Upon transition of plants from darkness to light the initiation of photosynthetic linear electron transfer (LET) from HO to NADP precedes the activation of CO fixation, creating a lag period where cyclic electron transfer (CET) around photosystem I (PSI) has an important protective role. CET generates ΔpH without net reduced NADPH formation, preventing overreduction of PSI via regulation of the cytochrome b f (cytb f) complex and protecting PSII from overexcitation by inducing non-photochemical quenching. The dark-to-light transition also provokes increased phosphorylation of light-harvesting complex II (LHCII). However, the relationship between LHCII phosphorylation and regulation of the LET/CET balance is not understood. Here, we show that the dark-to-light changes in LHCII phosphorylation profoundly alter thylakoid membrane architecture and the macromolecular organization of the photosynthetic complexes, without significantly affecting the antenna size of either photosystem. The grana diameter and number of membrane layers per grana are decreased in the light while the number of grana per chloroplast is increased, creating a larger contact area between grana and stromal lamellae. We show that these changes in thylakoid stacking regulate the balance between LET and CET pathways. Smaller grana promote more efficient LET by reducing the diffusion distance for the mobile electron carriers plastoquinone and plastocyanin, whereas larger grana enhance the partition of the granal and stromal lamellae plastoquinone pools, enhancing the efficiency of CET and thus photoprotection by non-photochemical quenching.

The cytochrome b 6 f (cytb 6 f) complex plays a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and conserving solar energy as a transmembrane proton gradient for ATP synthesis 1-3. Electron transfer within cytb 6 f occurs via the Q-cycle, which catalyses the oxidation of plastoquinol (PQH 2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation 2. In higher-plants cytb 6 f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting a n d c y c l i c e l e c t r o n t r a n s f e r t h at protect against metabolic and environmental stresses 3. Here we present a 3.6 Å resolution c r y o-e l e c t r o n m i c r o s c o p y (c r y o-E M) structure of the dimeric cytb 6 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 bound to one cytb 6 f monomer at 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 cytb 6 f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis. Photosynthesis sustains life on Earth by converting light into chemical energy in the form of ATP and NADPH, producing oxygen as a by-product. Two light-powered electron transfer reactions at photosystems I and II (PSI and PSII) are linked via the cytb 6 f complex to form the so-called 'Z-scheme' of photosynthetic linear electron transfer (LET) 1. Cytb 6 f catalyses the rate-limiting step in the LET chain, coupling the oxidation of PQH 2 and reduction of PC and PQ to the generation of a transmembrane proton gradient (p), used by ATP synthase to make ATP 2,3. The cytb 6 f complex is analogous to the cytochrome bc 1 (cytbc 1) complex found in mitochondria 4 and anoxygenic photosynthetic bacteria 5 and both operate via the modified Q

Prochlorococcus is a major contributor to primary production, and it is the most globally abundant photosynthetic genus of picocyanobacteria because it can adapt to highly stratified low-nutrient conditions that are characteristic of the surface ocean. Here we examine the structural adaptations of the photosynthetic thylakoid membrane that enable different Prochlorococcus ecotypes to occupy highlight (HL), low-light (LL) and nutrient-poor ecological niches. We used atomic force microscopy (AFM) to image the different photosystem I (PSI) membrane architectures of the MED4 (HL) Prochlorococcus ecotype acclimated to highlight and low-light conditions in addition to the MIT9313 (LL) and SS120 (LL) Prochlorococcus ecotypes acclimated to low-light conditions. Mass spectrometry quantified the relative abundance of PSI, photosystem II (PSII) and cytochrome b6f complexes and the various Pcb proteins in the thylakoid membrane. AFM topographs and structural modelling revealed a series of specialised PSI configurations, each adapted to the environmental niche occupied by a particular ecotype. MED4 PSI domains were loosely packed in the thylakoid membrane, whereas PSI in the LL MIT9313 is organised into a tightly-packed pseudo-hexagonal lattice that maximises harvesting and trapping of light. There are approximately equal levels of PSI and PSII in MED4 and MIT9313, but nearly twofold more PSII than PSI in SS120, which also has a lower content of cytochrome b6f complexes. SS120 has a different tactic to cope with low-light levels, and SS120 thylakoids contained hundreds of closely packed Pcb-PSI supercomplexes that economise on the extra iron and nitrogen required to assemble PSI-only domains. Thus, the abundance and widespread distribution of Prochlorococcus reflect the strategies that various ecotypes employ for adapting to limitations in light and nutrient levels.

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The reversible docking of small, diffusible redox proteins onto a membrane protein complex is a common feature of bacterial, mitochondrial and photosynthetic electron transfer (ET) chains. Spectroscopic studies of ensembles of such redox partners have been used to determine ET rates and dissociation constants. Here, we report a single-molecule analysis of the forces that stabilise transient ET complexes. We examined the interaction of two components of bacterial photosynthesis, cytochrome c2 and the reaction centre (RC) complex, using dynamic force spectroscopy and PeakForce quantitative nanomechanical imaging. RC–LH1–PufX complexes, attached to silicon nitride AFM probes and maintained in a photo-oxidised state, were lowered onto a silicon oxide substrate bearing dispersed, immobilised and reduced cytochrome c2 molecules. Microscale patterns of cytochrome c2 and the cyan fluorescent protein were used to validate the specificity of recognition between tip-attached RCs and surface-tethered cytochrome c2. Following the transient association of photo-oxidised RC and reduced cytochrome c2 molecules, retraction of the RC-functionalised probe met with resistance, and forces between 112 and 887 pN were required to disrupt the post-ET RC–c2 complex, depending on the retraction velocities used. If tip-attached RCs were reduced instead, the probability of interaction with reduced cytochrome c2 molecules decreased 5-fold. Thus, the redox states of the cytochrome c2 haem cofactor and RC ‘special pair’ bacteriochlorophyll dimer are important for establishing a productive ET complex. The millisecond persistence of the post-ET cytochrome c2[oxidised]–RC[reduced] ‘product’ state is compatible with rates of cyclic photosynthetic ET, at physiologically relevant light intensities.

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