Changes to the length of the flexible linker region of the Rieske protein impair the interaction of ubiquinol with the cytochrome bc1 complex

Crystal structures of the cytochrome bc(1) complex indicate that the catalytic domain of the Rieske iron-sulfur protein, which carries the [2Fe-2S] cluster, is connected to a transmembrane anchor by a flexible linker region. This flexible linker allows the catalytic domain to move between two positions, proximal to cytochrome b and cytochrome cl. Addition of an alanine residue to the flexible linker region of the Rieske protein lowers the ubiquinol-cytochrome c reductase activity of the mitochondrial membranes by one half and causes the apparent K-m for ubiquinol to decrease from 9.3 to 2.6 mu M. Addition of two alanine residues lowers the activity by 90% and the apparent K-m decreases to 1.9 mu M. Deletion of an alanine residue lowers the activity by approximate to 40% and the apparent K-m decreases to 5.0 mu M. Addition or deletion of an alanine residue also causes a pronounced decrease in efficacy of inhibition of ubiquinol-cytochrome c reductase activity by stigmatellin, which binds analogous to reaction intermediates of ubiquinol oxidation. These results indicate that the length of the flexible linker region is critical for interaction of ubiquinol with the bc(1) complex, consistent with electron transfer mechanisms in which ubiquinol must simultaneously interact with the iron-sulfur protein and cytochrome b.