Photosynthesis is a biological process that converts energy from sunlight into a form of chemical energy that supports almost all life on Earth. Over the course of evolution, photosynthesis has gone from being only performed by bacteria to appearing in algae and green plants. While this has given rise to a range of different machineries for photosynthesis, the process always begins the same way: with a structure called the reaction center-light harvesting (RC-LH) complex.

Two pigments in the light-harvesting (LH) region – known as chlorophyll and carotenoids – absorb light energy and transfer it to another part of the complex known as the quinone-type reaction center (RC). This results in the release of electrons that interact with a molecule called quinone converting it to hydroquinone. The electron-bound hydroquinone then shuttles to other locations in the cell where it initiates further steps that ultimately synthesize forms of chemical energy that can power essential cellular processes.

In photosynthetic bacteria, hydroquinone must first pass through a ring structure in the light harvesting region in order to leave the reaction center. Previous studies suggest that carotenoids influence the architecture of this ring, but it remains unclear how this may affect the ability of hydroquinone to move out of the RC-LH complex.

To investigate, Xin, Shi, Zhang et al. used a technique called cryo-electron microscopy to study the three-dimensional structure of RC-LH complexes in one of the first bacterial species to employ photosynthesis, Roseiflexus castenholzii. The experiments found that fully assembled complexes bind two groups of carotenoids: one nestled in the interior of the LH ring and the other on the exterior.

The exterior carotenoids work together with bacteriochlorophyll molecules to form a closed ring that blocks hydroquinone from leaving the RC-LH complex. To allow hydroquinone to leave, two groups of regulatory proteins, including a cytochrome and subunit X, then disrupt the structure of the ring to ‘open’ it up.

These findings broaden our knowledge of the molecules involved in photosynthesis. A better understanding of this process may aid the development of solar panels and other devices that use RC-LH complexes rather than silicon or other inorganic materials to convert energy from sunlight into electricity.

Carotenoids (Cars) are natural pigments that play important roles in light harvesting (LH), photoprotection, and assembly of the functional pigment-protein complexes required for photosynthesis. Specifically, Cars capture blue-green light (450–550 nm) and transfer it to chlorophyll or bacteriochlorophyll ((B)Chl) in the LH antenna. The excited energy is then transferred to the RC for primary photochemical reactions. In anoxygenic photosynthetic bacteria (PSB), Car-BChl interactions are essential for assembling the functional LH complexes (Davidson and Cogdell, 1981; Hashimoto et al., 2016; Lang and Hunter, 1994; Walz and Ghosh, 1997). The well-studied purple bacterium Rhodobacter (Rba.) sphaeroides contains a closed LH2 ring comprising nine αβ-polypeptides; each LHαβ non-covalently binds three BChls (two B850s and one B800) and one Car (Qian et al., 2021b). The Car-less strains of Rba. sphaeroides are unable to assemble an LH2 complex, indicating that Car-BChl interactions are essential for the maintenance of LH2 structural stability (Lang et al., 1995). In the LH1 ring of Rba. sphaeroides, a combination of two Car groups forms a tightly sealed, impenetrable fence-like structure that blocks the proposed quinone channel of the closed ring (Olsen et al., 2017; Qian et al., 2021c). However, there are fewer Cars in most LH1 structures, so in Thermochromatium (Tch.) tepidum and Rhodospirillum (Rsp.) rubrum for example, there are small gaps that allow quinones to shuttle cross the ring (Niwa et al., 2014; Qian et al., 2021a; Yu et al., 2018b). A point mutation in LHα (W24F) dramatically reduces the amounts of LH1-bound Car. However, in the pufX knockout strain of Rba. sphaeroides, which possesses a closed LH1 ring composed of 17 LHαβs, the same mutation promotes photosynthetic growth (Cao et al., 2022; McGlynn et al., 1994; Olsen et al., 2017). These observations indicate a correlation between the number of LH1-bound Cars and the architecture and photochemical functions of the RC-LH1. This phenomenon could be further studied using structural information about Car-depleted RC-LH (dRC-LH), but no such data have yet been reported.

Roseiflexus (R.) castenholzii is a chlorosome-less filamentous anoxygenic photosynthetic bacterium (Hanada et al., 2002). It contains only one LH, which forms an unusual RC-LH complex. This complex structurally resembles RC-LH1 but has similar spectroscopic characteristics that are similar to the peripheral LH2 of purple bacteria (Collins et al., 2010; Collins et al., 2009). We previously reported the cryo-electron microscopy (EM) structure of R. castenholzii RC-LH at 4.1 Å resolution. It revealed an RC composed of L, M, and cytochrome (cyt) c subunits surrounded by an opened elliptical LH ring of 15 LHαβs, with the tetraheme binding domain of cyt c protruding on the periplasmic side. The RC is compositionally larger in purple bacteria than in R. castenholzii, in which it does not contain an H subunit (Pugh et al., 1998; Qian et al., 2005; Yamada et al., 2005). However, it does contain a unique cyt c transmembrane (c-TM) helix and the newly identified subunit X, both of which flank the gap of the LH ring to form a novel quinone shuttling channel (Xin et al., 2018). Notably, the amino acid sequences of subunit X and TM7, a TM helix separated from the RC-L and RC-M subunits are unassigned. Pigment analyses have revealed a 2:3 Car:BChl molar ratio of R. castenholzii RC-LH (Collins et al., 2009). However, the cryo-EM structure resolved only one keto-γ-carotene (KγC) molecule spanning the interface of each LHαβ, coordinating two B880s and one additional B800 at the periplasmic and the cytoplasmic side, respectively. The lack of a clear cryo-EM density map leaves uncertainty about the presence of additional LH ring-bound Cars, the roles of which are unknown in maintaining the architecture and photochemical functions of the R. castenholzii RC-LH.

We here determined cryo-EM structures of native RC-LH (nRC-LH) complexes purified from R. castenholzii cells grown under high (180 μmol m^–2 s^–1), medium (32 μmol m^–2 s^–1), and low (2 μmol m^–2 s^–1) illuminations at 2.8 Å, 3.1 Å, and 2.9 Å resolutions, respectively. All three structures shared the same architecture, indicating that the Car composition and assembly are not affected by light intensities. From these high-resolution structures, we identified 14 additional KγC molecules in the exterior of the LH ring (KγC_ext). In combination with the B800 on the cytoplasmic side, the newly identified KγC_ext molecules blocked the proposed quinone channel between LHαβ subunits, forming a sealed LH ring conformation. We also assigned the full amino acid sequences of subunit X, TM7, and an additional TM helix that were derived from hypothetical proteins Y and Z, respectively, and demonstrated their roles in forming the quinone channel and stabilizing the RC-LH interactions. To investigate the role of Cars in the assembly of RC-LH, R. castenholzii cells were treated with Car biosynthesis inhibitor diphenylamine (DPA) to produce a dRC-LH; a 3.1 Å resolution cryo-EM structure of this complex resolved five KγC molecules bound in the interior of the LH ring (KγC_int). The absence of subunit X and exterior KγC (KγC_ext) molecules in the dRC-LH produced an LH ring with exposed LHαβ interface and a larger opening than that of nRC-LH. This conformation accelerated the in vitro quinone/quinol exchange rate of menaquinone-4, an analog of the native menaquinone-11, but did not affect the Car-to-BChl energy transfer efficiency of dRC-LH. This study thus revealed a previously unrecognized structural basis by which Car assembly regulates the architecture and quinone/quinol exchange rate of the bacterial RC-LH complex. These findings further our understanding of diversity and molecular evolution in the prokaryotic photosynthetic apparatus.

Unlike the well-studied purple bacteria, which contain two types of LH complexes, R. castenholzii contains only one RC-LH complex for LH and primary photochemical reactions. It does not contain the H subunit that is typically found in purple bacteria (Pugh et al., 1998; Qian et al., 2005; Yamada et al., 2005). Especially, R. castenholzii RC-LH contains a tetra-heme cyt c subunit that interrupts the LH ring, which is composed of 15 αβ-polypeptides, through a novel N-terminal TM helix; together with the newly identified subunit X, this forms a potential quinone shuttling channel on the LH ring. In the present study, we determined high-resolution cryo-EM structures of nRC-LH, from which we assigned the full amino acid sequence of subunit X, and two additional TM helices derived from hypothetical proteins Y and Z in the RC, which both functioned in stabilizing the RC-LH interactions. Most importantly, we identified 14 additional KγC molecules (KγC_ext) in the LH ring exterior, and one KγC inserted between LHαβ1 and c-TM, which generated a 2:3 Car:BChl molar ratio consistent with previous pigments analyses (Collins et al., 2009). Binding of the KγC_int and KγC_ext together with the B800s blocked the proposed quinone channel between LHαβ subunits. DPA treatment of the cells yielded a dRC-LH, referred to as dRC-LH; a 3.1 Å resolution cryo-EM structure resolved only five KγC_int molecules, and the absence of subunit X and the cytoplasmic region of c-TM. These alterations in the dRC-LH increased the size of the LH opening and exposed the LHαβ interface, accelerating the in vitro quinone/quinol exchange rate of menaquinone-4, but did not affect the Car-to-BChl energy transfer efficiency.

To maintain continuous photo-reaction and turnover of the electron transport chain, two quinone exchange/transport routes are required for the bacterial RC-LH1 complex. One is the exchange route for the free/bound quinone in the RC, which was represented by a newly identified MQc in our nRC-LH structure (Figure 3H), and also extra UQ molecules found in many purple bacterial RCs (Cao et al., 2022; Kishi et al., 2021; Qian et al., 2022; Qian et al., 2018; Swainsbury et al., 2021; Tani et al., 2022b; Yu et al., 2018a). The other one is shuttling channel between the inside and outside of the LH1 ring. For Tch. tepidum RC-LH1 that contains an almost symmetric and completely closed LH1 ring, except the ‘waiting’ UQ8 identified near Q_B, one UQ8 was found to be inserted between the LH1α and LH1β subunits (Yu et al., 2018a), representing a potential quinone exchange channel between the LHαβ interface. Therefore, the space between the LHαβ subunits can serve as quinone exchange channel for the closed LH1 ring (Qian et al., 2022; Yu et al., 2018b), and also for the opened LH1 ring bound only with interior Cars (Qian et al., 2021a; Swainsbury et al., 2021; Yu et al., 2018b). For most purple bacterial RC-LH1 complexes with an opened C-shaped LH1 ring, reduced quinones are also shuttled from the RC through a gap at the LH1 ring, which is disrupted by protein W, or PufX and PufY (or protein-U) (Cao et al., 2022; Jackson et al., 2018; Qian et al., 2021a; Tani et al., 2021a; Tani et al., 2022a; Tani et al., 2021b).

Distinct from the RC-LH1 of most purple bacteria, each LHαβ of R. castenholzii non-covalently bound an additional B800 BChl at the cytoplasmic side, which occupied the LHαβ interface at the cytoplasmic side (Figure 2C, Figure 2—figure supplement 1C). In addition, we identified KγC at three distinct positions in the nRC-LH ring: KγC_int and KγC_ext, and also an additional KγC near the LH opening (Figures 1D and 3B). The KγC_int molecules embedded between the LHαβs had a similar conformation as they do in the completely closed and also the opened LH1 ring of purple bacteria. In contrast, KγC_ext molecules occupied the space between adjacent LHβs, although they were not well aligned with the Tch. tepidum LHαβ-bound UQ8 molecule (Figure 2C, Figure 2—figure supplement 4D). Therefore, incorporation of the KγC_ext molecules and additional B800s in R. castenholzii nRC-LH most likely together blocked the LHαβ interface for putative quinone exchange (Figure 2F). Alternatively, R. castenholzii RC-LH incorporated a membrane-bound cyt c and a hypothetical protein X, which has the TM helices that interrupted the LH ring to form a potential channel for controlled quinone/quinol exchange (Figure 6). Superposition of R. castenholzii with purple bacterial RC-LH1s revealed distinct locations and orientations of subunit X and c-TM compared to PufX and PufY (Figure 2—figure supplement 4E), indicating R. castenholzii has evolved different structural elements for regulating quinone shuttling.

Figure 6

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Genetic depletion of the LH1-bound Cars promoted the photosynthetic growth of a PufX-knockout Rba. sphaeroides mutant with a closed LH1 ring (Cao et al., 2022; McGlynn et al., 1994; Olsen et al., 2017); this implies that disruption of Cars binding exposed the blocked quinone channel between LHαβ interface and facilitated the quinone exchange, thus promoting photosynthetic growth. In our study, depletion of the KγC_ext and most KγC_int molecules by DPA treatment could also expose the space between the Car-unbound LHαβ subunits. In addition, absence of the subunit X and cytoplasmic region of c-TM in dRC-LH broadened the dimensions of the LH ring opening, which most likely together accelerated the quinone/quinol exchange rate of the dRC-LH (Figure 6). This was consistent with a previous observation that the open form of the Rhodopseudomonas (Rps.) palustris RC-LH1 has a faster UQ2 diffusion rate than the closed form (Swainsbury et al., 2021). Notably, depletion of most LH-bound Cars only affected the stable conformation of the cytoplasmic region of c-TM, which was closely associated with subunit X to form the putative quinone channel (Figure 2I). Compared to cyt c subunit that formed extensive hydrogen bonding interactions with the L, M, and Y of the RC, the subunit X was characterized by high B-factors, fewer contacts with the RC-LH, and an easily disrupted conformation (Figures 3B and 4C, Figure 2—figure supplement 2B). Especially, the subunit X was derived from a hypothetical protein that inserted into the LH opening in an opposite orientation with LHαβ and c-TM, suggesting that it was likely the last subunit incorporated into the RC-LH. Therefore, R. castenholzii RC-LH could probably evolve the subunit X to control the conformation of the quinone shuttling channel.

Cars contribute to the self-assemble of natural α/β polypeptides to form LH1 complexes in vitro (Fiedor et al., 2004), Car-less Rsp. rubrum LH1 can be obtained by exogenous recombination (Parkes-Loach et al., 1988). In the Car-less Rba. sphaeroides mutant strain R26, the polymerized form of RC-LH is predominantly monomeric, and the curvature of the photosynthetic membrane is altered due to the lack of dimeric RC-LH (Ng et al., 2011). This implies that Cars assembly can regulate the conformation of the RC-LH complex. In our study, Car depletion also affected the LH opening conformation and the quinone/quinol exchange rate of the dRC-LH. Although the extensive interactions between subunit X, c-TM, and LHαβ15 and LHαβ1 were disrupted in dRC-LH (Figures 2I and 4C), the correlation between Car depletion and the absence of subunit X has not been adequately verified. Since DPA treatment is not a clean way to examine the effect of Cars, it left several interior Cars still bound to the LH ring. DPA is a broad-spectrum inhibitor that slows cellular metabolic processes and specifically affects Car biosynthesis by inhibiting phytoene desaturase (CrtI), an essential enzyme catalyzes conversion of the colorless Car precursor phytoene to the colored lycopene (Bramley, 1993). We here found that DPA treatment not only dramatically decreased the R. castenholzii proliferation rate but also depleted the LH-bound Cars in dRC-LH (Figures 1A and 4, Figure 1—figure supplement 1B). However, an efficient genetic manipulation system of R. castenholzii is required to obtain a Car-less RC-LH complex, for elucidating the correlations between Cars and the RC-LH assembly, as well as the photosynthetic growth of cells. To our current knowledge, genetic editing of R. castenholzii is restricted by its morphology as a multicellular filamentous bacterium with an optimal growth temperature ~50°C, and the lack of a well-studied genetic background that facilitates exogenous DNA introduction and replication.

In summary, this study revealed conformational changes of the R. castenholzii RC-LH in the presence and absence of KγC_ext and subunit X, which played a role in regulating the quinone/quinol exchange. KγC_ext incorporation results in a sealed conformation of the LH ring, whereas Car depletion and absence of the subunit X produces an exposed LH ring with larger opening, which together accelerate the in vitro quinone/quinol exchange of menaquinone-4. These results indicate a correlation between LH-bound Cars and the assembly and quinone/quinol exchange of R. castenholzii RC-LH. Overall, these findings deepen our understanding of the light absorption and photo-reaction mechanisms in prokaryotic photosynthesis and increase the feasibility of applying prokaryotic photosystems in synthetic microbiology approaches.

Cryo-EM maps and atomic coordinates of the native RC-LH (nRC-LH) and carotenoid depleted RC-LH (dRC-LH) complexes extracted from Roseiflexus castenholzii cells grown under high illumination (180 μmol m^-2 s^-1) have been deposited into the Electron Microscopy Data Bank (accession codes, EMD-34838 and EMD-34839) and the Protein Data Bank (PDB) (accession codes, 8HJU and 8HJV), respectively. Cryo-EM maps and atomic coordinates of the nRC-LH complexes extracted from cells grown under low (2 μmol m^-2 s^-1) and medium (32 μmol m^-2 s^-1) illuminations have been deposited into the Electron Microscopy Data Bank (accession codes, EMD-35988 and EMD-35989) and the Protein Data Bank (PDB) (accession codes, 8J5O and 8J5P), respectively. Cryo-EM maps and atomic coordinates of the four RC-LH complexes can also be accessed on Dryad (https://doi.org/10.5061/dryad.w6m905qv4).

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   Cryo-EM maps and atomic coordinates of RC-LH complexes from Roseiflexus castenholzii.

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2. 1. Xin J
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   (2023) Electron Microscopy Data Bank

   ID EMD-34838. Cryo-EM map of native RC-LH complex from Roseiflexus castenholzii at 180 μmol m^–2 s^–1.

   https://www.ebi.ac.uk/emdb/EMD-34838
3. 1. Xin J
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   (2023) Electron Microscopy Data Bank

   ID EMD-34839. Cryo-EM map of carotenoid-depleted RC-LH complex from Roseiflexus castenholzii at 180 μmol m^–2 s^–1.

   https://www.ebi.ac.uk/emdb/EMD-34839
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   https://www.rcsb.org/structure/8HJV
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   (2023) Electron Microscopy Data Bank

   ID EMD-35988. Cryo-EM map of native RC-LH complex from Roseiflexus castenholzii at 2 μmol m^–2 s^–1.

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   ID 8J5P. Cryo-EM structure of native RC-LH complex from Roseiflexus castenholzii at 32 μmol m^–2 s^–1.

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Author details

1. Jiyu Xin

   Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences and The Affiliated Hospital of Hangzhou Normal University, Hangzhou, China

   Contribution

   Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Purified the RC-LH complexes, Determined the cryo-EM structures, Performed the enzymatic and steady-state spectroscopic analyses, Analyzed the data, Assisted with preparing the manuscript

   Contributed equally with

   Yang Shi and Xin Zhang

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2354-2032

2. Yang Shi

   Liangzhu Laboratory, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Laboratory of Brain-machine Intelligence & Department of Neurobiology and Department of Pathology of the First Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang University, Hangzhou, China

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Assigned the subunit X, protein Y and Z in the RC-LH complex

   Contributed equally with

   Jiyu Xin and Xin Zhang

   Competing interests

   No competing interests declared

3. Xin Zhang

   1. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences and The Affiliated Hospital of Hangzhou Normal University, Hangzhou, China
   2. Photosynthesis Research Center, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Assisted with the cryo-EM sample preparation and data processing, Analyzed the structures

   Contributed equally with

   Jiyu Xin and Yang Shi

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6734-759X

4. Xinyi Yuan

   1. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences and The Affiliated Hospital of Hangzhou Normal University, Hangzhou, China
   2. Photosynthesis Research Center, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China

   Contribution

   Data curation, Formal analysis, Investigation, Extracted the pigments, Performed the HPLC-MS analyses, Analyzed the data

   Competing interests

   No competing interests declared

5. Yueyong Xin

   Photosynthesis Research Center, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China

   Contribution

   Resources, Data curation, Investigation, Assisted with the spectral and HPLC-MS experiments

   Competing interests

   No competing interests declared

6. Huimin He

   Photosynthesis Research Center, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China

   Contribution

   Data curation, Investigation, Assisted with the sample prepation and MS analysis

   Competing interests

   No competing interests declared

7. Jiejie Shen

   Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences and The Affiliated Hospital of Hangzhou Normal University, Hangzhou, China

   Contribution

   Data curation, Formal analysis, Assisted with the auracyanin oxidation experiments

   Competing interests

   No competing interests declared

8. Robert E Blankenship

   Departments of Biology and Chemistry, Washington University in St. Louis, St. Louis, United States

   Contribution

   Writing – review and editing, Assisted with preparing the manuscript

   Competing interests

   No competing interests declared

9. Xiaoling Xu

   1. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences and The Affiliated Hospital of Hangzhou Normal University, Hangzhou, China
   2. Photosynthesis Research Center, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing, Initiated the project, Supervised all experiments, Analyzed the data, Wrote the manuscript

   For correspondence

   xuxl@hznu.edu.cn

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8995-1213

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