Despite the detailed knowledge of photosystem I structure, the biogenesis of this energy-converting complex remains poorly understood. Here, we provide a 1.83 Å resolution model of a cyanobacterial PSI assembly intermediate isolated from a PsaC-less mutant. Our structure revealed the association of all small membrane subunits with the central PsaA/PsaB heterodimer except PsaL, which was missing together with PsaD and PsaE. The intermediate contained nearly all pigments and cofactors as the mature complex including the iron-sulfur cluster at the FX site, which was found incomplete; possibly due to conformational changes of PsaB. The data contravene previous assembly models initiated by the formation of PsaA/PsaB heterodimer and ending by incorporation of small membrane subunits. They rather support a parallel assembly of PsaA, PsaB, and PsaC, which is essential for the correct FX formation, while stable binding of small membrane subunits to the PsaA/PsaB heterodimer is mostly independent of the cytoplasmic subunits.
Letter
1.8 Å resolution structure of the cyanobacterial Photosystem I assembly intermediate lacking cytoplasmic subunits
https://doi.org/10.21203/rs.3.rs-4640615/v1
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Oxygenic photosynthesis, the fundamental bioenergetic process on our planet, is carried out by cyanobacteria, algae, and plants. The core components of this process are two sophisticated light-driven oxidoreductases - photosystem I (PSI) and photosystem II (PSII)1 that are composed of numerous protein subunits and cofactors, including chlorophylls (Chls),carotenoids, quinones, and lipids2. Photosystems fuel electrons from water to NADP+, which, together with concomitantly synthesized ATP, is used for the fixation of carbon dioxide. The PSI core complex typically comprises two large subunits - PsaA and PsaB, containing special Chls forming the primary electron donor P700 and primary acceptors, phylloquinones, and a [4Fe-4S] cluster involved in electron transfer 3. PsaA/B also binds most light-harvesting Chls. The cytoplasmic PsaC subunit is stabilized by its neighbour PsaD and PsaE, anchors additional two [4Fe-4S] clusters reducing ferredoxin2,4 while the remaining small transmembrane subunits play an important role in the function and organization of PSI5,6. The cyanobacterial PSI is typically organized as a trimer7 in contrast to the monomeric8 or dimeric9 forms in algae and plants.
Despite detailed knowledge of the active PSI structure, our understanding of its assembly is still incomplete. The formation of the PsaA/PsaB heterodimer is mostly considered an initial assembly step, which is followed by sequential incorporation of small subunits, although the order of their incorporation remains unclear 10,11. The whole process is assisted by numerous auxiliary assembly factors and includes insertion of pigments and redox cofactors, which are thought to be incorporated primarily during the formation of the PsaA/PsaB heterodimer. Interestingly, the iron-sulfur [4Fe-4S] cluster called FX, is coordinated by two Cys residues from PsaA and two from PsaB and the exact mechanism of its insertion in vivo remains elusive.
To isolate PsaC-less assembly intermediate of PSI we constructed a Synechocystis ΔpsaC mutant expressing FLAG-tagged PsaA protein. The psaC inactivation resulted in a significant reduction in the amount of PSI trimer, which was accompanied by an increased level of the monomeric complex when compared with the control strain containing only the FLAG-tagged PsaA (Extended Data Fig. 1). In contrast, the amount of PSII was the same in both strains as revealed by Chl fluorescence emitted mostly by PSII complexes (Extended Data Fig. 1).
To obtain detailed information about the oligomeric state and composition of the PSI complexes lacking PsaC (PSIΔC), we utilized FLAG-PsaA as a bait for the FLAG-immunoaffinity pulldown assay. The isolated FLAG-PSIΔC preparation was compared with the preparation isolated from the control strain expressing FLAG-PsaA instead of PsaA (FLAG-PSI). The absorption spectra of both preparations were practically identical showing a very similar overall pigment composition (Fig. 1a). The analysis of isolated complexes by clear native electrophoresis (CN PAGE) showed the prevalence of the monomeric PSI but substantial amounts of the trimer and dimer were also present in both FLAG-PSI and FLAG-PSIΔC preparations (Fig. 1b).
As expected, the isolated FLAG-PSIΔC also lacked the cytoplasmic PsaD and PsaE subunits, while PsaF and PsaK1 were clearly present in the nearly stoichiometric amount in all three oligomeric forms, monomer, dimer, and trimer (Fig. 1b, Extended Data Table 1). The complexes also contained a small amount of PsaK2, while the content of single-helix subunits PsaI, PsaJ, and PsaM remained unclear due to their small size and our inability to detect them by mass spectrometry (MS). Additionally, the absence of PsaL subunit and the presence of the dimeric and trimeric PSIΔC in the FLAG-PSIΔC preparation implies that this protein is not essential for the formation of PSI oligomers (Fig. 1b) which is in agreement with the CN gel analysis of the ΔpsaL strain (Extended Data Fig. 2). Nevertheless, PsaL protein was detected in the preparation using MS suggesting that it can weakly bind to the complexes lacking all stromal subunits (Extended Data Table 2). The same applies for subunits PsaI, PsaJ and PsaM, which were also detected in the preparation. In addition, the MS analysis of the FLAG-PSIΔC preparation identified other proteins such as Ycf3, an assembly factor, and Slr1796. The later protein is a transmembrane factor containing a putative thioredoxin-like domain on the lumenal side, which was previously found to be important for the accumulation of PSI12.
For solving a detailed 3D structure, the isolated PSIΔC complexes were subjected to the single-particle cryo-electron microscopic (cryo-EM) analysis. For the reference and template model, we used the Synechocystis trimeric PSI complex resolved by Malavath et al. (2018)13 (PDB 5OY0, referred to as mature PSI), which shows better resolution than later described structures of the mature PSI from the same cyanobacterium14. We collected 9,991 movies (Extended Data Fig. 3, Extended Data Table 3) and the well-defined density enabled us to build an atomic model of the monomeric PSIΔC(1) complex with a resolution of 1.83 Å while the dimeric and trimeric PSIΔC were not resolved, most probably due to their low abundance in the preparation. The PSIΔC(1) complex (for simplicity hereafter PSIΔC) consisted of seven protein subunits (PsaA, PsaB, PsaF, PsaI, PsaJ, PsaK, and PsaM) (Fig. 2a) and 147 cofactors, including 93 Chls, 20 carotenoids (14 β-carotenes, 5 echinenones, and one zeaxanthin), two phylloquinones, iron-sulfur cluster (but see below) and 31 aliphatic molecules modelled as three 1,2-dipalmitoyl-phosphatidyl-glyceroles, two 1,2-distearoyl-monogalactosyl-diglycerides and 26 dodecyl-β-D-maltosides (Table 1).
The overall organization of the photosynthetic pigments and other cofactors like P700, auxiliary Chls, and phylloquinones showed no major differences compared to the mature PSI from wild type (Fig. 3a). However, FA and FB iron sulfur clusters, coordinated by PsaC in the mature PSI, were absent and one β-carotene in the vicinity of PsaJ (Fig. 3b). In addition, two carotenoids were modelled as echinenones, whereas in the mature PSI, they were modelled as zeaxanthin and an isomer of canthaxanthin (Fig. 3c, 3d). Pigment profiling via high-performance liquid chromatography (HPLC) unveiled discrepancies in carotenoid counts (Extended Data Fig. 4), identifying 2 zeaxanthins and 9 echinenone molecules, versus the 1 zeaxanthin and 5 echinenone modelled based on reference structures and electron microscopy map fitting. The ambiguous occupation of carotenoids at certain sites is attributed to the uneven electron density across the ends of some carotenoids. This variability in mature PSI and the PSIΔC and these molecules were therefore modelled as β-carotenes.
Superimposing the PSIΔC with the mature PSI monomer confirmed that the stromal subunits PsaC, PsaD, and PsaE as well as membrane PsaL were absent in PSIΔC (Fig. 2c and 2d). This indicates that PsaL, PsaD, and PsaE are incorporated after or simultaneously with PsaC. Although PsaK was present in the PSIΔC complex like in the mature PSI (Fig. 1a, 2a, and 2e), it was clearly the PsaK1 homolog while the density in reference structure was ascribed to PsaK2. However, given the lower resolution, it cannot be excluded that the homologue present in the reference structure was also PsaK1.
An additional feature distinguishing PSIΔC and the reference model was the presence of the peripheral Chl 4003 molecule, coordinated by PsaK1. In line with the findings from14 (PDB: 6NWA), our analysis revealed that the PsaK2 cannot accommodate this Chl due to the region being structured as a loop, which clashes with the actual location of Chl 4003. Conversely, PsaK1 forms an alpha helix that effectively stabilizes Chl 4003 through the F51 residue (Fig. 2e). Our structure aligns with the results of the previous work showing that PsaK1 is the default subunit bound during the PSI assembly15 but can be replaced by PsaK2 under specific conditions such as high light16.
Our investigation of the PsaC-less PSI from Synechocystis has thus provided a new insight into the sequence of events during the PSI assembly process. By weakening of the PsaL binding, the absence of PsaC disrupts the usual trimeric assembly of PSI, favouring the monomeric complex lacking PsaL and the 2 additional cytoplasmic subunits PsaD and PsaE. The presence of small PsaF, I, J, K1, and M subunits in PSIΔC also shows that their attachment to the PsaA/PsaB heterodimer occurs much earlier than previously thought10,11. It cannot be excluded that some of them can be attached to large subunits before the formation of the heterodimer, which would imply the similarity to the modular principle of the PSII biogenesis. The early incorporation is surprising, especially for PsaK1 given the previous report15 that proposed the late attachment of PsaK to PSI. The only exception is the stable association of PsaL, which is most probably dependent on interaction with the cytoplasmic PsaD subunit7,13. Finally, the previously observed dependence of PsaD and PsaE binding on the presence of PsaC can also be easily justified based on the observed structural details. While PsaC mediates the binding of PsaD in PSI directly, PsaE is bound to PSI primarily via the cytoplasmically exposed part of PsaB. Normally, the backbone of residues Pro559 from PsaB forms hydrogen bonds with the backbone of residue Thr56 from PsaC, and the backbone of resides Gly560 and Arg561 from PsaB also forms hydrogen bonds with Gly48 and Ser50 from PsaE (Extended Data Fig. 5d). However, the absence of PsaC induces changes around the FX cluster in PsaB that prevent the stable interaction of PsaE with PsaB.
Table 1: Pigment and lipids composition of each subunit from the FLAG-PSIΔC. Abbreviations: BCR - β-carotene, ECH - 5 echinenone, ZEX - zeaxanthin, PQN - phylloquinone, LHG - 1,2-dipalmitoyl-phosphatidyl-glycerole, LMG - 1,2-distearoyl-monogalactosyl-diglycerides, and LMT - dodecyl-β-D-maltoside.
In mature PSI, the FX cluster is bound at the interface of PsaA and PsaB and consists of four ferrous cations coordinated by four Cys residues (Cys 574 and Cys583 of PsaA, and Cys556 and Cys 565 of PsaB), and four sulfide ions2,4 (Fig. 3e, Extended data Fig. 5a). While the FA and FB clusters were missing in the complex due to the absence of PsaC, the electron density of Fx was observed in the expected regions of PsaA and PsaB although it was weaker compared with the density of the mature FX in the reference structure, most probably due to a partial occupancy of the cluster. The detailed analysis of the region showed the presence of only two iron ions, thus, the iron-sulfur cluster in the FX site seems incomplete (Extended data Fig. 5b, 5c). Iron designated Fe1, which is normally coordinated by Cys565 of PsaB in the mature model, does not interact with this residue although it is still present in the cluster (Fig. 3e and Extended Data Fig. 5c for density). This is most probably due to the altered position of Cys565 which is replaced by a water molecule in PSIΔC. Fe2 is missing in PSIΔC although the residue Cys583 of PsaA, coordinating the Fe2, shows orientation analogous to the one in the mature PSI (Fig. 3e). Fe3 is coordinated by a cysteine (Cys574 of PsaA) similarly to the mature PSI (Fig. 3e). Fe4 is also missing in the PSIΔC and the residue Cys556 of PsaB, coordinating it in the mature model, is moved 2.8 Å away from its position in the mature structure. Finally, the S2 sulfide interacts with the backbone carbonyl of Gly562 and with the S-gamma of the Cys565, both in PsaB.
We cannot exclude that the observed loss of two irons from the FX cluster is a result of oxidation or other modification occurring during the purification of the complex and its embedding into the EM grid since in the absence of PsaC the FX cluster is closer to the cytoplasmic aqueous phase. Although is impossible to check the FX functionality on the EM grid, this cluster seems less susceptible to damage by chaotropic reagents than FA and FB 17 and we rather assume that the loss of two irons is related to a significant motion and partial disorder of the neighbouring cytoplasmically exposed PsaB region caused by the absence of PsaC (573-585). This region in the reference structure includes a β-turn (577-581) stabilized by interactions with PsaC and PsaE (Fig. 3f, Extended data Fig. 5d). This interaction ensures the correct positioning of Cys556 and Cys656 of PsaB to fully coordinate the complete Fx cluster. Thus, the results suggest an intrinsically encoded mechanism of the FX insertion and stabilization, working through synchronized structural rearrangement of PsaB, which accompanies the binding of PsaC and PsaE.
Construction of Synechocystis mutant strains
In this work, we used the Synechocystis GT-P sub-strain18 as a wild-type strain. To express the N-terminally 1xFLAG-tagged PsaA (FLAG-PsaA) from the psaA promoter in Synechocystis (replacing the native psaA gene), a set of DNA constructs was prepared by the NEBuilder HiFI DNA assembly kit, each containing the gentamycin resistance cassette downstream of the psaAB locus (Extended Data Fig. 6a). This construct was used to replace the whole psaAB operon to obtain strains expressing only FLAG-PsaA lacking both native PsaA and PsaB proteins (FLAG-psaA/ΔpsaB strain). The strain expressing FLAG-PsaA instead of the original PsaA (FLAG-psaA) was prepared by transforming the FLAG-psaA/∆psaB strain using the psaB-Gent construct and selecting for autotrophy (Extended Data Fig. 6b). The deletion of the psaC gene in the FLAG-psaA strain was achieved by replacing most of the psaC gene with chloramphenicol resistance cassette (Extended Data Fig. 6c). Segregation proceeded in low light (5 µmol photons m-2 s-1) on plates containing glucose and particular antibiotics19 and was confirmed by PCR using a specific set of primers (see Table S5). The correct sequence of the whole modified DNA region was confirmed by DNA sequencing in all newly constructed and fully segregated strains.
Cultivation conditions of Synechocystis strains
The strains were grown in 100 mL of liquid BG11 medium supplemented with 5 mM glucose using 250 mL conical flasks on a rotary shaker under 5 µmol photons m-2 s-1 at 29°C in liquid BG11 medium. For protein purification, 500 mL of cell culture in a 1-L cylinder was grown under 5 µmol of photons m-2 s-1 in BG11 medium supplemented with 5 mM glucose. The cell culture was agitated with a magnetic stirrer and bubbled with air.
Whole-cell absorption spectroscopy and pigment determination
Absorption spectra of whole cells were measured at room temperature using a UV-3000 spectrophotometer (Shimadzu, Japan) in the cultures with identical OD750nm. For routine Chl determination, pigments were extracted from cell pellets with 100% methanol and Chl concentration was determined spectroscopically (Wellburn 1994). The amount of Chl and carotenoids per a single PSI monomer was determined by HPLC (Agilent 1260) using the following method: The sample was mixed with an excess of 100% methanol and the extracted pigments were separated on a reverse-phase column Zorbax Eclipse Plus C18 (4.6 x 250 mm, 5 μm, Agilent) with 32% (v/v) methanol and 14% (v/v) acetonitrile in 0.25M pyridine (solvent A) and 20% (v/v) methanol, 20% (v/v) acetone, 60% (v/v) acetonitrile as solvent B. Pigments were eluted with a linear gradient of solvent B (60–100% (v/v) in 40 min) in solvent A followed by 100% of solvent B in solvent A for 30 min at a flow rate of 0.8 ml min−1 at 40 °C. Chl-a and carotenoids were detected by a diode-array detector (Agilent 1260) at 450 nm, the obtained peaks were integrated, and the molar stoichiometries calculated from calibration curves that were prepared using authentic standards. Chl-a C132 epimer was detected by a fluorescence detector (Agilent 1260) set to 435 nm and 670 nm excitation and emission wavelengths, respectively.
Low-temperature fluorescence spectroscopy
Low-temperature Chl fluorescence emission spectra of cells in the range 600-800 nm were measured at 77K in cultures with the identical OD750 nm using an SM 9000 spectrophotometer (Photon Systems Instruments, Czech Republic) at an excitation wavelength of 470 nm. The spectra of the isolated preparations (5 µg chlorophyll mL-1) were measured by the same method and the spectra were normalized for the red fluorescence maximum.
Preparation of membranes and FLAG-specific protein purifications
Small-scale membrane fractions were prepared by breaking the cells with zirconia/silica beads using a Mini-Beadbeater (BioSpec Products, USA) according to the procedure described in Komenda and Barber (1995)20, but the cells were resuspended and broken in 25 mM MES/NaOH, pH 6.5, 10 mM CaCl2, 10 mM MgCl2, and 25% glycerol (thylakoid buffer). Large-scale membrane preparations for the purification of proteins in the thylakoid buffer containing and a protease inhibitor cocktail (Roche or Sigma-Aldrich, USA) were isolated as described in Koskela et al. (2020)21 with the exception that membranes were broken using Precellys Evolution (Bertin technologies, France). The membranes were solubilized with 1% n-dodecyl-β-D-maltoside and FLAG-tagged proteins were isolated using the anti-FLAG M2 affinity gel (Sigma-Aldrich, USA) as described in detail in Koskela et al. (2020)21.
Analysis of membrane protein complexes and their subunit composition
The analysis of membrane protein complexes was performed as described in Komenda et al. (2012b)22 using CN-PAGE in a 4% to 14% gradient polyacrylamide gel. The subunit composition of PSI complexes in the second dimension was assessed using SDS-PAGE in a denaturing 12% to 20% gradient gel containing 7 M urea. The separated proteins were visualized by staining with Coomassie Brilliant Blue and detected by MS.
Protein identification by MS
Individual gel bands were treated for MS analysis as described in Solovchenko et al. (2006)23 with minor adaptation, where ammonium bicarbonate was replaced by triethylammonium bicarbonate. Obtained samples were analyzed by means of LC-MS with the same setup as in Koník et al. (2024)24. For identification of proteins in the purified preparations two ml of the FLAG-PSI and FLAG-PSIDC pull-downs were diluted into the 18ml of 50mM ammonium citrate pH 3.5 supplemented with 10% acetonitrile, 2M urea and 100mM TCEP. Subsequently, 50 ng of mucorpepsin was added to each sample and they were incubated at RT for 1hr. The desalting procedure and data-dependent mass spectrometry analysis were performed according to Felcikova et al. (2024)25.
Data were searched using PEAKS Studio 11 (Bioinformatics Solutions Inc., Waterloo, ON, Canada)26. The database consisted of 1,109 Synechocystis protein sequences (taxonomy ID: 1142, reviewed), obtained from UniProtKB27 on 14. June 2024, and 243 sequences of common contaminants. Protein digestion was set to unspecific, with allowed peptide lengths of 5-50. Protein N-term acetylation and Met oxidation were set as variable modifications, with a maximum of 2 modifications per peptide. The tolerance for precursors and fragments was set to 12 ppm and 0.05 Da, respectively. FDR thresholds were set to 1% on both the PSM and protein level. Only proteins with 2 and more unique peptides were accepted. The proteins were quantified by PEAKS as the total area of peptide features from unique supporting peptides
Cryo-EM single particle analysis:
Holey carbon grids for structural analysis were prepared using a PSIΔC sample with a chlorophyll concentration of 1.5mg/ml. The grids (Cu 300 mesh 1.2/1.3, Quantifoil) were glow discharged for 60 sec with 30mA using a GloQube (Quorum) instrument. 3ul of the protein sample were pipetted onto the glow-discharged grids, blotted for 2.5sec, and plunge frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) which was adjusted to a temperature of 4 ºC and a relative humidity of 100%. Clipped grids were imaged using a 300 kV Titan Krios G4 microscope (Thermo Fisher Scientific) that is equipped with a Selectris Energy filter and a Falcon 4i detector. Grid screening and data collection were carried out using EPU software (v3.5.1.6034). 9,999 movies were recorded at a nominal magnification of 165,000, which corresponds to a pixel size of 0.729 Å. The total dose per micrograph was 34.41 e/Å2, and a defocus range of -0.5 to -1.9 was collected. Movies were processed using the live version of cryoSPARC v4.4.028. Motion correction, CTF estimation, blob/template picking, and 2D classification were performed. Ab initio reconstruction was performed in cryoSPARC to get a low-resolution reconstruction. Subsequently, an intermediate-resolution reconstruction was generated using homogeneous refinement in cryoSPARC. This map and four additional junk classes were used as references in a heterogeneous refinement job using all picked particles as input. This enriched the number of good particles. The good particles were re-extracted (unbinned, 350px box) for a subsequent non-uniform refinement job in cryoSPARC. The particle information was then converted into a Star file using csparc2star of PyEM (UCSF pyem v0.5. Zenodo). The x-y coordinates were used to re-extract particles in RELION29. Following a local angular search in 3D refinement, a 3D classification with two classes was carried out. The class with a lower particle number (333,941) but higher resolution was considered to contain better-quality particles and was used in subsequent steps. Multiple rounds of 3D refinement, followed by CTF refinement and Bayesian polishing, were conducted. Finally, a 3D reconstruction was obtained with an overall resolution of 1.83 Å.
Model building and refinement:
Model building was carried out using Coot 9.8.930. The coordinates from the PDB entry 5OY0 were used as the starting model. The PDB was rigid-body fitted into our highest resolution map at 1.83 Å using UCSF Chimera31. All deviations from the starting model, such as chain deletion, correction of rotamers, rebuilding of local sections, building of water molecules, and other improvements in model quality, were carried out using Coot. The model-to-map weight was adjusted manually according to the local quality of the map. For all ligands, restraint files were generated using the Grade2 server (http://grade.globalphasing.org). The final model was then refined using PHENIX Real-Space-Refine32. The refinement protocol was optimized by variations of different weights (overall weight, Ramachandran weight). Several iterations of rebuilding, refinement, and validation were carried out using Coot, Real-space-Refine, and PHENIX32. The final model and its corresponding maps were validated using the PDB validation server (https://validate-rcsb-1.wwpdb.org/) and were subsequently deposited into the PDB databank. Structural figures were generated using UCSF ChimeraX33.
Data availability
Atomic coordinates of PSIΔC were deposited in the Protein Data Bank under accession code 8ZWB. The EM density map of PSIΔC was deposited in the Electron Microscopy Data Bank under accession code EMD-60522.
Acknowledgments
We are very grateful to the KAUST Opportunity Fund (AN, JK and R.S, project RFS-OFP2023-5561), the Czech Grant Agency (JK; project no. 24-10227S), ERC (JK; project no. 854126) and Ministry of Education, Youth and Sports (RS, PN, OP JAK project no. CZ.02.01.01/00/22_008/0004624) for funding. We thank Jan Pilný, Eva Prachová and Kateřina Novotná for excellent technical support. We would like to thank the cryo-EM facility at KAUST for their assistance with data collection.
Author Contributions Statement:
AN and JK designed the study. SG, RS, MT, PN and JK prepared and characterized the PSI complexes; AN processed the structural data; QC built the model and analyzed the structure; AA collected the cryo-EM data; ML and PK analyzed protein bands in the gel; PN and MZ performed MS analysis of the FLAG-PSIΔC preparation; QC, SG, AN, JK and RS jointly prepared the figures and wrote the manuscript.
Competing Interests Statement
The authors declare no competing interests.
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There is NO Competing Interest.
- PSIPEAKSCvsdC.xlsx
Mass Spectrometry Dataset
- ExtendedDataFigures.docx