A GENETIC ANALYSIS FOR THE FUNCTIONAL SITES OF THE

点击翻译按钮获取中文摘要

The ubiquinol:cytochrome c oxidoreductase (or the bc1 complex) is involved in the formation of the membrane potential and proton gradient, a vital process for living cells. The long term goal of this project is to understand, in molecular terms, how the structural subunits of the bc1 complex interact with their associated prosthetic groups to perform this role. As an initial step, the structural genes of the bc1 complex of Rhodopseudomonas capsulata, a photosynthetic prokaryote, have been isolated and their nucleotide sequences have been determined. The in vivo role of this complex has also been assessed by constructing null bc1 mutants. We now plan to undertake a detailed molecular genetic analysis to correlate the function sites of the bc1 complex (quinol oxidation, quinone reduction, and cytochrome c reduction) with its structural subunits (the FeS protein, cytochrome b, and cytochrome c1). For this purpose we will (1) select mutants resistant to specific inhibitors of the bc1 complex; (2) obtain new bc1 mutants that assemble a functionally altered complex; (3) map and sequence these mutations, including aer126 of R126, a mutant defective in the quinol oxidation site of the complex; and (4) change the amino acid residues thus defined to various other residues by sitedirected oligonucleotide mutagenesis to correlate the mutated sites with the perturbed functions. Insights gained in this prokaryotic system should be generally applicable to structurally more complex but functionally similar mitochondrial and chloroplast oxidoreductases of higher organisms, which are often refractory to genetic analysis. In general, this work should also contribute to a better understanding of functional interactions between the prosthetic groups and the structural subunits of membrane proteins.