Molecular basis of plastoquinone reduction in plant cytochrome b₆f

A multi-subunit enzyme, cytochrome b₆f (cytb₆f), provides the crucial link between photosystems I and II in the photosynthetic membranes of higher plants, transferring electrons between plastoquinone (PQ) and plastocyanin. The atomic structure of cytb₆f is known, but its detailed catalytic mechanism remains elusive. Here we present cryogenic electron microscopy structures of spinach cytb₆f at 1.9 Å and 2.2 Å resolution, revealing an unexpected orientation of the substrate PQ in the haem ligand niche that forms the PQ reduction site (Q_n). PQ, unlike Q_n inhibitors, is not in direct contact with the haem. Instead, a water molecule is coordinated by one of the carbonyl groups of PQ and can act as the immediate proton donor for PQ. In addition, we identify water channels that connect Q_n with the aqueous exterior of the enzyme, suggesting that the binding of PQ in Q_n displaces water through these channels. The structures confirm large movements of the head domain of the iron-sulfur protein (ISP-HD) towards and away from the plastoquinol oxidation site (Q_p) and define the unique position of ISP-HD when a Q_p inhibitor (2,5-dibromo-3-methyl-6-isopropylbenzoquinone) is bound. This work identifies key conformational states of cytb₆f, highlights fundamental differences between substrates and inhibitors and proposes a quinone-water exchange mechanism.