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Abstract

Crystal structures of the cytochrome bc₁ 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 c1. 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 µm. Addition of two alanine residues lowers the activity by 90% and the apparent K_m decreases to 1.9 µm. Deletion of an alanine residue lowers the activity by ≈ 40% and the apparent K_m decreases to 5.0 µm. 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 bc₁ complex, consistent with electron transfer mechanisms in which ubiquinol must simultaneously interact with the iron–sulfur protein and cytochrome b.