Overall structures of white PSI and far-red PSI

We purified the trimeric PSI cores from H. hongdechloris grown under white and far-red light conditions (Supplementary Fig. 1) and solved their structures by cryo-EM single particle analyses at resolutions of 2.35 and 2.41 Å, respectively (Supplementary Figs. 2–5 and Supplementary Table 1). Both PSI cores form homo-trimers (Supplementary Fig. 4a, b), and the overall structures are similar to that of the cyanobacterial PSI trimer (containing only Chl a) reported previously6,11, except for PsaF, PsaJ, and PsaX (and PsaK in the case of white PSI). Although the gene for PsaX was not found in the genome of H. hongdechloris, the genes for PsaF, PsaJ, and PsaK are present29. In the atomic model of the white PSI, each monomer contains five membrane-spanning subunits (PsaA, PsaB, PsaI, PsaL, and PsaM) and three stromal subunits (PsaC, PsaD, and PsaE) (Fig. 1a and Supplementary Fig. 6). The density map for PsaF, PsaJ, and PsaK of the white PSI are rather poor compared with that of the other assigned subunits, and hence, these three subunits were deleted in the structure (Supplementary Figs. 2g and 6). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified PSI solution sample showed that it contained PsaF, PsaJ, and PsaK (Supplementary Fig. 1d and supplementary Table 2). Therefore, the poor densities for these three subunits in the cryo-EM map may be owing to the weak association of these subunits with PSI and partial dissociation of them during the grid preparation for cryo-EM.

Fig. 1: Structure of white PSI and far-red PSI monomers. a Cryo-EM density map (left) and structure (right) of the white PSI monomer viewed along the membrane normal from the stromal side. b Cryo-EM density map (Left) and structure (Right) of the far-red PSI monomer viewed along the membrane normal from the stromal side. Full size image

In the atomic model of the far-red PSI, each monomer contains six membrane-spanning subunits (PsaA, PsaB, PsaI, PsaK, PsaL, and PsaM) and three stromal subunits (PsaC, PsaD, and PsaE) (Fig. 1b and Supplementary Fig. 3f). The densities for PsaF and PsaJ were practically not found even if we lowered the map contour to the noise level. As these two subunits were also detected in the solution sample by SDS-PAGE (Supplementary Fig. 1d), this result indicates an even weaker association of them with the PSI core. One of the reasons for this loose association of PsaF and PsaJ may be owing to the replacement of psaA, psaB, as well as psaF, psaI, psaL, and psaJ genes under the far-red light condition (see below)3,29. PsaF was previously suggested to be involved in binding of the electron donor, such as the c-type cytochrome30. We measured the light-induced difference absorption change of P700 for Thermosynechococcus elongatus PSI and far-red PSI, which showed that the difference absorption changes are almost the same between the two PSIs (Supplementary Fig. 7). This result supports the presence and functioning of PsaF in the purified far-red PSI core, and suggests that the absence of its density in the cryo-EM map may be caused by its loss during the grid preparation.

The root mean square deviation (RMSD) between the white and far-red PSI is 0.62 Å for 1855 C α atoms from subunits whose structures were built in both PSI cores, suggesting a large similarity in the overall structures of the two PSI cores. However, it is known that far-red light induces an extensive remodeling of the photosynthetic apparatus of H. hongdechloris by changing the expression of genes encoding the PSI core subunits as well as the synthesis of pigments (including change of Chl a to Chl f) of PSI22,23,24,25,29. Indeed, the sequences of some subunits in the structure of the far-red PSI were found to be different from that of the white PSI. In our structure, PsaA_2, PsaB_2, PsaI_3, and PsaL_2 subunits in the white PSI were changed to PsaA_1, PsaB_1, PsaI_2, and PsaL_1 subunits in the far-red PSI, respectively. These four subunits are encoded by different genes with different amino-acid sequences between the white and far-red PSI, whereas the other subunits are encoded by the same genes29. The sequence identities for the four subunits, PsaA, PsaB, PsaI, and PsaL between the white and far-red PSI are 74.6%, 80.9%, 36.8%, and 43.4%, respectively, in this cyanobacterium. Multiple sequence alignment of these four subunits from three species of cyanobacteria that are known to undergo remodeling between white and far-red light conditions (H. hongdechloris, Leptolyngbya sp. strain JSC-1 and Chroococcidiopsis thermalis PCC7203) shows high homologies for each pair of the subunits grown under the same conditions (either white or far-red light conditions), but lower homologies between the same subunits expressed under white or far-red light conditions (Supplementary Figs. 8–11 and Supplementary Table 3). This is particular apparent for the PsaA and PsaI subunits. For example, the PsaA subunit has an average identity of 84.4% and 83.2% among cyanobacteria grown under far-red and white light conditions, respectively, whereas the average identity of PsaA between cyanobacteria grown under far-red and white light conditions is 76.2% (Supplementary Table 3). Furthermore, the PsaI subunit has an average identity of 65.0% and 51.8% among cyanobacteria grown under far-red and white light conditions, respectively, whereas the average identity between cyanobacteria grown under far-red and white light conditions is only 38.6%.

Several regions with different sequences between the subunits expressed under white and far-red light conditions were identified to be important for the structural differences between the white and far-red PSI. In the PsaA subunit, loop1 (Ala232-Asp244) located at the lumenal side of white PSI was deleted in the far-red PSI, whereas loop2 (Pro331-Asn338) is inserted in the far-red PSI (Supplementary Fig. 8), which was found to be located at the stromal side (Fig. 2). An additional insertion (Gly503-Gly528) of PsaA was found to be disordered at the lumenal side in the far-red PSI. In the PsaL subunit, loop3 (Thr8-Leu28) and loop4 (Gln117-Pro134) are inserted in the far-red PSI (Supplementary Fig. 11), which are located at the stromal side in the structure (Fig. 2). Interestingly, all these insertions and deletion (loop1-4) are located at one side (PsaA-PsaL side) of the structure of a PSI monomer (Fig. 2a). Both loop1 and loop4 do not interact directly with other subunits (Fig. 2b, d), whereas loop3 is elongated to the interface between PsaA and PsaD, and contributes to the structural stability of loop2 (Fig. 2c). Chl a865 (a865A) is bound to a region in white PSI that is occupied by loop2 in far-red PSI, and it is lost in the far-red PSI. Apparently, the insertion of loop2 hindered the binding of Chl a865A (some of the numbering of pigments and other cofactors discussed in the text are different from those registered in the PDB file; for a complete correspondence of the numbering of pigments and cofactors in the text with those in the PDB file, refer to Supplementary Table 4) in the far-red PSI (Fig. 2c). The loop2 is conserved in the psaA gene expressed under far-red light condition but absent in the gene expressed under white light condition in the Chl f-containing species (Supplementary Fig. 8). This loop2 is also absent in PsaA from Synechocystis sp. PCC6803 (NCBI:WP_010872067) and T. elongatus (NCBI:WP_011056578.1), from which the PSI structure has been solved and the corresponding Chl a865A molecule is present. These results indicate that the far-red light-induced expression of different copies of the PSI genes psaA, psaB, psaI, and psaL, and differences in the sequences of these genes, especially the psaA gene, caused the structural changes of the PSI complex, especially with respect to the insertion (and deletion) of the four loop regions in the far-red PSI (Fig. 2 and Supplementary Figs. 8–11).

Fig. 2: Superposition of the structures of the white PSI and far-red PSI monomers. a Superposition of the white PSI (yellow) and far-red PSI (cyan) viewed along the membrane normal from the stromal side. The structural differences (loop insertions) are colored in red. The RMSD is 0.62 Å for 1855 Cα atoms from subunits commonly observed between white PSI and far-red PSI. b–d Close-up view of the loop insertions at A232-D244 (loop1) of PsaA in white PSI b, P331-N338 (loop2) of PsaA in far-red PSI c, T8-L28 (loop3) of PsaL in far-red PSI c, and Q117-P134 (loop4) of PsaL in far-red PSI d. c PsaA (dark cyan) and PsaD (orange) are also shown as surface model. Full size image

The cofactors of the white PSI monomer contained 90 Chl a, 16 β-carotene (BCR), 3 [4Fe-4S] cluster, 2 phylloquinones, and three lipid molecules (Supplementary Table 5). The locations of the cofactors are similar to those of the Chl a-containing cyanobacterial PSI6,11. However, we identified seven molecules of Chl f (f826A, f827A, f830A, f832A, f844A, f810B, and f825B) in the monomer of the far-red PSI based on the difference density maps calculated between the cryo-EM experimental maps of the white PSI and that of the far-red PSI, as well as between the cryo-EM experimental map of the far-red PSI and the calculated density map derived from the atomic models of the far-red PSI with all Chls assigned as Chl a (see Methods for details) (Fig. 3a and Supplementary Fig. 12). Other cofactors in the far-red PSI include 83 Chl a, 16 β-carotene, 3 [4Fe-4S] cluster, 2 phylloquinones and 2 lipid molecules in a monomer. This gives rise to a Chl a/f ratio of 11.8 in the far-red PSI, which is very similar to the results reported recently31. The relative areas after taking into consideration the molecular coefficients of Chl a and Chl f32 in the 80 K absorption spectrum of far-red PSI were 89.1 and 10.9, respectively (Supplementary Fig. 13a, insert). This value is also consistent with the number of Chls f found in the structure of the far-red PSI. Compared with the white PSI structure, the far-red PSI has one additional Chl a (a101K) in the PsaK subunit, as PsaK was lost in the white PSI. However, one Chl a (a865A) is depleted in the far-red PSI as described above. As a result, the total number of Chls are the same between the white PSI and far-red PSI.

Fig. 3: Arrangement of pigments in the far-red PSI monomer. a Arrangement of the pigments in the far-red PSI monomer with protein subunits depicted in surface models, with a viewed along the membrane normal from the stromal side. For clarity, PsaC, PsaD, and PsaE are not shown. Chls f are shown as magenta stick (the color of the Chl f molecules f826A/f827A/f830A/f832A look like purple owing to the overlap with the cyan color of the surface model of PsaA) and the other ligands are shown as cyan stick. b Arrangement of the Chls f in the far-red PSI monomer with the proteins depicted in cartoon models. The protein structures of white PSI (yellow) and the far-red PSI (cyan) are superposed with each other. The insertion loops found in far-red PSI are colored in red. Chls f are shown as magenta stick. c–e Close-up view of the binding environments for f826A c, f844A d and f810B e. The structure of white PSI (yellow) and the far-red PSI (cyan) are superposed with each other and shown as cartoons. Chls and residues different between white PSI and far-red PSI are shown as sticks. f–i, Close-up view of the binding environments for f827A f, f830A g, f832A h, and f825B i. The structure of the far-red PSI (cyan) is shown as cartoon, and Chls are shown as sticks. Full size image

As no novel binding site of Chl f was found, Chl f has substituted the binding sites of Chl a upon far-red light induction in the far-red PSI. In agreement with the previous studies, the RC Chls were a pair of Chl a/Chl a′ (epimer of C132–Chl a) in both white and far-red PSI6. However, Chl a865A was found in the white PSI but absent in the far-red PSI. As this Chl is located at the interface of monomers in the white PSI trimer, its absence in the far-red PSI trimer may suggest a different energy transfer behavior among monomers between the far-red PSI and white PSI trimers.

Binding sites of Chl f and their possible functions

The seven molecules of Chl f identified in the far-red PSI are located at the interface of each PSI protomer (Figs. 3a and 4a). Among them, five Chl f (f826A, f827A, f830A, f832A, and f844A) are coordinated by PsaA and the other two Chl f (f810B and f825B) are coordinated by PsaB. Except for one Chl f (f825B), five (f826A/f827A/f830A/f832A/f844A) form a Chl f network in a monomeric unit with edge-to-edge distances of 10.0–13.3 Å between the chlorin rings (Fig. 4). In addition, Chl f810B is also located at the same PsaA side with an edge-to-edge distance of 18.0 Å to Chl f844A. These two Chl f molecules are coupled through an intermediate BCR molecule BCR102I (Fig. 4c) (see below). Thus, Chl f810B can be considered to be part of the Chl f network.

Fig. 4: Arrangement of pigments in the far-red PSI trimer. a Arrangement of the pigments in a far-red PSI trimer viewed along the membrane normal from the stromal side. The Chls f are colored in magenta, and other cofactors are colored in cyan, yellow and green in three monomers, respectively. b Arrangement of Chl f in a far-red PSI monomer viewed along the membrane normal from the stromal side, with the protein subunits depicted in a cartoon model. For clarity, PsaC, PsaD, and PsaE are not shown in the model. Chls f and several Chls a that are assumed to form red Chls are colored in magenta and green, respectively. c, d Close-up views of nearby pigments of f844A and f810B c, f825B d. Full size image

Among the seven Chl f molecules, three (f826A, f832A, and f844A) are located near loop2–4 (Fig. 3b), suggesting the close correlation between the substitution of Chl a by Chl f and the structural changes occurred in the loop regions in the far-red PSI. At the Chl f826A-binding site, W367 and W371 of PsaA in the white PSI are changed to I362 and Q366 with relatively smaller side chains in the far-red PSI (Supplementary Fig. 8); among them, Q366 is hydrogen-bonded to the oxygen atom of the formyl group of f826A with a distance of 3.2 Å (Fig. 3c). At the f844A-binding site, V699 of PsaB in the white PSI is changed to G700 in the far-red PSI (Supplementary Fig. 9), with its main chain nitrogen associated with the oxygen atom of the formyl group of f844A at 2.9 Å (Fig. 3d). At the f810B-binding site, P94 of PsaB in the white PSI is changed to A94 in the far-red PSI (Supplementary Fig. 9), and its main chain nitrogen is associated with the formyl group at 3.2 Å (Fig. 3e). All these changes result in residues with rather small side chains in the far-red PSI, enabling them to be able to accommodate Chl f which has an formyl group in its chlorin ring instead of a methyl group in Chl a. These residues related to the Chl f-binding in PsaA and PsaB, are conserved in the two genes expressed under far-red light, but not in the white light, in the Chl f-containing species (Supplementary Figs. 8 and 9). These results indicate that these changes in the amino-acid sequences are important to accommodate the formyl group of the three Chl f molecules appeared under far-red light conditions. On the other hand, no changes in the amino-acid residues are observed in the binding sites for f827A, f830A, f832A, and f825B, and the formyl groups of these Chl f are associated with an oxygen atom of Chl a840A phytol (2.9 Å), an oxygen atom of the C132 group of Chl a829A (2.9 Å), a hydroxyl group of Thr443 of PsaA (3.5 Å), and an oxygen atom of Chl a839B phytol (2.8 Å), respectively (Fig. 3f–i).

It is well-known that cyanobacterial PSI core binds red-shifted Chls (red Chls) whose energy levels are lower than P700. It has been suggested that red Chls consist of 7–11 molecules of Chl a per 96 Chl a based on the absorption spectra of PSI at cryogenic temperatures33,34,35. At least four Chl a clusters are necessary to explain the absorption spectrum of red Chls. These red Chl a components are also seen in white and far-red PSI in the 80 K absorption spectra (Supplementary Fig. 13). Several red Chl a candidates have been reported based on hole-burning analysis33. In the present structure, Chls a836B–a835B–a834B and a841A–a842A form two Chl a clusters, and thus can be considered as candidates for the red Chl a clusters (Fig. 4b). These Chls a are conserved between white and far-red PSI. The triplet Chls a836B–a835B–a834B are also found in the T. elongatus PSI structure, and they are adjacent to PsaX but not directly related to PsaX6. This “red trap” may therefore also work in the H. hongdechloris PSI. Another candidate for the red Chl a is a839A–a840A33 (Fig. 4b), which is also conserved between white and far-red PSI. These two Chls a (a839A–a840A) are very close to f832A and f827A, suggesting that this red Chl a cluster is closely associated with the six Chl f network and these Chls may share the excitation energy. The other red Chl a candidates reported are Chls a840B–a844A and a810B–a835A. However, a844A and a810B are changed to f844A and f810B in far-red PSI, respectively. As a result, a840B and a835A also take part in the six Chl f network. This allows a835A and f844A to interact through BCR102I (Fig. 4c). Among them, f810B interacts with a835A at a distance of 3.3 Å, and Chl a840B interacts with f844A at a distance of 3.5 Å with an oblique dipole orientation of the Y axis between the two Chl molecules (Fig. 4c). The close Chl–Chl interaction and their dipole orientation appear to lower their energy levels, suggesting that the two Chl a/f hetero-dimers are the fluorescence-emitting Chls at 813 nm (Supplementary Fig. 13). One Chl f, f825B, is far from the Chl f network (Fig. 4b), and interacts with BCR847B at a distance of 4.3 Å (Fig. 4d). This seems to be a Chl-Car quencher by either charge transfer or excitation energy transfer36. These structural and spectroscopic observations suggest that Chl f molecules are responsible for excitation energy transfer and quenching events under far-red light conditions.

The absorption spectrum of the far-red PSI shows at least three components at long wavelength region, which are located at 737, 752, and 795 nm (Supplementary Fig. 13). Our time-resolved fluorescence study of the far-red PSI clearly showed that Chl f molecules serve as excitation energy transfer components in the early time range of 5–300 ps after excitation. In particular, fluorescence components appeared at 746, 752, 753, and 813 nm at 77 K can be ascribed to Chl f (Supplementary Fig. 13). This indicates that the red Chls a and Chls f are equilibrated within this time range. Thus, Chl f molecules function in uphill energy transfer for energy-trapping at the RC Chls. The candidates for the fluorescence components at 746–755 nm are likely f826A/f827A/f830A/f832A/f844A and f825B, suggesting the contribution of these Chl f to the uphill energy transfer to the RC Chls at physiological temperatures20,27,28.