Photosynthesis Research

, Volume 139, Issue 1–3, pp 499–508 | Cite as

Structure and function of photosystem I in Cyanidioschyzon merolae

  • Maya Antoshvili
  • Ido Caspy
  • Michael Hippler
  • Nathan NelsonEmail author
Original Article


The evolution of photosynthesis from primitive photosynthetic bacteria to higher plants has been driven by the need to adapt to a wide range of environmental conditions. The red alga Cyanidioschyzon merolae is a primitive organism, which is capable of performing photosynthesis in extreme acidic and hot environments. The study of its photosynthetic machinery may provide new insight on the evolutionary path of photosynthesis and on light harvesting and its regulation in eukaryotes. With that aim, the structural and functional properties of the PSI complex were investigated by biochemical characterization, mass spectrometry, and X-ray crystallography. PSI was purified from cells grown at 25 and 42 °C, crystallized and its crystal structure was solved at 4 Å resolution. The structure of C. merolae reveals a core complex with a crescent-shaped structure, formed by antenna proteins. In addition, the structural model shows the position of PsaO and PsaM. PsaG and PsaH are present in plant complex and are missing from the C. merolae model as expected. This paper sheds new light onto the evolution of photosynthesis, which gives a strong indication for the chimerical properties of red algae PSI. The subunit composition of the PSI core from C. merolae and its associated light-harvesting antennae suggests that it is an evolutionary and functional intermediate between cyanobacteria and plants.


Photosynthesis Photosystem I Crystal structure Synechocystis Cyanobacteria Membrane complexes 



Photosystem I


Photosystem II


Light-harvesting complex


Oxygenic photosynthesis, which takes place in cyanobacteria, algae, and plants, provides most of the food and fuel on earth (Nelson 2011; Barber 2004). Nearly half the world’s photosynthesis occurs in the oceans, performed by single-celled mesophilic cyanobacteria and algae. The architecture of oxygenic photosynthesis in cyanobacteria has been determined at high resolution for both photosystems, but only in thermophilic cyanobacteria (Jordan et al. 2001; Umena et al. 2011). Cyanidiales, eukaryotic red microalgae, represent the principle photosynthetic organisms in hot acid. While the Photosystem I structural information already exists for both cyanobacteria and a higher plant for over a decade, no structure exists from any eukaryotic algae (Vanselow et al. 2009). Cyanidioschyzon merolae is a primitive red alga, which lives in acidic hot water even at a pH < 2 and temperature of 45 °C. This organism is one of the simplest photosynthetic eukaryotes containing a single nucleus, a single mitochondrion, and a single chloroplast (Matsuzaki et al. 2004). Biochemical studies revealed that C. merolae PSI core is composed of 12 subunits from PsaA to PasF, PsaI to PsaM, and PsaO. Its genome lacks the genes for PsaG and PsaH (Matsuzaki et al. 2004), existing in green algae and vascular plants (Jensen et al. 2007). With respect to the light-harvesting complexes, the red algae are an intermediate between prokaryotic cyanobacteria and eukaryotic green algae and higher plants (Busch et al. 2010). The PSI antennae in C. merolae were shown to contain three gene products that are homologous to the green algae and plant light-harvesting complexes (Busch et al. 2010). They are equally related to PSI and PSII antenna complexes. In contrast to PSI that is related to the plant PSI, structural determination of red algae PSII revealed that it is highly related to the cyanobacterial PSII (Ago et al. 2016; Jordan et al. 2001). Therefore, structural determination of C. merolae PSI should be highly valuable for evolutionary studies of the photosynthetic apparatus.

Materials and methods

C. merolae cultivation

Cyanidioschyzon merolae De Luca, Taddei et Varano NIES-1084 was ordered through the National Institute of Environmental Studies (NIES), Japan and received on May 2012. C. merolae cells were cultivated in 10-L bottles at either 25 or 42 °C, in the presence of air and CO2, under approximately 50 µE m−2 s−1 continuous light, in 2× Allen medium at pH 2.8 (Allen 1959; Minoda et al. 2004).

Thylakoid preparation

Both thylakoid preparation and PSI purification were made according to Nechushtai and Nelson’s protocol from 1981 (Nechushtai and Nelson 1981) with slight modifications.

The cells were harvested after 9–10 days of growth when their OD730 reached 1.2–1.5, using Sorvall RC 6+ centrifuge with Fiberlite F10-4×1000 LEX Rotor at 6800×g for 5 min. After this step, the whole experiment was conducted at 4 °C and procedures were kept as short as possible. The cells were suspended with 150 ml STN buffer containing 0.4 M sucrose (Sigma-Aldrich), 0.03 M Tricine-NaOH pH 8 (Sigma-Aldrich), and 0.01 M NaCl (Merck), then centrifuged again using Sorvall centrifuge with ss-34 rotor at 4300×g for 5 min. The pellet was re-suspended again in 100 ml of the same buffer. Next, cells were disrupted using Avestin EmulsiFlex-C3 high-pressure homogenizer in 1000 bar three times. After disruption, cell debris was deposited using same rotor at 17,000×g for 5 min. The supernatant collected was loaded onto Ti-70 rotor and centrifuged for 2 h at 184,000×g in Beckman Coulter Optima XE-90 centrifuge. Pellet was then dissolved with 175 ml buffer 0.4 M sucrose, 0.02 M Tricine-NaOH pH 8, and 0.15 M NaCl, incubated for 30 min on ice and then centrifuged for 45 min using the same conditions as in the previous step. Pellet was then dissolved with 175 ml buffer, incubated, and centrifuged again for 25 min. The thylakoid membranes were dissolved in 0.4 M sucrose, 0.02 M Tricine-NaOH pH 8, to reach concentration of 2 mg of chl/ml and were frozen in − 80 °C for further purification.

Photosystem I purification

Frozen thylakoid membranes (15–20 ml) containing 2 mg of chl/ml were thawed in cold water and solubilized with 15 mg n-dodecyl-β-d-maltoside (β-DM, Anatrace) per 1 mg chlorophyll and incubated for 30 min on ice. Insolubilized material was removed by ultracentrifugation for 30 min at 184,000×g (Beckman Coulter, Ti70 rotor).

The supernatant was collected and applied to a 1.5 × 25 cm diethylaminoethyl-cellulose column (Toyopearl DEAE-650 C) for separation of the PSI from the other complexes. The column was equilibrated by 150 ml of 20 mM Tricine-Tris pH 8 and 0.5% β-DM solution, then the PSI containing material was loaded and the column was washed using 100 ml of the above buffer. PSI complex was eluted with 0–0.3 M tetraethyl ammonium chloride (4EACl, Sigma-Aldrich) linear gradient (125 ml in each chamber). Fractions containing PSI were precipitated by 10% polyethyleneglycol 4000 (PEG 4000, Hampton Research), followed by centrifugation at 4300×g for 5 min (ss-34, Sorvall). This pellet was dissolved in 20 mM Tricine-Tris pH 8 and 0.1% β-DM. The green suspension was applied onto a 10–35% sucrose gradient containing the same buffer and centrifuged at 170,000×g (SW40-Ti rotor, Beckman Coulter) for 17 h. The dark green band, containing PSI was collected and loaded onto a Tricorn 10/100 column (SOURCE 15Q-polystyrene/divinyl benzene polymer matrix, GE healthcare) using AKTA prime plus. The column was equilibrated with 20 mM Tricine-Tris pH 8 and 0.1% β-DM and eluted with 0–0.4 M 4EACl, linear gradient (30 ml in each chamber), dark green fractions were collected and applied onto a 10–35% sucrose gradient and centrifuged at 364,000×g for 4 h (SW60-Ti rotor, Beckman Coulter). The purified PSI appeared as a dark band in the middle of the tube, only the central part of the band was collected. The material was precipitated with 100 mM 4EACl and 12% PEG 4000, then centrifuged at 20,000×g for 4 min. The pellet was dissolved in a solution containing 2 mM Tricine-NaOH pH 8 and 0.05% β-DM and adjusted to a chlorophyll concentration of 2.0 mg/ml.

Triton treatment and electron transfer measurements

Previous studies have shown that PSI subunits F, J, K, L, and I can be deleted without causing severe growth defects in cyanobacteria (Mazor et al. 2014). To test the minimal structure that can sustain PSI activity in C. merolae, the dark green fraction (9 ml containing 1.3 mg chlorophyll per ml) of the first sucrose gradient was incubated with 4% Triton X-100 for 4 h at room temperature and loaded onto a Toyopearl DEAE column (1 × 8 cm), that was prewashed with 16 ml wash buffer containing 30 mM Tricine-Tris pH 8, 0.1% β-DM, and 0.4% Triton X-100. It was then washed with 40 ml of the same buffer. Subsequently, the column was washed with 16 ml buffer without Triton. The protein was eluted with the same buffer containing 200 mM Tetraethylammonium chloride and the dark green peak was loaded onto small sucrose gradient and centrifuged at 84,000×g for 16 h (SW60-Ti rotor, Beckman Coulter).

For measuring the rate of P700 reduction, PSI or Triton washed PSI was supplemented with plastocyanin or cytochrome c6. The P700 reduction rate was measured by a JTS-10 pump probe spectrometer, by following absorbance changes at 705 nm. The reaction mixture contained 1 ml of 30 mM Tricine-NaOH pH 8, 5 mM MgCl2, 0.05% β-DM, 20 µM methyl viologen, 10 mM ascorbate, and PSI preparation containing 17 µg chlorophyll. It was supplemented by either 0.6 µM pea plant plastocyanin or 0.7 µM Synechocystis cytochrome c6.

Importance of detergents and lipids

Integral membrane proteins are often crystallized in a complex with detergents, which requires pure and stable preparations and identification of detergents compatible with crystal formation (Michel and Ostermeier 1997). Another important aspect to consider is lipid content that may be part of the membranal protein. Aggressive purification may lead to lipid depletion which may harm the complex integrity. Thus, the problem of protein purification may not center on obtaining the highest possible purity, but rather an optimum purity that does not cause depletion of the native lipid content (Zhang et al. 2003). Using a lipid augmentation strategy, as described in Zhang et al., lipid content was optimized by adding ten equivalents of synthetic lipid, DOPC (dioleoylphosphatidylcholine), per monomer (final concentration 0.01%), and better diffracting crystals were obtained.

PSI crystallization

Crystallization was conducted immediately after purification of PSI in 24-well plates using the sitting drop variant of the vapor-diffusion technique at 277 K (Charles Supper Company). Aliquots of (4–5 µl) PSI solution were mixed with equal volumes of reservoir solution (80 mM MgCl2, 50 mM citric acid, 4–7.5% PEG 4000, 0.025% n-dodecyl-α-d-maltoside (α-DM, Anatrace), pH 4.8-5.0 or 80 mM MgCl2, 50 mM sodium cacodylate pH 5–6, 4–7.5% PEG 4000, 0.025% α-DM) and equilibrated against 0.5 ml of reservoir solution. Long rectangular-shaped crystals of dark green color and varying thickness appeared after 2–3 weeks and reached maximum size within 5–6 weeks.

Cryogenic protection of the crystals

For cryogenic protection, the solution was changed in three steps of increasing PEG 4000 concentrations, from PEG 4000 10%, then 15%, and then to a final concentration of 20% PEG 4000 and 10% glycerol (Hampton Research). The crystals were frozen and stored in liquid nitrogen untill used in the synchrotron beam line.

Crystal diffraction measurements

The preliminary diffraction quality of the crystal was measured with the Rigaku Microfocus rotating anode X-ray generator, MicroMax-007, in Tel Aviv University, life sciences faculty. Crystals that diffracted the X-ray beam around 10 Å or further were sent to one of the following European synchrotrons, The European Synchrotron Radiation Facility (ESRF, Grenoble, French, station ID23-1, ID23-2, ID29), Paul Scherrer Institute (PSI, Villigen, Switzerland, station PXI, PXII, and PXIII), or Helmholtz- Zentrum Berlin für Materialien und Energie (HZB, Germany station BESSYII) for a diffraction measurement by a more focused beam.

Data collection

X-ray diffraction data were collected from crystals that diffracted around 4 Å, at the European Synchrotrons. Image data were processed using the HKL programs suite (Otwinowski and Minor 1997) and XDS (Kabsch 2010). Table 1 shows data collection and statistics of the C. merolae PSI crystal structure.

Table 1

Data collection and refinement statistics

Data collection



 Wavelength (Å)


 Resolution (Å)


 Space group


Unit cell dimensions

 a, b, c (Å)

163.13, 213.52, 349.58

 α, β, γ

90, 90, 90

 Measured reflections

1,707,215 (142,577)

 Unique reflections

51,806 (4451)

 Rpim (%)

0.036 (1.236)


10.2 (1.3)


0.998 (0.855)

 Completeness (%)

99.8 (99.9)


33 (32)

Refinement statistics



 No. of chains


 No. of ligands


 Average B-factor (Å2)


R.M.S deviations

 Bond angles


 Bond lengths


Ramachandran statistics

 Favored region (%)


 Allowed region (%)


 Outliers region (%)




Structure determination

The structure of PSI was determined by molecular replacement (Vaguine et al. 1999) using the theoretical model (PDB ID: 1YO9). The model was built using the program Coot (Emsley et al. 2004) and refined using the REFMAC program (Vagin et al. 2004) and PHENIX (Adams et al. 2010). Additional amino acid sequences and cofactors were built in Coot. Structure figures were generated and rendered with PyMOL (DeLano 2002). The structure of C. merolae photosystem I supercomplex was deposited in Protein Data bank (PDB 6FOS).


Purification and crystallization of C. merolae PSI supercomplex

During the past 15 years, our lab accumulated a large amount of information on the growth conditions and purification procedures towards obtaining homogeneous PSI preparations from different organisms (Pisum sativum and Synechocystis sp. PCC 6803) amenable to yield good crystals (Ben-Shem et al. 2003; Amunts et al. 2007, 2010; Mazor et al. 2014, 2015, 2017). These procedures have been implemented towards revealing the PSI structure of one of the most primitive photosynthetic eukaryote C. merolae, which represents a structural intermediate in evolution of eukaryotic PSI and LHC antennae (Busch et al. 2010; Nikolova et al. 2017). Altering growth conditions and improvement of purification procedure, together with finding better conditions for crystal formation, were critical for getting high-resolution diffracting crystals. Growth conditions affected dramatically the quality of the crystals. Once the cells’ growth was shifted from 42 to 25 °C, better diffracting crystals were obtained. Figure 1 shows sucrose gradient of the last step prior to crystallization and SDS gel of the crystallized material. Dissolved crystals that were analyzed by SDS electrophoresis exhibited identical polypeptide pattern indicating that the crystals represent the entire PSI preparation. Figure 2 shows typical crystals in the crystallization plate. The morphology of the better diffracting crystals is highlighted.

Fig. 1

Purified C. merolae PSI on sucrose gradient and SDS-PAGE gel. A sample of 0.4 ml containing 0.4 mg chlorophyll was loaded onto a sucrose gradient tube that was centrifuged at 84,000×g for 4 h (SW60-Ti rotor, Beckman Coulter). The sample in the gel contains 2.5 µg chlorophyll

Fig. 2

Large photosystem I crystals from C. merolae (the circled crystal’s length is 1 mm). a The circled crystal represents the “gutter-like” shape that diffracted to 8 Å (old type crystals). b Some changes in purification steps as well as crystallization procedure, resulted in a new type of rhombic-like crystals, which diffracted to 6 Å and altering growth conditions resulted the current type of rectangle-like crystals, which belong to a space group (C 2221) and diffract to resolution 4 Å (c)

The various preparations were subjected to mass spectroscopy (MS) analysis. The purified concentrated PSI preparation was fractioned via SDS-PAGE into four identical, side-by-side lanes and in-gel digested the protein bands. Independently, purified PSI complexes were digested in solution and analyzed by LC–MS/MS. The MS data for the purified PSI complexes agreed with the recently published data (Tian et al. 2017). We detected no difference in the subunit of PSI supercomplex isolated from cells grown at 25 or 42 °C.

The genome sequence of C. merolae lacks the plastocyanin gene, suggesting that the red algae exclusively use cytochrome c6 as electron donor for PSI (Matsuzaki et al. 2004). Triton-wash experiment on isolated C. merolae PSI particles, resulted in the removal of LHCI and PsaF subunits from the supercomplex (Fig. 3) as shown before for plant PSI (Bengis and Nelson 1977). Interestingly, the rate of P700 reduction by cytochrome c6 was not affected by the removal of LHCI and PsaF subunits; however, the reduction of P700 by plastocyanin was strongly inhibited in the Triton-treated particles. This suggests that in C. merolae, the eukaryotic N-terminal helix-loop-helix motif for efficient binding of plastocyanin already evolved in PsaF (Hippler et al. 1996), as revealed by the structure (PDB 6FOS), while it uses a cyanobacterial-type cytochrome c6 which is capable of binding and transfering electrons to PSI in the absence of PsaF (Chitnis et al. 1991; Hippler et al. 1996, 1999).

Fig. 3

The effect of antennae and PsaF depletion on the rate of P700 reduction by electron donors. a P700 reduction rate by cytochrome c6 with or without Triton treatment. b P700 reduction rate by plastocyanin with or without Triton treatment. c Subunit depletion by Triton treatment shown by SDS-PAGE. (a) The native PSI was taken from the first sucrose gradient (see “Materials and methods”). (b) The column Triton-wash contains light-harvesting and PsaF subunits. (c) The green material eluted from the column. (d) The material in “c” after sucrose gradient

Crystal structure of PSI supercomplex from C. merolae

The crystal structure of cyanobacteria and plant PSI supercomplexes have been solved at high resolution revealing most of the amino acids and numerous prosthetic groups at atomic level (Jordan et al. 2001; Qin et al. 2015; Mazor et al. 2015, 2017; Malavath et al. 2018). Low-resolution cryo-EM structures of Nannochloropsis gaditana, Chlamydomonas reinhardtii, and C. merolae suggest varying arrangements of LHCI belt compared to higher plants crescent-shaped PSI-LHCI, which consists of four Lhca subunits (Drop et al. 2014; Alboresi et al. 2017; Haniewicz et al. 2017). Remarkably, so far, no high-resolution structure of algal PSI is available, Biochemical and electron microscopy studies indicated that the algal PSI is highly complex and versatile (Kargul et al. 2003; Busch et al. 2010).

Most of the studies on algal PSI supercomplex were performed using the model green alga Chlamydomonas reinhardtii. In C. reinhardtii, there are 20 LHC antenna genes that encode nine Lhcas and 11 Lhcbs, nine of which (LhcbM1–9) code for the major antenna complex LHCII and two for the monomeric antennae CP26 (Lhcb5) and CP29 (Lhcb4) (Elrad and Grossman 2004; Merchant et al. 2007; Busch and Hippler 2011). The antenna size of both PSI and PSII was shown to be larger in C. reinhardtii as compared with higher plants (Germano et al. 2002; Kargul et al. 2003; Stauber et al. 2009; Liguori et al. 2013; Drop et al. 2011, 2014). The structure of C. merolae PSI supercomplex was studied by combination of biochemical, spectroscopic, and mass spectrometry methods (Busch et al. 2010; Nikolova et al. 2017; Tian et al. 2017). The studies revealed a versatile complex formed of a reaction center that is similar to the cyanobacterial complex containing subunits PsaA to F, PsaK to M as well as PsaO. The complex includes also eukaryotic-type three LHCs. Very recently, an electron microscopy single-particle analysis demonstrated large and versatile particle that is changing in response to light intensity and growth conditions (Haniewicz et al. 2017). Structural remodeling of the LHCI antenna and adjustment of its effective absorption cross-section as well as a dynamic re-adjustment of the stoichiometry of PSI-LHCI isomers were observed.

In this study, we used X-ray crystallography to determine the structure of PSI-LHCI supercomplex. We purified, crystallized, and solved the structure of the complex from C. merolae grown at 25 and 42 °C. The best solution was obtained so far for PSI isolated from 25 °C grown cells. Typical crystals are shown in Fig. 2. We had to screen several hundreds of crystals that were obtained from over hundred modified conditions to obtain the reported 4 Å resolution. Under all conditions tested, the crystals exhibited C2221 symmetry, with cell dimension of a 163.13, b 213.52, c 349.58 and α, β, γ 90, 90, 90. Unfortunately, the diffraction pattern was highly anisotropic. Anisotropic correction was performed according to Strong et al. (2006). The structure of the PSI supercomplex at 4 Å resolution is shown in Fig. 4 and a comparison with Synechocystis sp. PCC 6803 and plant PSI structures is shown in Fig. 5. Into the structure, we modeled in 12 core subunits A, B, C, D, E, F, I, J, K, L, M, and PsaO as well as the three LHC complexes (Lhcr) as peripheral antenna systems. The latter are situated in positions close to the corresponding Lhca4, Lhca2, and Lhca3 in plant PSI. C. merolae contains 3 Lhcr subunits encoded by three genes CMQ142C, CMN235C, and CMN234C (Matsuzaki et al. 2004; Busch et al. 2010). We tentatively modeled cmq142C as Lhcr1, cmn234C as Lhcr2, and cmn235 as Lhcr3 in relation to the order of plant Lhca subunits. The resolution did not permit positive identification of the above light-harvesting proteins. The structure revealed that all of them moved towards PsaK, assumed a distinct position, and consequently adapted excitation transfer pathways.

Fig. 4

A view from the stromal side on the C. merolae PSI-LHCI supercomplex

Fig. 5

Comparison of Synechocystis, C. merolae, and plant PSI crystal structures. The two PSI subunits G and H are exclusively present in plants and green algae but non-existing in C. merolae and Synechocystis structures. The light-harvesting antenna Lhca1 from plant is missing in C.merolae. PsaM is present in C. merolae and cyanobacteria but missing in plants, while PsaO is missing in cyanobacteria and plant structure

The amino acid sequences and the structure of the main core subunits PsaA, PsaB, and PsaC as well as the chlorophyll molecules are highly conserved. As expected, most of the changes occurred in the peripheral subunits. PsaD is shorter in both N and C termini than the corresponding cyanobacterial and plant subunits. PsaE has a longer C terminus but according to the published reading frame (Matsuzaki et al. 2004) is missing 25 amino acids at the end, that presumably removed due to transcriptional processing or posttranslational modification. While PsaF, PsaI, PsaJ, and PsaL maintained their cyanobacterial position, they contain several modifications that may be linked with their role in the binding of Lhcr1–3 or related to the formation of binding sites for additional membrane proteins. Even though the general structure of PsaF is highly conserved, its amino acid sequence varies considerably in comparison with cyanobacteria and plants. Its single transmembrane segment that is in close contact with PsaA and PsaB is highly conserved, while its N- and C-termini sequences are poorly conserved (see Fig. 6). Regardless the poor conservation their structure is strictly conserved. The N-terminus functions in the binding of electron donors and the C-terminus in eukaryotes interact with the light-harvesting complex.

Fig. 6

Superposition of Synechocystis, C. merolae, and plant PsaFs and PsaBs with chlorophyll molecules in their vicinity. Synechocystis PsaF is shown in purple and PsaB in yellow, C. merolae PsaF is shown in orange and PsaB in blue, plant PsaF and chlorophyll J1102 are shown in teal and PsaB in red

The most interesting addition to the core complex is the presence of PsaO. This protein was reported to be one of the plant PSI subunits but it was never detected in the solved structures of the supercomplex (Jensen et al. 2007; Mazor et al. 2017a, b). According to our model, PsaO contains two transmembrane helices connected in the lumen by a loop with a small apparent alpha-helix. Our resolution does not allow amino acid tracing, therefore the sequence in the model is only tentative. We modeled PsaO alongside PsaA and close to PsaK, as well as three chlorophylls. In close excitation transfer distance to three chlorophylls of PsaA (1120, 1121, and 1141), it appeared that BCR A4007 is in close proximity of PsaO chlorophyll. This position between PsaA, PsaK, and PsaL was suggested to provide the binding site for LHCII during state-transition (Amunts et al. 2007). The presence of PsaO in this location might prevent interaction with Lhcr and leave only PsaB as a potential binding site for light-harvesting complexes in addition to the three ones reported above.

Most of the regions connecting the LHCI belt to the C. merolae PSI core resemble those observed in plant PSI, with two notable differences: one in the vicinity of Lhca1 and subunit PsaG which are both absent in C. merolae and the second close to PsaA, PsaJ, and PsaK which possibly occurred by Lhcr 1–3 displacement towards the K-pole (see Fig. 7).

Fig. 7

Stromal view of LHC subunits interactions with PSI core in C. merolae and plant. Lhca1, yellow; lhca4/Lhcr1, blue; lhca2/Lhcr2, orange; lhca3/Lhcr3, green. Contact sites of PsaA magenta, PsaB dark green, PsaJ raspberry, PsaF teal, PsaG cyan, PsaK red. a The contact regions between the Lhcr subunits and the reaction center of C. merolae in the vicinity of subunit F and Lhcr1, PsaJ and Lhcr2, PsaA and Lhcr2, PsaA and Lhcr3, PsaK and Lhcr3. b The contact regions between the Lhca subunits and the reaction center of plant, in the vicinity of subunit G and Lhca1, PsaB and Lhca1, PsaF and Lhca4, PsaJ and Lhca2, PsaA and Lhca3, PsaK and Lhca3

Due to the shift in the position of Lhcr 1–3 and the lack of Lhca1, the evolution of new excitation transfer pathways is expected. Indeed, the structure suggests new pathways for this function. Lhcr1 contains 13 chlorophylls, 11 of them in similar locations to those observed in plant Lhca4, which includes 15 chlorophylls in total. Chlorophyll 616, which is missing in Lhca4, together with 601 Lhcr1 makes up a chlorophyll pair close to the Lhcr1 edge. Chlorophyll 603 shows significant displacement and is located closer to PsaJ J1301, connecting Lhcr1 to the PSI core. Despite the large movement of Lhcr1 towards the K-pole, it exhibits the highest similarity to the corresponding plant Lhca4, Lhca2, and Lhca3. The plant PsaF contains two chlorophylls F1301 and F1302, which may function in excitation transfer from Lhca4 and Lhca1 to the core, respectively. Examination of C. merolae PsaF shows these two chlorophyll molecules—F1301 and F1302. Figure 6 shows superimposed Synechocystis, C. merolae, and plant PsaFs with chlorophyll molecules in their vicinity. Chlorophyll F1301 may together with chlorophyll 605 of Lhcr1 function in excitation transfer from Lhcr1 to the PSI core. In plants, chlorophyll F1302 connects Lhca1 to the plant PSI core. Interestingly, this chlorophyll is conserved from Synechocystis to higher plants. Although present in all three organisms, it appears that this chlorophyll has adapted a role in excitation energy transfer only in plant PSI. Comparison of Lhca2 and Lhcr2 reveals that the latter has 15 chlorophylls, 13 of them found in similar positions to those in plant Lhca2. Two new chlorophylls 615 and 616 were identified in C. merolae Lhcr2—chlorophyll 616 is of particular interest, since its position is occupied by a beta-carotene A4019 in cyanobacteria. Chlorophylls 614 of Lhcr2 and 609 Lhcr1 are located between Lhcr2 and Lhcr1, thus enabling excitation energy transfer between the two complexes. In contrast to Lhcr2, in Lhcr3 two chlorophylls are missing from C. merolae structure. Lhcr3 seems to be more parsimonious than Lhca3—the chlorophyll pair 605 and 612, found in the stromal part of Lhca2 is replaced by a single chlorophyll 612 in C. merolae. Examination of the luminal area of Lhca3 reveals that chlorophylls 606, 610, and 613 are substituted by 606 and 610 in Lhcr3 as chlorophyll 610 shifts towards the space occupied by 613 of plant Lhca3. Chlorophyll 614 of Lhcr3 is found in high proximity to PsaA chlorophylls A1108, A1110, and A1111 serving as a facilitator of excitation energy transfer from Lhcr3 to PsaA, whereas in plant this chlorophyll (614) is far from PsaA. Inspection of PsaK reveals that two chlorophylls are present in C. merolae as opposed to four in plant PSI. Each of the chlorophylls has a unique role—K1401 mitigates excitation energy transfer from Lhcr3 603 and 608 to the PSI core, whereas K1402 serves the same purpose for PsaO 1603 on the other side of PsaK. Superposition of C. merolae and cyanobacterial PSI reveals that PsaO is occupying the space of PsaM from an adjacent PSI monomer and possibly interrupting trimerization in this region. The PsaM subunit is required for the formation of stable Photosystem I trimers (Naithani et al. 2000).


The photosynthetic apparatus of a red microalga C. merolae has gained a considerable interest, due to the unique evolutionary positioning of this species near the root of the red algal lineage (Nozaki et al. 2003; Reeb and Bhattacharya 2010). It is considered as an evolutionary intermediate link between the photosynthetic systems of cyanobacteria and higher eukaryotes like green algae and plants. The most distinct difference of the red algal PSI from the cyanobacterial PSI is the presence of a crescent-like peripheral light-harvesting antenna complex (LHCI), composed of a variable number of chlorophyll-binding complexes (Tan et al. 1997; Ben-Shem et al. 2003; Nelson and Ben-Shem 2004; Busch et al. 2010; Thangaraj et al. 2011; Tian et al. 2017). The interesting features of the red algal PSI are the retention of the cyanobacterial PsaM subunit and the lack of higher plant and green algal PsaH and PsaG subunits (Busch et al. 2010) (Table 2). The prokaryotic characteristics include the presence of cyanobacterial-like phycobilisomes functioning as the peripheral light-harvesting antenna, as well as the presence of cyanobacterial-like PSII (Busch and Hippler 2011). The red algal PSI-LHCI supercomplex is reminiscent of its higher plant and green algal counterparts by being monomeric reaction center core complex composed of 12 subunits PsaA–PsaF and PsaI–PsaO, (Vanselow et al. 2009; Jensen et al. 2007; Tian et al. 2017) and is associated with an asymmetrically arranged LHC complexes (Ben-Shem et al. 2004). The presence of the two core subunits PsaF and PsaL in cyanobacterial, algae and higher plant supports the evolutionary intermediate character of the red algal PSI-LHCI supercomplex (Busch and Hippler 2011; Kargul et al. 2012). It is interesting to note that in marine viruses operon encoding PSI the gene for PsaL subunit is missing (Sharon et al. 2009; Mazor et al. 2014). This suggests a pivotal function of PsaF, and in particular of its eukaryotic N-terminal helix-loop-helix motif, for the evolution of the LHC belt in the higher organisms. This is also apparent as this N-terminal helix-loop-helix motif is present in C. merolae and required for efficient electron between eukaryotic plastocyanin and PSI, although the endogenous electron donor, a cyanobacterial-type cytochrome c6, does not require this motif for efficient electron transfer to PSI (Fig. 3). This suggests that the eukaryotic N-terminal helix-loop-helix motif of PsaF was recruited later by plant PSI electron transfer donors, making electron transfer more efficient, although its primary function was related to the evolution of the LHCI belt.

Table 2

The presence of specific subunits, PSI oligomeric state, light-harvesting antennae, and electron donors in photosynthetic organisms



C. merolae



Peripheral antennae











An alternative point of view is that red algae did not precede green plants, but rather red algae and green plants have a common ancestor. A significant support for a sisterhood of green plants and red algae was based on a multi-gene analysis of a fusion of 13 nuclear markers (Moreira et al. 2000). These authors suggested that a single primary endosymbiosis took place before the separation of red algae and green plants and even predated the divergence of Glaucophytes. Other researchers suggest that Glaucophytes may be similar to the original algal type that led to green plants and red algae evolution (Keeling 2004; Kim and Graham 2008). Another possible pathway suggests that the shared features between red algae and other photosynthetic organisms are caused by horizontal gene transfer between cyanobacteria and primitive red and green algae, coupled with direct or indirect endosymbiosis (Chan and Bhattacharya 2013; Keeling and Palmer 2008).

The crystal structure of PSI-LHC supercomplex, reported in this communication, contains a core complex that includes twelve subunits A to F and I, J, K, L, M, and O. Very recently, a low-resolution cryo-EM structure was published revealing several distinct large complexes containing a core complex surrounded by up to eight light-harvesting complexes (Haniewicz et al. 2017). A dynamic re-adjustment of the stoichiometry of the two PSI-LHCI isomers and changes in the oligomeric state of the PSI-LHCI supercomplex, accompanied by dissociation of the PsaK subunit was reported. Our structure contains only three Lhcr subunits and there is no evidence of possible dissociation of PsaK subunit. Regardless to the extensive purification, the latter is present in all the reported crystal structures of PSI supercomplexes (Jordan et al. 2001; Ben-Shem et al. 2003; Mazor et al. 2014, 2017; Malavath et al. 2018). Obviously, our work could not rule out the existence of larger complexes with two rows of Lhcr complexes or alternative binding on PsaB at the PsaL pole. It can also not rule out a transient binding of phycobilisome proteins. The distinct position of the Lhcr complexes suggests an intermediate evolutionary step towards the arrangement of the Lhca complexes in green algae and plants.



The authors would like to thank the ESRF, SLS, and BESSYII synchrotrons for beam time and the staff scientists for excellent guidance and assistance. This work was supported by a grant (No. 293579 – HOPSEP) from the European Research Council, by The Israel Science Foundation (Grant No. 569/17), and by the I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation (Grant No. 1775/12). M.H acknowledges funding by the German Science Foundation (DFG HI 739/13.1).


  1. Adams PD et al (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D 66:213–221CrossRefGoogle Scholar
  2. Ago H, Adachi H, Umena Y et al (2016) Novel features of eukaryotic photosystem II revealed by its crystal structure analysis from a red alga. J Biol Chem 291:5676–5687CrossRefPubMedPubMedCentralGoogle Scholar
  3. Alboresi A, Le Quiniou C, Yadav SK, Scholz M, Meneghesso A, Gerotto C, Simionato D, Hippler M, Boekema EJ, Croce R, Morosinotto T (2017) Conservation of core complex subunits shaped the structure and function of photosystem I in the secondary endosymbiont alga Nannochloropsis gaditana. New Phytol 213, 714–726CrossRefPubMedGoogle Scholar
  4. Allen MB (1959) Studies with Cyanidium caldarium, an anomalously pigmented chlorophyte. Arch Mikrobiol 32:270–277CrossRefPubMedGoogle Scholar
  5. Amunts A, Drory O, Nelson N (2007) The structure of a plant photosystem I supercomplex at 3.4 A resolution. Nature 447:58–63CrossRefPubMedGoogle Scholar
  6. Amunts A, Toporik H, Borovikova A, Nelson N (2010) Structure determination and improved model of plant photosystem I. J Biol Chem 285:3478–3486CrossRefPubMedGoogle Scholar
  7. Barber J (2004) Engine of life and big bang of evolution: a personal perspective. Photosynth Res 80:137–155CrossRefPubMedGoogle Scholar
  8. Bengis C, Nelson N (1977) Subunit structure of chloroplast photosystem I reaction center. J Biol Chem 252:4564–4569PubMedGoogle Scholar
  9. Ben-Shem A, Frolow F, Nelson N (2003) Crystal structure of plant photosystem I. Nature 426:630–635CrossRefPubMedGoogle Scholar
  10. Ben-Shem A, Frolow F, Nelson N (2004) Evolution of photosystem I—from symmetry through pseudosymmetry to asymmetry. FEBS Lett 564:274–280CrossRefPubMedGoogle Scholar
  11. Busch A, Hippler M (2011) The structure and function of eukaryotic photosystem I. Biochim Biophys Acta 1807:864–877CrossRefPubMedGoogle Scholar
  12. Busch A, Nield J, Hippler M (2010) The composition and structure of photosystem I-associated antenna from Cyanidioschyzon merolae. Plant J 62:886–897CrossRefPubMedGoogle Scholar
  13. Chan CX, Bhattacharya D (2013) Analysis of horizontal genetic transfer in red algae in the post-genomics age. Mob Genet Elem 3:e27669CrossRefGoogle Scholar
  14. Chitnis PR, Purvis D, Nelson N (1991) Molecular cloning and targeted mutagenesis of the gene psaF encoding subunit III of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem 266:20146–20151PubMedGoogle Scholar
  15. Croce R, van Amerongen H (2013) Light-harvesting in photosystem I. Photosynth Res 116:153–166CrossRefPubMedPubMedCentralGoogle Scholar
  16. DeLano WL (2002) Pymol: an open-source molecular graphics tool. Scientific, San CarlosGoogle Scholar
  17. Drop B, Webber-Birungi M, Fusetti F, Kouril R, Redding KE, Boekema EJ, Croce R (2011) Photosystem I of Chlamydomonas reinhardtii contains nine light-harvesting complexes (Lhca) located on one side of the core. J Biol Chem 286:44878–44887CrossRefPubMedPubMedCentralGoogle Scholar
  18. Drop B, Yadav KNS, Boekema EJ, Croce R (2014) Consequences of state transitions on the structural and functional organization of photosystem I in the green alga Chlamydomonas reinhardtii. Plant J 78:181–191CrossRefPubMedGoogle Scholar
  19. Elrad D, Grossman AR (2004) A genome’s-eye view of the light-harvesting polypeptides of Chlamydomonas reinhardtii. Curr Genet 45:61–75CrossRefPubMedGoogle Scholar
  20. Emsley P, Cowtan K, Coot (2004) Model-building tools for molecular graphics. Acta Crystallogr Sect D 60:2126–2132CrossRefGoogle Scholar
  21. Germano M, Yakushevska AE, Keegstra W, van Gorkom HJ, Dekker JP, Boekema EJ (2002) Supramolecular organization of photosystem I and light-harvesting complex I in Chlamydomonas reinhardtii. FEBS Lett 525:121–125CrossRefPubMedGoogle Scholar
  22. Haniewicz P, Abram M, Nosek L, Kirkpatrick J, El-Mohsnawy E, Janna Olmos JD, Kouril R, Kargul JM. (2017) Molecular mechanisms of photoadaptation of photosystem I supercomplex of in an evolutionary cyanobacterial/algal intermediate. Plant Physiol 01022Google Scholar
  23. Hippler M, Reichert J, Sutter M et al (1996) The plastocyanin binding domain of photosystem I. EMBO J 15:6374–6384CrossRefPubMedPubMedCentralGoogle Scholar
  24. Hippler M, Drepper F, Rochaix JD, Mühlenhoff U (1999) Insertion of the N-terminal part of PsaF from Chlamydomonas reinhardtii into photosystem I from Synechococcus elongatus enables efficient binding of algal plastocyanin and cytochrome c6. J Biol Chem 274:4180–4188CrossRefPubMedGoogle Scholar
  25. Jensen PE et al (2007) Structure, function and regulation of plant photosystem I. Biochim Biophys Acta 1767:335–352CrossRefPubMedGoogle Scholar
  26. Jordan P, Fromme P, Witt HT, Klukas O, Saenger W, Krauss N (2001) Three-dimensional structure of cyanobacterial photosystem I at 2.5 A resolution. Nature 411:909–917CrossRefPubMedPubMedCentralGoogle Scholar
  27. Kabsch W (2010) Xds. Acta Crystallogr Sect D 66:125–132CrossRefGoogle Scholar
  28. Kargul J, Nield J, Barber J (2003) Three-dimensional reconstruction of a light-harvesting complex I-photosystem I (LHCI-PSI) supercomplex from the green alga Chlamydomonas reinhardtii – insights into light harvesting for PSI. J Biol Chem 278:16135–16141CrossRefPubMedGoogle Scholar
  29. Kargul J, Janna Olmos JD, Krupnik T (2012) Structure and function of photosystem I and its application in biomimetic solar-to-fuel systems. J Plant Physiol 169:1639–1653CrossRefPubMedGoogle Scholar
  30. Keeling PJ (2004) Diversity and evolutionary history of plastids and their hosts. Am J Bot 91:1481–1493CrossRefPubMedGoogle Scholar
  31. Keeling PJ, Palmer JD (2008) Horizontal gene transfer in eukaryotic evolution. Nat Rev Genet 9:605–618CrossRefPubMedGoogle Scholar
  32. Kim E, Graham LE (2008) EEF2 analysis challenges the monophyly of archaeplastida and chromalveolata. PLoS ONE 3(7):e2621CrossRefPubMedPubMedCentralGoogle Scholar
  33. Liguori N, Roy LM, Opacic M, Durand G, Croce R (2013) Regulation of light harvesting in the green alga Chlamydomonas reinhardtii: the C-terminus of LHCSR is the knob of a dimmer switch. J Am Chem Soc 135:18339–18342CrossRefPubMedGoogle Scholar
  34. Malavath T, Caspy I, Netzer-El SY et al (2018) Structure and function of wild-type and subunit-depleted photosystem I in Synechocystis. Biochim Biophys Acta 0–1Google Scholar
  35. Matsuzaki M et al (2004) Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature 428:653–657CrossRefGoogle Scholar
  36. Mazor Y, Nataf D, Toporik H, Nelson N (2014) Crystal structures of virus-like photosystem I complexes from the mesophilic cyanobacterium Synechocystis PCC 6803. Elife 3:e01496CrossRefPubMedCentralGoogle Scholar
  37. Mazor Y, Borovikova A, Nelson N (2015) The structure of plant photosystem i super-complex at 2.8 Å resolution. Elife 4:1–18CrossRefGoogle Scholar
  38. Mazor Y, Borovikova A, Caspy I, Nelson N (2017a) Structure of the plant photosystem i supercomplex at 2.6 Å resolution. Nat Plants 3:1–9CrossRefGoogle Scholar
  39. Mazor Y, Borovikova A, Caspy I, Nelson N (2017b) Structure of the plant photosystem I supercomplex at 2.6Å resolution. Nat Plants 3:17014CrossRefPubMedGoogle Scholar
  40. Merchant SS, Prochnik SE, Vallon O et al (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245–250CrossRefPubMedPubMedCentralGoogle Scholar
  41. Michel H, Ostermeier C Crystallization of membrane proteins. Biophys Methods 697–700 (1997)Google Scholar
  42. Minoda A, Sakagami R, Yagisawa F, Kuroiwa T, Tanaka K (2004) Improvement of culture conditions and evidence for nuclear transformation by homologous recombination in a red alga, Cyanidioschyzon merolae 10D. Plant Cell Physiol 45:667–671CrossRefPubMedGoogle Scholar
  43. Moreira D, Guyader HL, Phillippe H (2000) The origin of red algae and the evolution of chloroplasts. Nature 405:69–72CrossRefPubMedGoogle Scholar
  44. Naithani S, Hou JM, Chitnis PR (2000) Targeted inactivation of the psaK1, psaK2 and psaM genes encoding subunits of photosystem I in the cyanobacterium Synechocystis sp. PCC 6803. Photosynth Res 63:225–236CrossRefPubMedGoogle Scholar
  45. Nechushtai R, Nelson N (1981) Purification properties and biogenesis of Chlamydomonas reinhardii photosystem I reaction center. J Biol Chem 256:11624–11628PubMedGoogle Scholar
  46. Nelson N (2011) Photosystems and global effects of oxygenic photosynthesis. Biochim Biophys Acta 1807:856–863CrossRefPubMedGoogle Scholar
  47. Nelson N, Ben-Shem A (2004) The complex architecture of oxygenic photosynthesis. Nat Rev Mol Cell Biol 5:971–982CrossRefPubMedGoogle Scholar
  48. Nelson N, Junge W (2015) Structure and energy transfer in photosystems of oxygenic photosynthesis. Annu Rev Biochem 84:659–683CrossRefPubMedGoogle Scholar
  49. Nikolova D, Weber D, Scholz M, Bald T, Scharsack JP, Hippler M (2017) Temperature-induced remodeling of the photosynthetic machinery tunes photosynthesis in the thermophilic alga Cyanidioschyzon merolae. Plant Physiol 174:35–46CrossRefPubMedPubMedCentralGoogle Scholar
  50. Nozaki H, Matsuzaki M, Takahara M et al (2003) The phylogenetic position of red algae revealed by multiple nuclear genes from mitochondria-containing eukaryotes and an alternative hypothesis on the origin of plastids. J Mol Evol 56:485–497CrossRefPubMedGoogle Scholar
  51. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326CrossRefPubMedGoogle Scholar
  52. Qin X, Suga M, Kuang T, Shen JR (2015) Structural basis for energy transfer pathways in the plant PSI-LHCI supercomplex. Science 348:989–995CrossRefPubMedGoogle Scholar
  53. Reeb V, Bhattacharya D (2010) The thermo-acidophilic Cyanidiophyceae (Cyanidiales). In: Seckbach J, Chapman DJ (eds) Red algae in the genomic age. Springer Netherlands, Dordrecht, pp 409–426CrossRefGoogle Scholar
  54. Sharon I, Alperovitch A, Rohwer F, Haynes M, Glaser F, Atamaa-Ismaeel N, Pinter RY, Partensky F, Koonin EV, Wolf YI, Nelson N, Oded Béjà O (2009) Photosystem I gene cassettes are present in marine virus genomes. Nature 461:258–262CrossRefPubMedPubMedCentralGoogle Scholar
  55. Sommer F, Drepper F, Haehnell W, Hippler M (2004) The hydrophobic recognition site formed by residues PsaA-Trp651 and PsaB-Trp627 of photosystem I in Chlamydomonas reinhardtii confers distinct selectivity for binding of plastocyanin and cytochrome c6. J Biol Chem 279:20009–20017CrossRefPubMedGoogle Scholar
  56. Stauber EJ, Busch A, Naumann B, Svatos A, Hippler M (2009) Proteotypic profiling of LHCI from Chlamydomonas reinhardtii provides new insights into structure and function of the complex. Proteomics 9:398–408CrossRefPubMedGoogle Scholar
  57. Strong M, Sawaya MR, Wang S et al (2006) Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc Natl Acad Sci USA 103:8060–8065CrossRefPubMedGoogle Scholar
  58. Tan S, Ducret a, Aebersold R, Gantt E (1997) Red algal LHC I genes have similarities with both Chl a/b- and a/c-binding proteins: a 21 kDa polypeptide encoded by LhcaR2 is one of the six LHC I polypeptides. Photosynth Res 53:129–140CrossRefGoogle Scholar
  59. Thangaraj B, Jolley CC, Sarrou I et al (2011) Efficient light harvesting in a dark, hot, acidic environment: the structure and function of PSI-LHCI from Galdieria sulphuraria. Biophys J 100:135–143CrossRefPubMedPubMedCentralGoogle Scholar
  60. Tian L, Liu Z, Wang F, Shen L, Chen J, Chang L, Zhao S, Han G, Wang W, Kuang T, Qin X, Shen JR (2017) Isolation and characterization of PSI-LHCI super-complex and their sub-complexes from a red alga Cyanidioschyzon merolae. Photosynth Res 133:201–214CrossRefPubMedGoogle Scholar
  61. Umena Y, Kawakami K, Shen J-R, Kamiya N (2011) Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 A ̊. Nature 473:55–60CrossRefPubMedGoogle Scholar
  62. Vagin AA et al (2004) REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr Sect D 60:2184–2195CrossRefGoogle Scholar
  63. Vaguine AA, Richelle J, Wodak SJ (1999) SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model. Acta Crystallogr Sect D 55:191–205CrossRefGoogle Scholar
  64. Vanselow C, Weber APM, Krause K, Fromme P (2009) Genetic analysis of the photosystem I subunits from the red alga, Galdieria sulphuraria. Biochim Biophys Acta 1787:46CrossRefPubMedGoogle Scholar
  65. Zhang H, Kurisu G, Smith JL, Cramer WA (2003) A defined protein-detergent-lipid complex for crystallization of integral membrane proteins: the cytochrome b6f complex of oxygenic photosynthesis. Proc Natl Acad Sci USA 100, 5160–5163CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Maya Antoshvili
    • 1
  • Ido Caspy
    • 1
  • Michael Hippler
    • 2
  • Nathan Nelson
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life SciencesTel Aviv UniversityTel AvivIsrael
  2. 2.Institute of Plant Biology and BiotechnologyUniversity of MünsterMünsterGermany

Personalised recommendations