Distinct ubiquinone binding at the oxidation and reduction sites of cytochrome bc₁

The function of cytochrome bc₁, a widespread energy-conserving enzyme, requires the coordinated activity of two quinone-binding sites (Q_o catalyzing oxidation of ubiquinol and Q_i catalyzing reduction of ubiquinone). The operation of Q_o, but not Q_i, involves large-scale movement of the head domain of iron-sulfur protein (ISP-HD). How the respective sites accommodate quinone molecules for efficient catalysis remains elusive. Here, we present high-resolution cryoelectron microscopy structures of bacterial cytochrome bc₁ with native ubiquinone molecules in various states. They show that the quinone headgroup occupies a catalytically competent position in Q_o only when the ISP-HD interacts with cytochrome b. When the ISP-HD does not interact with this subunit, quinone is present in the hydrophobic groove, however its headgroup is prevented from reaching the catalytic cavity by steric hindrance. In this state, the position of quinone headgroup is clearly not fixed. In contrast, all structures show Q_i in the same state with a well-resolved and catalytically competent quinone headgroup, but with its tail not fixed. These distinctly different ubiquinone binding modes for Q_o and Q_i secure the smooth operation of cytochrome bc₁.