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In response to a sudden increase in light intensity, plants must cope with absorbed excess photon energy to protect photosystems from photodamage. Under fluctuating light, PSI is severely photodamaged in the Arabidopsis (Arabidopsis thaliana) proton gradient regulation5 (pgr5) mutant defective in the main pathway of PSI cyclic electron transport (CET). Here, we aimed to determine how PSI is protected by two proposed regulatory roles of CET via transthylakoid ΔpH formation: (1) reservation of electron sink capacity by adjusting the ATP/NADPH production ratio (acceptor-side regulation) and (2) down-regulation of the cytochrome b₆f complex activity called photosynthetic control for slowing down the electron flow toward PSI (donor-side regulation). We artificially enhanced donor- and acceptor-side regulation in the wild-type and pgr5 backgrounds by introducing the pgr1 mutation conferring the hypersensitivity of the cytochrome b₆f complex to luminal acidification and moss Physcomitrella patens flavodiiron protein genes, respectively. Enhanced photosynthetic control partially alleviated PSI photodamage in the pgr5 mutant background but restricted linear electron transport under constant high light, suggesting that the strength of photosynthetic control should be optimized. Flavodiiron protein-dependent oxygen photoreduction formed a large electron sink and alleviated PSI photoinhibition, accompanied by the induction of photosynthetic control. Thus, donor-side regulation is essential for PSI photoprotection but acceptor-side regulation also is important to rapidly induce donor-side regulation. In angiosperms, PGR5-dependent CET is required for both functions.

The cytochrome b  f (cytb  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 . Electron transfer within cytb  f occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH ) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation . In higher plants, cytb  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 stresses . Here we present a 3.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb  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 cytb  f monomer near the PQ oxidation site (Q ) adjacent to haem b and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Q 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 ) on the PQ1 side and committing the electron transfer network to turnover at the occupied Q site in the neighbouring monomer. A conformational switch involving the haem c propionate promotes two-electron, two-proton reduction at the Q 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 and Q sites in opposite monomers during the Q cycle. The spinach cytb  f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis.

The structure and function of the cytochrome b6 f complex is considered in the context of recent crystal structures of the complex as an eight subunit, 220 kDa symmetric dimeric complex obtained from the thermophilic cyanobacterium, Mastigocladus laminosus, and the green alga, Chlamydomonas reinhardtii. A major problem confronted in crystallization of the cyanobacterial complex, proteolysis of three of the subunits, is discussed along with initial efforts to identify the protease. The evolution of these cytochrome complexes is illustrated by conservation of the hydrophobic heme‐binding transmembrane domain of the cyt b polypeptide between b6 f and bc1 complexes, and the rubredoxin‐like membrane proximal domain of the Rieske [2Fe‐2S] protein. Pathways of coupled electron and proton transfer are discussed in the framework of a modified Q cycle, in which the heme cn, not found in the bc1 complex, but electronically tightly coupled to the heme bn of the b6 f complex, is included. Crystal structures of the cyanobacterial complex with the quinone analogue inhibitors, NQNO or tridecyl‐stigmatellin, show the latter to be ligands of heme cn, implicating heme cn as an n‐side plastoquinone reductase. Existing questions include (a) the details of the shuttle of: (i) the [2Fe‐2S] protein between the membrane‐bound PQH2 electron/H+ donor and the cytochrome f acceptor to complete the p‐side electron transfer circuit; (ii) PQ/PQH2 between n‐ and p‐sides of the complex across the intermonomer quinone exchange cavity, through the narrow portal connecting the cavity with the p‐side [2Fe‐2S] niche; (b) the role of the n‐side of the b6 f complex and heme cn in regulation of the relative rates of noncyclic and cyclic electron transfer. The likely presence of cyclic electron transport in the b6 f complex, and of heme cn in the firmicute bc complex suggests the concept that hemes bn‐cn define a branch point in bc complexes that can support electron transport pathways that differ in detail from the Q cycle supported by the bc1 complex.

The diffusion efficiency of oxygen in the atmosphere, like that of CO₂, is approximately 10⁴ times greater than that in aqueous environments. Consequently, terrestrial photosynthetic organisms need mechanisms to protect against potential oxidative damage. The liverwort Marchantia polymorpha, a basal land plant, has habitats where it is exposed to both water and the atmosphere. Furthermore, like cyanobacteria, M. polymorpha has genes encoding flavodiiron proteins (FLV). In cyanobacteria, FLVs mediate oxygen-dependent alternative electron flow (AEF) to suppress the production of reactive oxygen species. Here, we investigated whether FLVs are required for the protection of photosynthesis in M. polymorpha. A mutant deficient in the FLV1 isozyme (∆MpFlv1) sustained photooxidative damage to photosystem I (PSI) following repetitive short-saturation pulses of light. Compared with the wild type (Takaragaike-1), ∆MpFlv1 showed the same photosynthetic oxygen evolution rate but a lower electron transport rate during the induction phase of photosynthesis. Additionally, the reaction center chlorophyll in PSI, P700, was highly reduced in ∆MpFlv1 but not in Takaragaike-1. These results indicate that the gene product of MpFlv1 drives AEF to oxidize PSI, as in cyanobacteria. Furthermore, FLV-mediated AEF supports the production of a proton motive force to possibly induce the nonphotochemical quenching of chlorophyll fluorescence and suppress electron transport in the cytochrome 𝑏₆/𝑓 complex. After submerging the thalli, a decrease in photosystem II operating efficiency was observed, particularly in ∆MpFlv1, which implies that species living in these sorts of habitats require FLV-mediated AEF.

The cyanobacterial cytochrome b₆f complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric b₆f complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this complex. Moreover, the efficient transformability of this strain allowed the generation of a △petP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700⁺ reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and cytochrome b₆f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated △petP complex in comparison with the dimeric wild-type complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of cytochrome b₆f complexes with different functions that might be correlated with supercomplex formation.

The cytochrome b6f complex provides the electronic connection between the photosystem I and photosystem II reaction centers of oxygenic photosynthesis and generates a transmembrane electrochemical proton gradient for adenosine triphosphate synthesis. A 3.0 angstrom crystal structure of the dimeric b6f complex from the thermophilic cyanobacterium Mastigocladus laminosus reveals a large quinone exchange cavity, stabilized by lipid, in which plastoquinone, a quinone-analog inhibitor, and a novel heme are bound. The core of the b6f complex is similar to the analogous respiratory cytochrome bc1complex, but the domain arrangement outside the core and the complement of prosthetic groups are strikingly different. The motion of the Rieske iron-sulfur protein extrinsic domain, essential for electron transfer, must also be different in the b6f complex.

Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b₆f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast.

Starving microalgae for nitrogen sources is commonly used as a biotechnological tool to boost storage of reduced carbon into starch granules or lipid droplets, but the accompanying changes in bioenergetics have been little studied so far. Here, we report that the selective depletion of Rubisco and cytochrome b₆f complex that occurs when Chlamydomonas reinhardtii is starved for nitrogen in the presence of acetate and under normoxic conditions is accompanied by a marked increase in chlororespiratory enzymes, which converts the photosynthetic thylakoid membrane into an intracellular matrix for oxidative catabolism of reductants. Cytochrome b₆f subunits and most proteins specifically involved in their biogenesis are selectively degraded, mainly by the FtsH and Cip chloroplast proteases. This regulated degradation pathway does not require light, active photosynthesis, or state transitions but is prevented when respiration is impaired or under phototrophic conditions. We provide genetic and pharmacological evidence that NO production from intracellular nitrite governs this degradation pathway: Addition of a NO scavenger and of two distinct NO producers decrease and increase, respectively, the rate of cytochrome b₆f degradation; NO-sensitive fluorescence probes, visualized by confocal microscopy, demonstrate that nitrogen-starved cells produce NO only when the cytochrome b₆f degradation pathway is activated.

As much as two-thirds of the proton gradient used for transmembrane free energy storage in oxygenic photosynthesis is generated by the cytochrome b₆f complex. The proton uptake pathway from the electrochemically negative (n) aqueous phase to the n-side quinone binding site of the complex, and a probable route for proton exit to the positive phase resulting from quinol oxidation, are defined in a 2.70-Å crystal structure and in structures with quinone analog inhibitors at 3.07 Å (tridecyl-stigmatellin) and 3.25-Å (2-nonyl-4-hydroxyquinoline N-oxide) resolution. The simplest n-side proton pathway extends from the aqueous phase via Asp20 and Arg207 (cytochrome b₆ subunit) to quinone bound axially to heme cn. On the positive side, the heme-proximal Glu78 (subunit IV), which accepts protons from plastosemiquinone, defines a route for H⁺ transfer to the aqueous phase. These pathways provide a structure-based description of the quinone-mediated proton transfer responsible for generation of the transmembrane electrochemical potential gradient in oxygenic photosynthesis.

Oxygenic respiration and photosynthesis based on quinone redox reactions face a danger of wasteful energy dissipation by diversion of the productive electron transfer pathway through the generation of reactive oxygen species (ROS). Nevertheless, the widespread quinone oxido-reductases from the cytochrome bc family limit the amounts of released ROS to a low, perhaps just signaling, level through an as-yet-unknown mechanism. Here, we propose that a metastable radical state, nonreactive with oxygen, safely holds electrons at a local energetic minimum during the oxidation of plastohydroquinone catalyzed by the chloroplast cytochrome b₆f. This intermediate state is formed by interaction of a radical with a metal cofactor of a catalytic site. Modulation of its energy level on the energy landscape in photosynthetic vs. respiratory enzymes provides a possible mechanism to adjust electron transfer rates for efficient catalysis under different oxygen tensions.