Matthew S. Proctor

The chloroplast F ₁ F _o ATP synthase is a rotary motor that converts the light-driven proton-motive force into the chemical energy of ATP. The number of c-subunits in its rotor fixes the number of protons translocated per ATP formed, a fundamental parameter of bioenergetic systems. The reference spinach enzyme possesses fourteen c-subunits and a H ⁺ /ATP ratio of 4.67. Green algae additionally operate a carbon-concentrating mechanism that sustains CO ₂ fixation in water at a substantial cost in ATP, yet the structure of the algal motor, and whether its bioenergetic parameters differ from those of vascular plants, remains unresolved. Here, a 2.2 Å structure of the ATP synthase of Chlamydomonas reinhardtii reveals that the enzyme carries a thirteen-membered c-ring, the first departure from c ₁₄ in a chloroplast, and with a lower predicted H ⁺ /ATP ratio of 4.33. Ordered waters trace a Grotthuss proton relay through the membrane, where an insulating triad separates the proton loading and unloading sites and couples flux to rotation. A single substitution in the redox switching γ-subunit abolishes the contact with the catalytic β-subunit that idles the enzyme in darkness in vascular plants. These unique features of the algal ATP synthase lower the H ⁺ /ATP cost of carbon fixation in the light and facilitate acetate metabolism in the dark.

In oxygenic and anoxygenic photosynthesis, excitation energy migrates from a surrounding antenna to specialised chlorophyll (Chl) or bacteriochlorophyll (BChl) pigments housed within a reaction centre (RC) complex. Here, a charge-separated state is formed within a few picoseconds, and an electron moves along a series of cofactors until it arrives at a quinone or iron–sulphur centre acceptor. Further photochemical cycles rely on rapid re-reduction in the photo-oxidised RC, usually by small, soluble metalloproteins which vary considerably between different phototrophic clades. In the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides, the electron carrier cytochrome c2 (cyt c2) shuttles between the periplasmic faces of the cytochrome bc1 complex and the reaction centre-light harvesting 1 (RC-LH1) core complex, the location of the BChl special pair (P865). By contrast, in the model cyanobacterium Synechocystis sp. PCC 6803, electrons are transferred from cytochrome b6f to photosystem I (PSI) via two isofunctional redox carrier proteins, cytochrome c6 (cyt c6) or plastocyanin (Pc). In the present study, we demonstrate that both cyt c6 and Pc can substitute for cyt c2in silico, in vitro and in vivo, even though their electrostatic properties may be counter-productive for binding the RC-LH1 complex. Interestingly, whilst P865+ reduction was highest with cyt c2 and the full physiological RC-LH1 complex, both Synechocystis proteins were more compatible with the RC-only complex lacking the surrounding LH1 antenna. Taken together, this suggests the subunits that constitute the LH1 ring improve both the donor side activity and selectivity of the Rba. sphaeroides RC complex.

Fixing CO2 via photosynthesis requires ATP and NADPH, which can be generated through linear electron transfer (LET). However, depending on the environmental conditions, additional ATP may be required to fix CO2, which can be generated by cyclic electron transfer (CET). How the balance between LET and CET is determined remains largely unknown. Ferredoxin-NADP+ reductase (FNR) may act as the switch between LET and CET, channeling photosynthetic electrons to LET when it is bound to photosystem I (PSI) or to CET when it is bound to cytochrome b6f. The essential role of FNR in LET precludes the use of a direct gene knock-out to test this hypothesis. Nevertheless, we circumvented this problem using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing in Chlamydomonas reinhardtii. Through this approach, we created a chimeric form of FNR tethered to PSI via PSAF. Chimeric FNR mutants exhibited impaired photosynthetic growth and LET along with enhanced PSI acceptor side limitation relative to the wild type due to slower NADPH reduction. However, the chimeric FNR mutants also showed enhanced ΔpH production and NPQ resulting from increased CET. Overall, our results suggest that rather than promoting LET, tethering FNR to PSI promotes CET at the expense of LET and CO2 fixation.

The light reactions of photosynthesis couple electron and proton transfers across the thylakoid membrane, generating NADPH, and proton motive force (pmf) that powers the endergonic synthesis of ATP by ATP synthase. ATP and NADPH are required for CO₂ fixation into carbohydrates by the Calvin–Benson–Bassham cycle. The dominant ΔpH component of the pmf also plays a photoprotective role in regulating photosystem II light harvesting efficiency through nonphotochemical quenching (NPQ) and photosynthetic control via electron transfer from cytochrome b₆f (cytb₆f) to photosystem I. ΔpH can be adjusted by increasing the proton influx into the thylakoid lumen via upregulation of cyclic electron transfer (CET) or decreasing proton efflux via downregulation of ATP synthase conductivity (gH⁺). The interplay and relative contributions of these two elements of ΔpH control to photoprotection are not well understood. Here, we showed that an Arabidopsis (Arabidopsis thaliana) ATP synthase mutant hunger for oxygen in photosynthetic transfer reaction 2 (hope2) with 40% higher proton efflux has supercharged CET. Double crosses of hope2 with the CET-deficient proton gradient regulation 5 and ndh-like photosynthetic complex I lines revealed that PROTON GRADIENT REGULATION 5 (PGR5)-dependent CET is the major pathway contributing to higher proton influx. PGR5-dependent CET allowed hope2 to maintain wild-type levels of ΔpH, CO₂ fixation and NPQ, however photosynthetic control remained absent and PSI was prone to photoinhibition. Therefore, high CET in the absence of ATP synthase regulation is insufficient for PSI photoprotection.

Singlet exciton fission is the spin-allowed generation of two triplet electronic excited states from a singlet state. Intramolecular singlet fission has been suggested to occur on individual carotenoid molecules within protein complexes, provided the conjugated backbone is twisted out-of-plane. However, this hypothesis has only been forwarded in protein complexes containing multiple carotenoids and bacteriochlorophylls in close contact. To test the hypothesis on twisted carotenoids in a 'minimal' one-carotenoid system, we study the orange carotenoid protein (OCP). OCP exists in two forms: in its orange form (OCPo), the single bound carotenoid is twisted, whereas in its red form (OCPr), the carotenoid is planar. To enable room-temperature spectroscopy on canthaxanthin-binding OCPo and OCPr without laser-induced photoconversion, we trap them in trehalose glass. Using transient absorption spectroscopy, we show that there is no evidence of long-lived triplet generation through intramolecular singlet fission, despite the canthaxanthin twist in OCPo.

Carotenoids are crucial photosynthetic pigments utilized for light harvesting, energy transfer, and photoprotection. Although most of the enzymes involved in carotenoid biosynthesis in chlorophototrophs are known, some are yet to be identified or fully characterized in certain organisms. A recently characterized enzyme in oxygenic phototrophs is 15-cis-zeta(ζ)-carotene isomerase (Z-ISO), which catalyzes the cis-to-trans isomerization of the central 15–15′ cis double bond in 9,15,9′-tri-cis-ζ-carotene to produce 9,9′-di-cis-ζ-carotene during the four-step conversion of phytoene to lycopene. Z-ISO is a heme B-containing enzyme best studied in angiosperms. Homologs of Z-ISO are present in organisms that use the multi-enzyme poly-cis phytoene desaturation pathway, including algae and cyanobacteria, but appear to be absent in green bacteria. Here we confirm the identity of Z-ISO in the model unicellular cyanobacterium Synechocystis sp. PCC 6803 by showing that the protein encoded by the slr1599 open reading frame has ζ-carotene isomerase activity when produced in Escherichia coli. A Synechocystis Δslr1599 mutant synthesizes a normal quota of carotenoids when grown under illumination, where the photolabile 15–15′ cis double bond of 9,15,9′-tri-cis-ζ-carotene is isomerized by light, but accumulates this intermediate and fails to produce ‘mature’ carotenoid species during light-activated heterotrophic growth, demonstrating the requirement of Z-ISO for carotenoid biosynthesis during periods of darkness. In the absence of a structure of Z-ISO, we analyze AlphaFold models of the Synechocystis, Zea mays (maize), and Arabidopsis thaliana enzymes, identifying putative protein ligands for the heme B cofactor and the substrate-binding site.

In oxygenic photosynthesis, the cytochrome b₆f (cytb₆f) complex links the linear electron transfer (LET) reactions occurring at photosystems I and II and generates a transmembrane proton gradient via the Q-cycle. In addition to this central role in LET, cytb₆f also participates in a range of processes including cyclic electron transfer (CET), state transitions and photosynthetic control. Many of the regulatory roles of cytb₆f are facilitated by auxiliary proteins that differ depending upon the species, yet because of their weak and transient nature the structural details of these interactions remain unknown. An apparent key player in the regulatory balance between LET and CET in cyanobacteria is PetP, a ~10 kDa protein that is also found in red algae but not in green algae and plants. Here, we used cryogenic electron microscopy to determine the structure of the Synechocystis sp. PCC 6803 cytb₆f complex in the presence and absence of PetP. Our structures show that PetP interacts with the cytoplasmic side of cytb₆f, displacing the C-terminus of the PetG subunit and shielding the C-terminus of cytochrome b₆, which binds the heme c_n molecule that is suggested to mediate CET. The structures also highlight key differences in the mode of plastoquinone binding between cyanobacterial and plant cytb₆f complexes, which we suggest may reflect the unique combination of photosynthetic and respiratory electron transfer in cyanobacterial thylakoid membranes. The structure of cytb₆f from a model cyanobacterial species amenable to genetic engineering will enhance future site-directed mutagenesis studies of structure-function relationships in this crucial ET complex.

(Bacterio)chlorophylls are modified tetrapyrroles that are used by phototrophic organisms to harvest solar energy, powering the metabolic processes that sustain most of the life on Earth. Biosynthesis of these pigments involves enzymatic modification of the side chains and oxidation state of a porphyrin precursor, modifications that differ by species and alter the absorption properties of the pigments. (Bacterio)chlorophylls are coordinated by proteins that form macromolecular assemblies to absorb light and transfer excitation energy to a special pair of redox-active (bacterio)chlorophyll molecules in the photosynthetic reaction centre. Assembly of these pigment–protein complexes is aided by an isoprenoid moiety esterified to the (bacterio)chlorin macrocycle, which anchors and stabilizes the pigments within their protein scaffolds. The reduction of the isoprenoid ‘tail’ and its addition to the macrocycle are the final stages in (bacterio)chlorophyll biosynthesis and are catalysed by two enzymes, geranylgeranyl reductase and (bacterio)chlorophyll synthase. These enzymes work in conjunction with photosynthetic complex assembly factors and the membrane biogenesis machinery to synchronize delivery of the pigments to the proteins that coordinate them. In this review, we summarize current understanding of the catalytic mechanism, substrate recognition and regulation of these crucial enzymes and their involvement in thylakoid biogenesis and photosystem repair in oxygenic phototrophs.