Cryo-EM structure of the spinach cytochrome b6 f complex at 3.6 Å resolution.

The cytochrome b₆ f (cytb₆ f ) complex has a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and converting solar energy into a transmembrane proton gradient for ATP synthesis^1-3. Electron transfer within cytb₆ f occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH₂) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation². In higher plants, cytb₆ f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses³. Here we present a 3.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb₆ f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb₆ f monomer near the PQ oxidation site (Q_p) adjacent to haem b_p and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Q_p site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Q_n) on the PQ1 side and committing the electron transfer network to turnover at the occupied Q_n site in the neighbouring monomer. A conformational switch involving the haem c_n propionate promotes two-electron, two-proton reduction at the Q_n site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Q_p and Q_n sites in opposite monomers during the Q cycle. The spinach cytb₆ f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis.