Photosynthesis is performed by two types of photoreaction systems, namely type I and type II reaction centers. Photosystems I 1 and II 2 (PSI and PSII, belonging to type I and type II, respectively) work sequentially in oxygenic photosynthesis in plants and cyanobacteria 3. They typically use chlorophyll (Chl) a (Extended Data Fig. 1) as the major pigment that absorbs visible light (670–700 nm) (Extended Data Fig. 2). Anoxygenic photosynthetic bacteria use bacteriochlorophylls (BChl) that absorb 800–900 nm light with either a type I or type II reaction center. Acaryochloris marina (A. marina) is a cyanobacterial species that uses the novel pigment, Chl d (Extended Data Fig. 1), which absorbs 700–750-nm light in vivo 4 (Extended Data Fig. 2). A. marina was isolated from colonial ascidians, which harbor mainly Chl a-type cyanobacteria, resulting in an environment with low visible light and high far-red light. A. marina has exploited this with the niche-filling introduction of Chl d 5,6. The discovery of A. marina prompted intensive research into the mechanism of this low-energy- driven system. Spectroscopic and pigment analyses revealed that Chl d indeed plays a central role in the photoreaction, although the organism also contains Chl a at around 5% relative abundance 4,5,7. With the acquisition of Chl d, A. marina, uniquely harnesses far-red light, which is the lowest energy light that can be used in all oxygenic photosynthetic organisms. The cyanobacterium can be seen as a missing-link between organisms possessing visible light/Chl a-based oxygenic photosynthesis and 800–900 nm light/BChl-based anoxygenic photosynthesis. Chl d has a peak wavelength of absorption at around 697 nm in methanol (Extended Data Fig. 3) (so-called Qy band; 700–750 nm in vivo, Extended Data Fig. 2), which is longer than the 665.2 nm of Chl a (670–700 nm in vivo, Extended Data Fig. 2 and 3) by ~30 nm, which means that the photon energy absorbed is ~80 mV (10%) lower. How PSI and PSII of A. marina drive the similar photochemical reactions that occur in the Chl a- dependent systems of other oxygenic photosynthetic organisms has been a long-standing puzzle.
PSI generates reducing power for NADPH production by accepting electrons originating from PSII 3. PSI of A. marina has a similar subunit composition to that of the PSIs of other oxygenic photosynthetic organisms 8,9 (Extended Data Table 1). However, the peak wavelength of the light-induced redox difference absorption spectrum of A. marina PSI showing that of reaction center is longer (740 nm) than that of Chl a (700 nm) in the PSI of plants and typical cyanobacteria 10 (Fig. 1A). Reaction center P740 thus named has been assumed to be a heterodimer of Chl d and d′ 11,12 by analogy to P700, which is the reaction center composed of a heterodimer of Chl a/Chl a' in all other PSIs (Fig. 1A) 1,13. Cofactors of the PSI electron transfer chain in A. marina (Fig. 1B) and the reduction potential of P740 are also similar to those of P700 in other organisms 8,9,14.
To date, several structures of type I reaction centers have been determined from higher plants 15,16, green algae 17,18, red algae 19, diatoms 20, cyanobacteria 1,21,22 and an anoxygenic bacterium 23. All structures have Chl a in the reaction center with the exception of the one from the anoxygenic bacterium which has BChl. The PSI of A. marina with Chl-d in its reaction center and its ability to utilize such low energy light is thus unique. Here, we present the first structure of the PSI trimer isolated from A. marina revealed by cryo-electron microscopy (cryo-EM) at 2.48 Å resolution to understand the mechanism for utilization of far-red light (Fig. 1B).
Overall structure, protein subunits, and cofactors of A. marina PSI
The PSI trimer complex was isolated from A. marina. Detailed methods of sample preparation, data processing, and structural refinement by cryo-EM, and biochemical data, are presented in Supplementary Materials (Extended Data Figs. 4–11, Extended Data Tables 1–4). The model of the PSI trimer was refined to give a correlation coefficient and Q-score, which are indices for validating the correctness of a model, of 0.80 and 0.678, respectively (Extended Data Table 4). These index values can be considered reasonable at 2.48 Å resolution (Extended Data Fig. 10).
The PSI trimer had dimensions of 100 Å depth, 200 Å length, and 200 Å width, surrounded by detergent micelles (Fig. 2). The overall structure resembles those of PSI trimers reported for other cyanobacteria 1,22,24. The root-mean-square deviations using secondary structure matching between the monomer model (chains A–M) and Protein Data Bank (PDB) structures 1JB0 of Thermosynechococcus elongatus 1, 5OY0 of Synechocystis sp. PCC 6803 22, and 6KMX of Halomicronema hongdechloris 24 are 0.90 Å, 0.90 Å, and 0.87 Å, respectively.
The A. marina PSI monomer contains 11 subunits [PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaJ, PsaK, PsaL, PsaM, and Psa27], all of which could be assigned to the cryo-EM density map (Fig. 2, Extended Data Figs. 5 and 12, and Extended Data Table 1). PsaX (a peripheral subunit in other cyanobacteria such as T. elongatus), PsaG and PsaH (subunits in higher plants) were missing in the density map, consistent with the absence of their genes from the A. marina genome 6. The unique name Psa27 was given to a novel subunit protein in A. marina that has low sequence similarity (29.4%) with PsaI of T. elongatus (Extended Data Table 5) 8. Here, we found that Psa27 is in the same location as PsaI in T. elongatus and has a similar fold. Psa27 also contributes to the structural stabilization of the PSI trimer just as PsaI does in T. elongatus. Thus, we concluded that Psa27 and PsaI are in effect the same subunit.
The loop regions of PsaB (residues 290−320 and 465−510) were not identified due to disorder because of the absence of PsaX, suggesting that PsaX stabilizes these regions in other cyanobacteria. Most of subunits PsaE, PsaF, and PsaK were modeled using polyalanine because of poor regional map quality (Extended Data Figs. 12, 13, and Extended Data Table 1), possibly suggesting that some part of those subunits dissociated during sample preparation. The cofactors assigned (Extended Data Fig. 14) are 71 Chl d (Extended Data Table 6), one Chl d′ (an epimer of Chl d), 11 α-carotenes (α-Car) but no β-Car (Extended Data Table 7) 6,10, two pheophytins (Pheo) (a derivative of Chl; however, unlike Chl, no Mg2+ ion is coordinated by the tetrapyrrole ring), two phylloquinones (PhyQs), three iron–sulfur clusters, two phosphatidylglycerols, one monogalactosyl diacylglycerol, and 84 water molecules. The Chls and α-Car pigments are mainly involved in the electron transfer reactions and light-harvesting.
Electron transfer components
The important cofactors involved in electron transfer (Fig. 1B) are four Chls (three Chl d and one Chl d′), two Pheos, two PhyQs (A1), and three iron–sulfur clusters (FX, FA, and FB) (Fig. 3A). The Chls and PhyQs are arranged in two branches, the A-branch and the B-branch, which are related by a pseudo-C2 axis as in other type I reaction centers, and are stabilized by amino acid residues of subunits PsaA and PsaB. The reaction center, P740, is composed of Chl d′ (PA) and Chl d (PB) (Fig. 4A), which are coordinated by residues His678/A and His657/B, respectively (capitalized letters following the slash (/) indicate the subunit names such as PsaA or PsaB) (Fig. 3B and C). The distance between the ring planes (the π–π interaction distance) of Chl d′ and Chl d is 3.5 Å (Fig. 3D) (3.6 Å in T. elongatus). Two Tyr residues, Tyr601/A and Tyr733/A, are positioned within hydrogen bonding distance of PA. These configurations are similar to those in the PSI from T. elongatus (Fig.1), although cofactor compositions are different.
In A. marina PSI, we uniquely identified three water molecules (W1−W3) around PA, forming hydrogen bonds with surrounding amino acid residues (Tyr601/A, Ser605/A, Asn608/A, Ser741/A, and Try745/A) and one Chl d (Chl d 32) (Fig. 3B and C). For the T. elongatus PA, only one water molecule forms hydrogen bonds with surrounding amino acid residues (Tyr603/A, Ser607/A, Ile610/A, Thr743/A, and Phe747/A) and Chl a′ (PA). There are no water molecules around PB in A. marina, but some water molecules surround PB in T. elongatus without forming hydrogen bonds. This difference around PB may come from the difference in two amino acid residues: Val594/B in A. marina vs. Thr597/B in T. elongatus, and Asn598/B in A. marina vs. His601/B in T. elongatus. The hydrogen bonding pattern around PA probably contributes to the charge distribution ratio (PA˙+/PB˙+) 1,25, and therefore, is likely different in the two organisms. The midpoint potential values, Em (PA) and Em (PB), are influenced by the protein environment, in particular, by the presence of charged residues, and the Em difference, Em (PA) − Em (PB) (= ΔEm), is an important factor in determining the PA˙+/PB˙+ ratio 26.
Previous studies have suggested that the primary electron acceptor A0 in A. marina PSI (Fig. 1A) is Chl a 9,27, in line with other species 1,28. However, we found that there was no mass corresponding to Mg2+ at the center of the tetrapyrrole rings of A0A and A0B, but rather a hole in the cryo-EM density map (Fig. 4A). This indicates that A0 is actually Pheo, a derivative of Chl, which is consistent with pigment analysis identifying the presence of Pheo a (Extended Data Table 7 and Extended Data Fig. 15). Furthermore, the position of Leu665/B (Extended Data Fig. 16B) 29, pointing to A0B and in the same place as conserved Met688/B that ligates A0B in T. elongatus PSI 1,29, supports Pheo a as A0B in A. marina, because Leu cannot serve as a ligand to Mg2+ in Chl. Thus, surprisingly, A0A and A0B are assigned as Pheo a in A. marina PSI (Fig. 1B; Fig. 3A; Fig. 4A–C).
The amino acid residue nearest to A0A in A. marina is Met (Met686/A) (Extended Data Fig. 16A) 29 and the identity of close amino acid residues may modify the reduction potentials of the two A0 and their absorption peak wavelength(s). Two mechanisms have been proposed for electron transfer in PSI reaction centers, one using both A- and B-branches
30,31, and the other using the A-branch preferentially 32,33. Only the A-branch may be active in A. marina PSI 29. Details of the electron transfer mechanism in relation to the PA˙+/PB˙+ ratio can now be filled in using the precise structure of A. marina PSI.
Why is Pheo a the A0 in A. marina PSI? Pheo a has not been identified in any PSI before, although it is the primary electron acceptor in all type II reaction centers 3. While the reaction center P740, which is composed of Chl d/d’, uses low-energy far-red light of 740 nm (1.68 eV), it generates reducing power almost equivalent to that of the P700 (Chl a/a', 1.77 eV) in plants and most cyanobacteria 8. Then, the excited state of P740 (P740*) produces 0.09 eV-weaker reducing power than P700* (i.e., 77 – 1.68 eV). This could result in a slower electron transfer rate to A0 and an increase in reverse reaction, due to the smaller driving force. However, the rates of electron transfer from P740* to A0, and to PhyQ are actually comparable to those from P700* to A0 and to PhyQ 27, therefore, Pheo a as A0 must achieve the same electron transfer efficiency as the Chl a-type PSI.
The midpoint potential value (Em) vs. the normal hydrogen electrode (NHE) of P740 is 439 mV 8,9,14, whereas that of P700 is 470 mV 34. However, most of the amino acid residues around A0 in A. marina PSI are similar to those in T. elongatus, so it is unlikely that the protein structure around A0 influences the Em value. We obtained Em (vs. NHE) values of purified Chl a, Chl d, and Pheo a in acetonitrile of −1100, −910, and −750 mV, respectively (Extended Data Fig. 17) 34,35. Therefore, according to the Em value, it seems reasonable for the Chl d-driven PSI to use Pheo a as A0, as the energy gap is sufficient for the primary electron transfer step. However, a possible effect on the rate of the following A0 to PhyQ step should be investigated as the rate is also known to be comparable to that in Chl a-type PSI.
A. marina contains a limited but distinct amount of Chl a 8,10,29, and we found a small amount in the PSI (1–2 Chl a per PSI monomer) (Extended Data Table 7). One possible locality is as the accessory Chls, AccA and AccB (Figs. 1 and 4). However, unfortunately, Acc could not be conclusively identified as Chl d or Chl a, because the functional group containing C31 of Acc Chl could not be precisely defined from the cryo-EM density map. While quantum mechanical/molecular mechanical calculations show that the formyl group of Chl d adopts two orientations (Extended Data Fig. 1C, D) 36, the oxygen atom of the formyl group on the Chl d molecule in a vacuum is more stable when oriented towards the C5 H atom, suggesting the conformer in Extended Data Fig. 1C. In contrast, the vinyl group of Chl a can adopt either orientation. The assignment of Chl d to the Accs in this study is actually due to the difference spectrum of P740, which suggests that the large electrochromic shift of Acc at 710 nm arises from Chl d 7.
The Mg2+ in AccA and AccB are coordinated by water molecules forming hydrogen bonds with Asn588/B and Asn602/A, respectively (Fig. 4B and C). In T. elongatus PSI, the methyl ester groups of the Chl a in AccA and AccB affect the charge and spin distributions on P700 26,37–39. The distance between the carbonyl O atom in AccA and the Mg2+ in PA, an epimer of Chl a, is 5.4 Å, whereas in AccB it is 7.1 Å. In contrast, in A. marina, these distances were 6.2 Å (AccA−PA) and 6.6 Å (AccB−PB), respectively (Fig. 4D), suggesting a different charge distribution with that in T. elongatus.
The amino acid residues surrounding PhyQ (A1A and A1B) are switched from those in T. elongatus PSI - Met720/A and Leu665/B in A. marina vs. Leu722/A and Met668/B in T. elongatus (Extended Data Fig. 18A, B). The arrangement of the phytol chain of A1B is also different from that in other cyanobacteria (Extended Data Fig. 18B). These structural differences may suggest that the protein environment within A. marina PSI is modified to optimize electron transfer involving Chl d. However, cofactors FX, FA, and FB and their surrounding structures in A. marina PSI are nearly identical to those in other cyanobacteria (Fig. 3A and Extended Data Fig. 18), indicating that the reduction potentials are likely the same. We suggest that FX, FA, FB, and their surrounding structures must be invariant for the reduction of NADP+.
Pigment environments: antennas and red chlorophylls
In the PSI, a large number of Chl d and α-Car harvest low energy far-red light and finally transfer the energy to P740. The arrangements of Chl d and α-Car in A. marina are similar to those of Chl a and β-Car, respectively, in T. elongatus, as shown in Extended Data Fig. 19, in which, the Chl ds are numbered according to the nomenclature of Chl a in T. elongatus 40. However, there are differences in some amino acid residues surrounding the cofactors, slight gaps in the arrangement of the cofactors, and lack of some Chls, which, we assume, reflects optimization for efficient use of far-red light.
According to the theoretical study of Adolphs et al. 40, the changes in site-energy of the three Chl a molecules whose band positions are most significantly shifted in T. elongatus PSI are produced by the following factors: (1) interaction with the backbone of α-helices, in particular with that of residues Gly316/B−Met320/B, causing Chl a 65 to be the third-lowest site-energy site of all the pigments; (2) the amide dipole in the side-chain of Gln115/A causing a decrease of the site-energy of Chl a 12; and (3) the positive charge of Arg24/M that induces a significant increase in the site-energy of Chl a 94.
The first factor is related to what are known as “red Chls” 1,41. Red Chls are a dimer or trimer of Chls, whereby the peak wavelengths of their Qy bands are shifted to wavelengths longer than the 700-nm peak of P700. In T. elongatus PSI, 9−11 Chl a per PSI monomer are considered to be red Chls. In particular, the trimer of Chl a 77–79 positioned in the region of Gly316/B−Met320/B is proposed to be a red Chl (Extended Data Fig. 19A) 1. In A. marina, this latter region seems highly flexible, and Chl d 55, 56, 63–67, and 77–79 could not be assigned as counterparts of Chl a molecules. The high flexibility of this region likely comes from the absence of PsaX. The seeming absence of Chl d 77–79 may be the reason why the A. marina PSI intrinsically does not exhibit features of a long-wavelength Chl d (Extended Data Figs. 2, 6, and 7) 7,42.
Regarding the second factor, in A. marina PSI, the Mg2+ ions of two Chls, namely Chl d 13 and Chl d 32, are within 10 Å of those of Chl d 12 (Extended Data Fig. 19B). The amino acid residue that is an axial ligand of Chl d 12, corresponding to Gln115/A of T. elongatus, is His (His116/A) in A. marina. This difference may affect the site-energy of Chl d 12 and surrounding Chls (Chl d 13 and 32).
Regarding the third factor above, Arg24/M in T. elongatus PSI corresponds to Asn (Asn24/M) in A. marina, which has a polar uncharged side-chain and no Chl d that corresponds to Chl a 94 of T. elongatus PSI could be identified in the vicinity of Asn24/M (Extended Data Fig. 19C). The A. marina PSI appropriately lacks the Chl with most increased site-energy that captures high-energy, short-wavelength light in T. elongatus.
Differences in some of the amino acid residues in the A. marina PSI compared with those of T. elongatus (Extended Data Table 5), result in different arrangements of some cofactors. For example, Phe49/J in A. marina, the counterpart of His39/J that is an axial ligand of Chl a 88 in T. elongatus PSI, explains the absence of the corresponding Chl d (Extended Data Fig. 19D). Owing to the absence of PsaX in A. marina, the Chl d corresponding to Chl a 95 in T. elongatus whose axial ligand is Asn23/X is also missing. These differences help explain the unique features of light harvesting in A. marina.
The arrangements of pigments adjacent to Psa27 (PsaI) and PsaL, whose sequence identity with other cyanobacterial PsaLs is low, are specific to A. marina (Extended Data Fig. 19A, black dashed line region, and Extended Data Fig. 20, and 21). Chl d 38, 52, 53, and the ring (α or ε) of Car4007 near Chl d 53 are set within the surrounding structure differently from those in T. elongatus (Extended Data Fig. 20A). This impacts on the distances between Car4010 and Chl 92 (8.1 Å in A. marina vs. 4.9 Å in T. elongatus), and Car4010 and Chl 47 (7.9 Å in A. marina vs. 6.9 Å in T. elongatus) (Extended Data Fig 20B and C). In the A. marina PSI, Chls 37 and 91−93 are in close proximity to PsaL, and Gln92/L (Ala94/L of T. elongatus) and Trp27/L (Phe30/L of T. elongatus) are positioned within hydrogen-bonding distance of Chl d 91 and 92, respectively (Extended Data Fig. 20D and E). Moreover, the formyl group of Chl d 37 and 93 and Ser62/L may form hydrogen bonds via one water molecule (Extended Data Fig. 21). Again, these divergences must affect energy transfer efficiencies in the two organisms.
A. marina PSI is a trimer as is the case for other cyanobacterial PSIs, but the amino acid sequence identity of each protein subunit is low, and, not unexpectedly, the divergence is most pronounced around the unique pigments, for example Chl d, α-Car, and Pheo a-A0 (Extended Data Table 5). The structure exhibits unexpectedly high flexibility of photosynthetic machinery. Again, the alterations must reflect optimization to drive efficient photochemistry. The structure would also bridge the evolutionary gap between the Chl a-type oxygenic reaction centers (adapted to visible light) and the BChl-type reaction centers (adapted to 800–900-nm light).
In conclusion, the structure and identification of electron carriers and key light- harvesting pigments provide the architecture for understanding how low energy far-red light is utilized by A. marina PSI. However, the full picture must wait for the structure of PSII, which is responsible for oxygen evolution through water-splitting, to be elucidated 3.