoreductases, and are relevant to the understanding of the molecular basis of mitochondrial diseases. This work also contributes to a better recognition of the interactions between the subunits of membrane proteins and their prosthetic groups during their biogenesis and assembly.
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