The ubiquinol: cytochrome c oxidoreductases (the bc1-and b6f-complexes) are the central enzymes of respiratory and photosynthetic electron transfer chains, catalyzing the oxidation of quinol and the reduction of cytochrome c, and the coupling of these redox reactions to proton transfer across the membrane. The complex has three catalytic sites connected through two separate internal electron transfer chains. At one site, cytochrome c1 is oxidized by cytochrome c. The other two catalytic sites are involved in oxidation or reduction of ubiquinone and associated photolytic reactions. They work like conventional enzymic sits, but with the quinone substrates interacting from the lipid phase. The two sites are connected by a b- cytochrome chain which transfers electrons between them and carries the electrogenic flux across the membrane. Many inhibitors have been shown to act competitively at these latter sites, displacing the quinone substrates. In addition to its central role in energy metabolism nd intrinsic interest as an electron transfer protein, the enzyme provides a useful model system for studies of the processing of lipid soluble substrates, and ligand binding from the lipid phase. The bc1-complex of Rhodobacter sphaeroides presents many advantages as a research tool, including a simple structure, a well developed molecular engineering technology, and sell established protocols linked to photoactivation, for rapid kinetic evaluation of partial reactions, and of changes arising from mutation. Our previous work has provided a map of the main features of the complex. These include a tertiary structural model for cytochrome b, potential membrane domains in the Rieske protein and cytochrome c1, the catalytic sites for quinone processing, heme ligands in cytochrome b, and for the 2Fe.2S center in the Rieske protein, and a set of residues where mutation modifies the catalytic sites through changes in substrate or inhibitor binding, electron transfer rates for quinol oxidation or quinone reduction, or intermediates states in catalysis (semiquinone stability). We will extend this work to provide a more complete map of the relation between structure and function. We aim to better understand catalysis by measuring physico-chemical parameters associated with specific structural changes. Detailed characterization of strains mutated at specific residues will provide a 'free-energy' map for the role of each residue, which can be compared with the 3-D structural map given by the atomic coordinates of the model. To understand molecular mechanism we need to explore more completely the tertiary structure for cytochrome b and the other subunits of the complex. We will use casette mutagenesis to identify residues involved in the interfaces between helices, the packing of residues in loops, and the interactions between subunits. A justifiable structure will emerge only if constraints can be introduced by experiment, and we anticipate that for the next few years these will be provided in main by our analysis of mutants.
NIH ExPorter
NIH ExPorter