The ubiquinol-cytochrome c oxidoreductase (cytochrome bc1 complex) is among the most highly conserved constituents of energy transducing membranes. Although complexes from phylogenetically diverse species are functionally interchangeable and their cytochrome c1 cytochrome b and iron-sulfur protein components are homologous, isolated oxidoreductases differ markedly with respect to additional polypeptide constituents. Mitochondrial complexes contain as many as eight additional subunits which are required in the enhancement of electron transport and in the assembly process. The cytochrome bc1 complex of Rhodobacter sphaeroides contains a tightly bound forth subunit which provides sa unique opportunity to elucidate the role of a single noncatalytic component. Partial sequence data will be used to construct hybridization probes to facilitate the isolation of the subunit IV structural gene for sequence comparisons. Topological analyses in which either the cytoplasmic or the periplasmic side of the membrane is digested with proteinase K will determine the location of subunit IV to assist in elucidating its functional role. Electron transfer activity and changes in the EPR signals of the iron-sulfur and Qc components will be assessed in the purified cytochrome bc1 complex following limited proteolytic digestion and after exposure to anti-subunit IV antibodies. Mutants in which the structural gene for subunit IV has been interrupted by interposon mutagenesis will be constructed and the composition and functional properties of the altered complex will be examined. These integrated approaches should prove valuable in elucidating important structure-function and assembly relationships in the bc1 complex.
