Molecular basis of plastoquinone reduction in plant cytochrome bf.

A multi-subunit enzyme, cytochrome bf (cytbf), 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 cytbf is known, but its detailed catalytic mechanism remains elusive. Here we present cryogenic electron microscopy structures of spinach cytbf 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). PQ, unlike Q 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 with the aqueous exterior of the enzyme, suggesting that the binding of PQ in Q 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) and define the unique position of ISP-HD when a Q inhibitor (2,5-dibromo-3-methyl-6-isopropylbenzoquinone) is bound. This work identifies key conformational states of cytbf, highlights fundamental differences between substrates and inhibitors and proposes a quinone-water exchange mechanism.