Structural insights into blue-green light utilization by marine green algal light harvesting complex II at 2.78 Å

Highlights

• •A marine green macroalga light-harvesting complex II Cryo-EM structure was assessed
• •Two hydrogen bonds between the carbonyl oxygen and 19-OH of siphonaxanthin were found
• •The polarity of the binding pockets of siphonaxanthin and its ester was heterogeneous
• •Structurally, siphonaxanthin should show a more redshifted absorption than its ester
• •Two established chlorophyll a binding sites were replaced by chlorophyll b.

Abstract

Light-harvesting complex II (LHCII) present in plants and green algae absorbs solar energy to promote photochemical reactions. A marine green macroalga, Codium fragile, exhibits the unique characteristic of absorbing blue-green light from the sun during photochemical reactions while being underwater owing to the presence of pigment-altered LHCII called siphonaxanthin–chlorophyll a/b-binding protein (SCP). In this study, we determined the structure of SCP at a resolution of 2.78 Å using cryogenic electron microscopy. SCP has a trimeric structure, wherein each monomer containing two lutein and two chlorophyll a molecules in the plant-type LHCII are replaced by siphonaxanthin and its ester and two chlorophyll b molecules, respectively. Siphonaxanthin occupies the binding site in SCP having a polarity in the trimeric inner core, and exhibits a distorted conjugated chain comprising a carbonyl group hydrogen bonded to a cysteine residue of apoprotein. These features suggest that the siphonaxanthin molecule is responsible for the characteristic green absorption of SCP. The replaced chlorophyll b molecules extend the region of the stromal side chlorophyll b cluster, spanning two adjacent monomers.

1. Introduction

Although sunlight is a renewable source of energy, its low photon density makes its use as a source of chemical energy difficult. Photosynthetic organisms have developed light-harvesting systems that collect and supply energy to photosynthetic reaction centers [1]. However, sunlight occasionally becomes sufficiently intense to cause damage (similar to an overdose) to photosynthetic systems [1,2]. Thus, photosynthetic organisms are required to not only use sunlight efficiently (light harvesting) but also compensate during overdose circumstances via a rapid energy dissipation system (excess energy quenching) [1,2]. Rapid energy dissipation mechanisms are collectively termed as nonphotochemical quenching (NPQ), which is dominated by energy-dependent quenching (qE) [2]. In plants, light-harvesting complex II (LHCII) is critical in light harvesting and excess energy quenching [2]. Atomic resolution X-ray structure analyses [3,4] have provided accurate coordinates of the pigments bound to the plant-type LHCII. Each monomeric protein contains the following molecules: eight chlorophyll (Chl) a, six Chl b, two lutein, one 9′-cis neoxanthin (Nx), and one violaxanthin. The function of LHCII in photosynthesis has been spectroscopically examined, and several excitonic models have been proposed [5,6] to explain the primary features of the spectroscopic results [2,[7], [8]], although some questions remain [2,5,[8], [9]]. For NPQ generation, several mechanisms have been proposed based on local interactions of Chl-Chl and Chl-carotenoid in LHCII, but whether they fully explain the experimental results is under debate, and no consensus has been reached [2,5,10]. To further elucidate the mechanism of these functions, it is promising to apply advanced spectroscopic techniques to pigment-altered LHCII based on high-resolution structural data [2,5,9].

Marine macroalgae constitute the primary producers of organic carbon in coastal ecosystems and contribute to the global sequestration of atmospheric carbon in deep sea owing to their rapid growth in numerous habitats [11]. Siphonous green macroalgae, a group of marine macroalgae, grows well in the intertidal zone [12] where sunlight conditions change regularly and sometimes drastically, from weak blue-green light to intense white light, depending on the water depth [13]. Among various siphonous green macroalgae, Codium (C.) fragile, which is native to Japan but has invaded intertidal ecosystems in harbors worldwide over the past century [12], contains a unique LHCII that efficiently utilizes the blue-green light available underwater [14], [15], [16], [17], [18]. This is because of the pigments bound to LHCII: carbonyl carotenoids siphonaxanthin (Sx) and siphonein (Sn), which are substituted with two lutein molecules, absorb green light, and a high proportion of Chl b molecules induces a larger absorption cross section for blue light [14,15]. This is collectively referred to as the Sx–Chl a/b-binding protein (SCP) [14]. However, siphonous green algae do not exhibit qE-type NPQ [19]. Moreover, as they lack photoprotective light-harvesting complex-family proteins (LHCSR and PSBS) and a light-dependent Chl biosynthetic enzyme, the photoprotective mechanisms of siphonous green algae remain largely unknown [20].

SCP differs from spinach LHCII in two aspects: a 2-nm blueshift of the fluorescence peak and the presence of the so-called long-lived Chl b responsible for an additional Qy absorption peak at 659 nm between the well-characterized Qy bands of Chl b and Chl a in the spinach LHCII. Moreover, recent two-dimensional electronic [21,22] and single molecular [23,24] spectroscopic observations have indicated that SCP demonstrates drastically different excitation energy dynamics from those of spinach LHCII. Recently, the pigment composition of SCP, especially of carotenoids, was found to be significantly altered in C. fragile depending on the intensity and color (wavelength) of the irradiated light during culture [25]. Particularly, blue-green light enhances the substitution of Sx and Sn in SCP with lutein and 19-deoxysiphonaxanthin [25]. Similar changes in pigment composition due to high-intensity light irradiation have been reported for a naturally occurring siphonous green alga, Caulerpa racemosa [26], and a photoacclimation mechanism involving Sx–lutein interconversion has been proposed [25,26]. However, the lack of information regarding the structure of SCP limits further discussion.

Here, we determined the cryogenic electron microscopy (cryo-EM) structure of SCP, purified from C. fragile grown in dim light to avoid heterogeneity in carotenoid composition, at a resolution of 2.78 Å. The locations of Sx, Sn, and the two substituted Chl b molecules in SCP were determined. The structural differences from spinach LHCII were primarily detected in the periphery of the SCP trimer. In this study, we described the main differences from the spinach LHCII that appeared in SCP, which could affect the energy dynamics.

2. Materials and methods

2.1. Sample preparation

SCP was purified from laboratory cultivated C. fragile (KU-0654, KU-MACC, Kobe, Japan) in a filamentous form as described in ref [25]. Briefly, the crude SCP band collected from the initial sucrose density gradient was purified twice using an ion exchange column chromatograph and collected again by the sucrose density gradient centrifugation. After final purification with a gel filtration column, the resultant SCP solution was concentrated to be ∼7.0 mg ml⁻¹ and frozen at 77 K until use.

2.2. Spectroscopy

Optical spectra, namely absorption, fluorescence and fluorescence excitation, and circular dichroism spectra were recorded in 20 mM Tris-HCl pH 8.2 containing 0.03% β-DDM using UV-1800 (Shimadzu, Kyoto, Japan), FP-8500 (JASCO, Tokyo, Japan), and J720W (JASCO, Tokyo, Japan), respectively (SFig. 1a−b).

2.3. High-performance liquid chromatography (HPLC) analysis

All pigments were extracted from whole cells or purified SCP, and subjected to the HPLC system as described in ref [25]. Briefly, whole pigments were extracted from a lyophilized sample and then re-dissolved into dimethyl formamide. HPLC analyses were performed using an ODS column (Cosmosil _2.5C₁₈-MS-II, 2.0 mm ID x 75 mm, Nacalai tesque, INC., Kyoto, Japan) and detected by a photodiode array detector (SPD-40M, Shimadzu, Kyoto, Japan) using gradient elution. Pigment compositions were calculated using the molar extinction coefficient given in [25,27]. The standard errors were calculated from three biological replicants (SFig 1c).

2.4. Cryo-EM data collection

A 3.0 μl aliquot of the purified SCP (∼7.0 mg ml⁻¹) was applied to a glow-discharged holey carbon grid (Quantifoil grid R1.2/1.3, 300 mesh Cu). The grid was plunged into liquid ethane cooled by liquid nitrogen using a FEI Vitrobot 4 under a following condition: blotting time 2.5 seconds, blotting force 1, sample chamber humidity 99%, sample chamber temperature 4 °C. The frozen grid was stored in liquid nitrogen before use. Data were recorded on a Thermofisher Scientific Titan Krios G3i Cryo-EM equipped with a Falcon 4 direct electron detector at the Cambridge Pharmaceutical Cryo-EM Consortium [28]. The microscope was operated at 300 kV accelerating voltage and a nominal magnification of 120 k, corresponding to a pixel size of 0.65 Å at the specimen level. The detector was operated in a counting mode. The total dose of 50.75 electrons per Ų was fractionated on 42 frames within 10 seconds of exposure time, resulting in a unit area electron dose of 1.21 electrons per Ų per frame. In total, 8935 movies were collected with defocus values varying from 0.8 to 2.2 μm using aberration-free image shift protocol (AFIS). Typical cryo-EM images averaged from motion-corrected movie frames are shown in SFig. 2.

2.5. Cryo-EM data processing

The movie frames were subsequently aligned to correct for beam-induced movement and drift using MotionCor2 [29], and the contrast transfer function (CTF) was evaluated using Gctf [30]. In total, 2,336,076 particle images were automatically picked from 8,250 images using Gautomatch (http://www.mrc-lmb.cam.ac.uk/kzhang/), and then several rounds of 2D classifications were performed using RELION-3.1 [31]. A total of 1,807,516 particles were selected for building the initial model of the SCP using cryoSPARC2 [32] and subjected to 3D classification into 10 classes using RELION-3.1 (SFig. 2). The best 3D class representing a trimmer SCP was selected for further refinement. In total, 250,633 particles were re-extracted with a pixel size of 0.65 Å/pixel and subjected to five 3D refinements, three CTF refinements, and Bayesian polishing. For the cryo-EM images of SCP, the movie frames were collected using two different cryo-EM. Since the pixel size is slightly different, the pixel size was corrected using the value of the anisotropic magnification estimated by the CTF refinement process. The corrected pixel size value was 0.662 Å/pixel. Final 3D refinement and post-processing yielded maps with global resolutions of 3.13 Å (C1) and 2.78 Å (C3), according to the 0.143 criterion of the FSC. No obvious difference could be observed between the C1 and C3 maps in terms of structural features. Since the C3 symmetrized map reduced noise level, it was used for model building. The local resolution was estimated using RELION-3.1. The processing strategy is described in SFig. 2.

2.6. Modeling and refinement

The atomic model of SCP was constructed with using WinCOOT 0.9.447 [33], Phenix 1.19-415848 [34], and both Chimera and ChimeraX [35] software packages. The coordinate of spinach LHCII (PDB: 1RWT) was directly docked as a template into the C3 symmetry imposed cryo-EM map of SCP using Chimera such that the trans-membrane helices were roughly matched to the electrostatic potential profile of the cryo-EM map. Amino acids in the template were replaced one by one using WinCOOT to fit the SCP sequence (DDBJ: LC536571). Then a rigid body refinement was performed in Chimera, forming an atomic model of the SCP. CIF files for Sx and Sn were constructed, and two lutein molecules were replaced with Sx and Sn, respectively, in WinCOOT to match the cryo-EM derived density profile (which is referred to simply as the density hereafter). Each Chl molecule obtained from the template was examined for its density to determine whether it matched Chl a or Chl b molecule, and two Chl a molecules (602 and 610) were replaced with Chl b molecules in WinCOOT. After real space refinement in WinCOOT, the geometry-optimized model was globally refined in Phenix to achieve a global resolution of 2.73 Å. After further refinement to acquire better density around Chl molecules, the global resolution became slightly poor to be 2.78 Å in the final density map. The refinement statistics are summarized in supporting material (STable 1). The refined model and its cryo-EM map were deposited in the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB) with codes 7WLM and EMD-32588, respectively.

2.7. Peptide fingerprint analyses of the C terminus of SCP

Peptide mass fingerprint analyses were performed for the SCP band after SDS-PAGE gel. In-gel digestion was performed by Digest Pro 96 (INTAVIS Bioanalytical instruments AG, Cologne, Germany) with trypsin (Sequencing Grade Modified Trypsin, Promega Corporation, Madison, WI, USA). A concentrated peptide solution was placed onto MALDI AnchorChip Targets (Bruker), and then performed MALDI-MS measurements using Autoflex Speed (Bruker, Germany). Digested peptides were assigned using Biotools ver 3.2 (Bruker) according to the SCP amino acid sequences (Lhcbm11 and Lhcbm10, DDBJ: LC536571 and LC536572, respectively). Peptide sequences for two peptide ions in the C-terminal region were assigned using MS/MS measurements (LIFT) (see SFig. 3).

3. Results and discussion

3.1. Overall structure and pigment assignment

A functionally intact SCP was prepared from cultivated C. fragile (SFig. 1). An electron potential map of SCP was reconstructed at a global resolution of 2.78 Å from 8935 cryo-EM movies of 250,633 particles (SFig. 2) (Fig. 1a–f). SCP is a trimer with a molecular weight of 118 kDa. Each monomer comprises an apoprotein (223 amino acids), pigments (3 carotenoid and 14 Chl molecules), and a lipid molecule (assuming dipalmitoylphosphatidylglycerol [PG]). We sequenced the cDNA of five lhcb-family proteins from C. fragile and identified that two proteins, Lhcbm11 and Lhcbm10, constitute the SCP trimer using peptide mass fingerprinting analysis (SFig. 3). The apoprotein in the cryo-EM map was determined to be Lhcbm11 as its density profile fits better with the amino acids of Lhcbm11 than with those of Lhcbm10 at three specific positions unique to each sequence (SFig. 4b, c). Starting from the N-terminus (Ile 1) at the stromal side, three transmembrane (A, B, and C) and two amphipathic (D and E) helices were observed in the order of B, E, C, A, and D (Fig. 1g, h). The density profile continued to the His 201 residue in the center of helix D. However, the peptide chain continues up to a Phe 223 residue, the C-terminus (SFig. 3). Although the apoprotein likely continues to the C-terminus, it is hard to directly identify it in the cryo-EM map owing to its high flexibility.

Fig. 1.

Cryo-EM structure of SCP.

Panels a−c show the SCP model surface on the cryo-EM map. Panels d−h show the model coordinates with cofactors. Panels a and d are viewed from the stromal side, panels b and e from the membrane plane, and panels c and f from the lumenal side. Each monomer is numbered from 1 to 3 in panels d–f. Panels g and h show the side views of a monomer from the outer (left) and the inner (right) sides of the trimer, respectively. The red parts represent missing densities displayed against the structure of spinach LHCII (1RWT). The figures were drawn using ChimeraX [35].

The cryo-EM map indicates the presence of 3 carotenoid molecules and 13 Chl molecules in the monomer. The Chl molecules were named after the spinach LHCII [3]. The carotenoid molecules were determined to be Sx, Sn, and Nx based on the cryo-EM map and HPLC analyses (SFig. 1c). The carotenoid-binding sites corresponded well to those of spinach LHCII: Sx binds to the inner core of the trimer corresponding to the L2 site in spinach LHCII, while Sn binds to the periphery of the trimer corresponding to the L1 site in spinach LHCII (Fig. 1g, h). For clarity, the Sx and Sn binding sites have been referred to as SxL2 and SnL1, respectively. The cryo-EM map clearly demonstrates the s-cis conformation of the carbonyl group for the polyene backbone of both Sx and Sn (SFig. 5a, b). The locations and structures of the hydroxymethyl and acyloxy groups of Sx and Sn, respectively, were clearly identified in the cryo-EM map. Notably, the amount of Sx and Sn decreased slightly during the purification process (SFig. 1c). One plausible reason for this is that the presence of the V1 site in the natural environment (see below) allows SCP to bind an additional Sx or Sn to this site. However, it was difficult to identify a carotenoid in the V1 site on the cryo-EM map. Nevertheless, biochemical analysis detected a trace amount of violaxanthin, which often occupied the V1 site in plant-type LHCII, in C. fragile unlike purified SCP (SFig. 1c).

The cryo-EM map indicated the presence of 13 Chl macrocycles located in the transmembrane region with a relatively high local resolution (SFig. 2d and SFig. 4a). Despite the intrinsic difficulty in distinguishing Chl a and Chl b in the cryo-EM map [36,37], we identified 11 Chl molecules: 4 Chl a (603, 611, 612, 613) and 7 Chl b (601, 602, 605, 606, 607, 608, 609) molecules (SFigs. 6 and 7b). For example, the Chl at site 602 was determined to be Chl b based on the presence of a distinct density profile of the C7 formyl oxygen (SFig. 7b). For the remaining two sites (610 and 604), the density profile indicated a protrusion from the 7¹ carbon toward the formyl oxygen when the cutoff level fully covered the macrocycle (SFig. 7c and d, left panels). However, after lowering the cutoff level, the protrusion remained only in Chl 610 and not in Chl 604 (SFig. 7c and d, middle and right panels). Additionally, the cutoff level of Chl 604 was lower than that of Chl 610, indicating that the former exhibited a lower local resolution (SFig. 7a). Overall, site 610 appeared to be occupied by Chl b; however, a map of a higher resolution is required for conclusive determination. In SCP, the amino acid residues at sites 602 and 610 are well conserved with those in spinach LHCII (SFig. 8) and do not provide additional hydrogen bonds to the C7 formyl oxygen of the Chl b molecules. Moreover, these sites are exclusively occupied by Chl a molecules in various LHC-family proteins, such as the green lineage Lhcb and Lhca proteins [38] and the diatom Lhcf protein [39].

The Chl a/b ratio of purified SCP was determined to be ∼0.71. This value lies between the ideal ratio of 0.75 (Chl a/b = 6/8) and 0.625 (Chl a/b = 5/8) (SFig. 1c), suggesting that some SCP lost one Chl a molecule after purification, consistent with the poor density profile corresponding to Chl 614 in spinach LHCII (indicated with red in Fig. 1g, h). Because the axial ligand (His 201) of Chl 614 was conserved in SCP, the unidentified site 614 in SCP could be occupied by a Chl a molecule.

Based on direct hydrogen bonds with cofactors (4 Chl and 1 PG molecules) and the hydrogen bond networks involved, 18 water molecules were identified. Owing to the difficulty in distinguishing water molecules from noise on the cryo-EM map [40], the X-ray crystal structure of spinach LHCII [3] was used as a reference to identify water molecules in SCP.

The overall structure of the apoprotein is almost identical to that of spinach LHCII [3], with a few differences (Fig. 2 and SFig. 8a). The Tyr 76 residue inserted at the lumenal terminus of helix B introduced a small expansion toward the outer side of SCP (Fig. 2a). The insertion of a Glu 17 residue into the N-terminal loop forced two neighboring residues to expand, protruding toward the adjacent monomer and deforming the stromal terminus of helix C (helix C' in Fig. 2b). Replacing a Trp residue with a Phe residue in a part of the “trimerization motif” could reduce the hydrophobic steric hindrance and correspondingly the repulsive force toward the hydrophilic headgroup of PG (Fig. 2c) [41]. Replacing two Leu residues with Phe in a part of the so-called “carotenoid-binding motif” [42] (Fig. 2d) and a Leu residue with a Trp residue at the center of helix D (Fig. 2e) increases hydrophobicity, stabilizing the hydrophobic Sn from both ends (See below). The outer surface of the SCP trimer, which governs the interaction of other proteins within the thylakoid membranes, was almost identical to that of spinach LHCII [3], except for a small protrusion on the lumenal side of helix B (Fig. 2a). In spinach LHCII, a Trp residue in helix C' faces the V1 xanthophyll, whereas in SCP, the corresponding Trp 118 residue is oriented in a different direction because it is shifted one residue toward the C-terminus (SFig. 8a). Conversely, the geometric relationship of helix C' in the trimer agrees well with that of spinach LHCII (Fig. 2f). This suggests that SCP has a V1 carotenoid-binding pocket with a different binding affinity from that of spinach LHCII.

Fig. 2.

Differences in coordinates between spinach LHCII and Codium SCP: enlarged views from the boxed windows (a−e).

Coordinates of spinach LHCII (cyan, 1RWT) and Codium SCP (yellow, 7WLM) were superimposed based on the A, B, and C helices using CCP4MG version 2.10.11. Panels a−f show enlarged views: a, lumenal edge of the helix B; b, proximal region of the N-terminal loop and helix C of the neighboring monomer; c, trimeric motif with proximal PG; d, e, around the 3′- and 3-hydroxy end-rings of the carotenoid, respectively; and f, a vertical projection from the lumenal side of the helix C' adjacent to a xanthophyll (Xant) associated with spinach LHCII. The figures were drawn using ChimeraX [35].

3.2. Structures of Sx and Sn and their binding environments

Sx and Sn have a carbonyl group asymmetrically attached to the polyene backbone (SFig. 5). Such carbonyl carotenoids can form an intramolecular charge-transfer (S_ICT) state [43,44]. For peridinin and fucoxanthin bound to the light-harvesting complex, the S_ICT state is well-characterized and allows highly efficient energy transfer to Chl a [7,43,44]. All fucoxanthin molecules exhibit s-trans conformation in the light-harvesting complexes of diatoms [39,45], different from the s-cis conformation of both Sx and Sn in SCP. However, the influence of the s-cis or s-trans conformation on optical properties remains unclear [46].

Comparing the Sx and Sn binding sites revealed that the SnL1 site of the latter is more hydrophobic than the SxL2 site of the former owing to the accumulation of the nonconserved amino acid residues at the SnL1 site (Fig. 3 and SFig. 8a). Nonconserved hydrophobic residues appear at the intervals of one or two residues in helix B (Val 64, Val 65, and Leu 68), in the middle of the stromal loop between helices C and A (Phe 153 and Phe 155), and in the middle of the lumenal amphipathic helix D (Trp 198) (SFig. 8a). These residues face Sn, generating a large hydrophobic cavity, which helps to preferentially and stably retain a bulky hydrophobic carotenoid. Conversely, the amino acid residues that face Sx are conserved (Fig. 3b). A relatively hydrophilic belt is observed on the protein surface of the SxL2 site along the Sx molecule because of the absence of hydrophobic side chains directed toward Sx (Fig. 3e). Furthermore, the C7 formyl oxygen of Chl b602, located between the protein surface and Sx, is proximal to the conjugate plane of Sx (Fig. 3e, Fig. 4c). Thus, we conclude that the SxL2 site is relatively hydrophilic and polarized. Since polar solvents promote the S_ICT characteristics of carbonyl carotenoids [43,44,47], Sx at the SxL2 site is expected to exhibit stronger S_ICT characteristics than Sn at the hydrophobic SnL1 site.

Fig. 3.

Siphonein (Sn) and siphonaxanthin (Sx) binding pockets (a−e) and their conformations (f).

Panels a, b, d, and e show the SCP model surfaces of the carotenoid-binding pockets of Sn (a and d) and Sx (b and e). Amino acid residues are color coded for a and b, homology to spinach LHCII (white, conserved; cornflower blue, strongly conserved; blue, weakly conserved; and midnight blue, not conserved, as in SFig. 8), and for d and e, polarity (yellow, hydrophobic; blue, hydrophilic), respectively. Panel c represents the enlarged view of the area enclosed by the dashed square in b. The hydrogen bonds are shown in broken lines with distances in Å. Panel f represents coordinates of Sx and Sn superimposed at the central part of the molecule (C10−C10′ and methyl groups therein). In d and e, Chl b610 and b602 are shown, respectively. The figures were drawn using ChimeraX [35].

Fig. 4.

Pigment arrangements at the stromal side of the trimer (a, b), and around the carotenoids (c, d).

Panels a and b represent the pigment coordinates of SCP from the stromal side. a, Each Chl molecule is indicated by a central Mg atom (gray) and two nitrogen atoms (lime green for Chl a and cyan for Chl b) along the Qy axis. The distances between the Mg atoms are shown in bold in Å. b, Putative Chl clusters are enclosed by broken lines (see text). Panels c and d represent the coordinates of Sx and Sn with the proximal Chl molecules: the closest C–C distances between each Chl macrocycle and the conjugated carbon of Sx or Sn are shown in bold in Å. The figures were drawn using Chimera X [35].

Sx accepts two newly introduced hydrogen bonds from the backbone of Trp 59 and the thiol group of Cys 67 to the 19-hydroxymethyl and the 8-carbonyl groups, respectively, in addition to the hydrogen bonds of the hydroxy groups at the rings at both ends (Fig. 3c). The observed distance of 3.6 Å between a sulfur atom of Cys 67 and the carbonyl oxygen of Sx was slightly longer than the typical hydrogen bond lengths (2.7–3.3 Å) but reasonable in terms of hydrogen bond lengths between a Cys residue and carbonyl group (3.5 ± 0. 22 Å between O and S atoms [48]). Trp 59 is highly conserved among other light-harvesting complex-family proteins, such as lhca and lhcb, whereas Cys 67 is only conserved among the trimeric lhcb proteins in vascular plants [38]. The resonance Raman spectra of SCP differed significantly from those of spinach LHCII [18]. Specifically, when excited at 441.6–488.0 nm, SCP presents a unique band at 1623 cm^–1 in a fingerprint region of C=O stretching vibrational modes from hydrogen-bonded carbonyl groups. As Sx, Sn, as well as Chl b contribute to resonant absorption in this wavelength region, this signal probably originates from the hydrogen-bonded 8-carbonyl group of Sx. Akimoto et al. [16,49,50] suggested that the unusual fluorescence anisotropy of the green band is attributable to the hydrogen bonding to the 8-carbonyl group of Sx. This observation is consistent with our findings.

Sx at the SxL2 site is distorted from C9′ to C7′ compared with Sn at the SnL1 site (Fig. 3f and SFig. 5c). The steric hindrance of Chl a603′ from the neighboring monomer may cause this distortion, as observed in spinach LHCII [51,52]. This is consistent with the results of resonance Raman spectroscopy wherein two nonequivalent Sx-type molecules resemble the optical behavior of two lutein molecules of spinach LHCII [18]. Therefore, we conclude that the redshifted absorption band at ∼535 nm is due to Sx at the SxL2 site while the band at ∼501 nm is due to Sn at the SnL1 site.

Furthermore, the conjugated chains of both Sx and Sn face the macrocycles of Chl a603 and a612 and Chl b602 and b610, respectively (Fig. 4). At distances of 3.8-3.4 Å, strong dipole interactions enhance the excitation energy transfer between them. Hence, Sx at the SxL2 site primarily absorbs green light and efficiently transfers the energy to Chl molecules through the S_ICT state. Further experiments are warranted to clarify this hypothesis.

Since the SxL2 site of SCP is identical to the L2 site of LHCII to which lutein binds, SCP can likely accept lutein at its SxL2 site. This is consistent with previous findings stating that lutein binds to SCP when the algae are grown under intense blue-green light [25]. Additionally, lutein at the L2 site (lut2) has been proposed as a quenching site in spinach LHCII [2,52], suggesting that lutein binding to SCP instead of Sx may contribute to mitigating photodamage through quenching.

3.3. Plausible generation of the intermonomeric macrocluster of Chl b at the stromal side

The structure of SCP revealed that Chl b molecules replaced Chl a molecules at sites 602 and 610 on the stromal side. However, all Chl arrangements, including the Mg–Mg distance and dipole orientation (Fig. 4a), were nearly identical to those in spinach LHCII [3]. The excitation energy transfer between Chl molecules in spinach LHCII has been well described by the concept of Chl clusters [8]. The replaced Chl b molecules approach from both sides of the b608-b609-b601′ cluster defined for spinach LHCII [[5], [8]], expanding the area of the Chl b cluster (Fig. 4b). The resultant Chl b macrocluster (610-608-609-601′-602′) spans two adjacent monomers, potentially contributing to trimer-specific energy transfer.

The Qy band energy of each Chl molecule bound to LHCII shows a specific shift from that in organic solvents due to the environmental heterogeneity of each binding site, called site energy [8]. In spinach LHCII, the calculated site energies for both 602 and 610 show a large redshift [5,6,9]. The charged residues in those binding sites, mainly responsible for the site energy shift [6], are identical to those in spinach LHCII [3] (SFig. 8). Therefore, in SCP, the extent of the redshift of the site energies of both Chl molecules at sites 602 and 610 should remain the same. Consequently, in SCP, b602 and b610 exhibit the lowest Qy energy in the Chl b macrocluster. Besides forming the Chl b macrocluster, the strongly excitonically coupled Chl a dimer (611–612) and relatively isolated Chl a (603) remain (Fig. 4b). Hence, a plausible picture is that the initially delocalized excitation energy in the Chl b macrocluster is transferred via the low-energy Chl b molecules (602 and 610) to adjacent Chl a molecules (603 and 612), respectively. The long-lived Qy state of Chl associated with absorption at 659 nm detected by low-temperature absorption [18,21,53] and two-dimensional electronic spectroscopy [21] of SCP has recently been proposed to be due to a weakly-coupled excitonic state between Chl b and Chl a molecules [22]. This would correspond to the two candidate pairs: the b602–a603 pair and/or the b610–a612 pair in SCP. Thus, SCP exhibits significantly different Chl clustering on the stromal side than spinach LHCII.

Another spectroscopic feature of SCP is the 2-nm blueshift of its emission maximum compared with that of spinach LHCII (SFig. 1a). The Chl a cluster of 610–611–612 has been attributed as the terminal emitter of spinach LHCII at the stromal side [2,5,6,9]. This cluster forms the a611–a612 dimer in SCP (Fig. 4b). The blueshift of absorption is thus attributable to the change from the Chl a cluster (610-611-612) to the Chl a dimer (611-612). However, this observation somewhat differs from that of Nguyen et al. [22]. Using spinach LHCII-based exciton models with all possible combinations of Chl a-to-b two-site substitutions, they concluded that only the substitution pair of 602 and 611 or 612 represents the blueshift of the terminal emitter and that the substitution pair of 602 and 610 does not induce a sufficient blueshift [22]. The calculated site energy is highly dependent on assumptions such as the methodology and environmental factors [7,9], and spinach LHCII-based exciton models do not include the influence of Sx and Sn, which have static dipoles and potentially large transition dipoles. Therefore, further calculation using an elaborate model based on the structure determined here is warranted to solve this slight discrepancy.

Notably, despite the apparent lack of qE-type NPQ in species containing SCP [19], some of the proposed quenching sites for spinach LHCII [[2], [8]] are conserved in SCP, i.e., the a603–b609 and a613–a614 pairs. Recently, we applied single-molecule spectroscopy to SCP [24] and observed that the single SCP exhibited a signal that somewhat resembled the “moderately redshifted” emission observed in a single spinach LHCII. This signal was observed when excited at 639 nm but not at 561 nm and was associated with a substantially reduced lifetime [24]. To explain the redshifted emission observed in the single LHCII, Krüger et al. proposed switching between two quenching sites in the lut2-a603-b609 and lut1-a610-a611-a612 domains of spinach LHCII by changing the protein conformation [[8], [54]]. The applicability of this model to SCP will be examined in follow-up experiments that are currently underway. The newly determined structure of SCP may be beneficial in improving the understanding of and give new insight into the quenching mechanism of the trimeric LHCII framework.

4. Conclusion

We successfully determined the three-dimensional (3D) structure of SCP from C. fragile at 2.78 Å resolution using cryo-EM. The overall 3D structure of SCP is almost identical to that of spinach LHCII, except for the slight modifications from the two insertions (Tyr 76 and Glu 17) and one mutation (Phe 3). The structure reveals the location of two carbonyl carotenoids, Sx and Sn and two replaced Chl-b molecules. Sx and Sn are located at sites SxL2 and SnL1, corresponding to the sites L2 and L1 in plant-type LHCII, respectively, and the Chl b molecules are located at sites 602 and 610, which were both occupied by Chl a molecules in plant-type LHCII [3,4]. Both Sx and Sn bind with the s-cis carbonyl conformation. Sx forms two hydrogen bonds with amino acids in helix B, between the C8 carbonyl group and thiol residue of Cys 67 and between the C19 hydroxy group and peptide carbonyl of Trp 59. Sx at site SxL2 is substantially distorted from C9′ to C7′, much like lutein at the L2 site of plant LHCII [3,4]. Six nonconserved hydrophobic amino acids face Sn in the SnL1 site, while the amino acids surrounding Sx are highly conserved in the SxL2 site. Additionally, the formyl oxygen in the substituted Chl b at site 602 faces Sx. These results indicate that the binding pocket of Sn is highly hydrophobic, whereas that of Sx is relatively hydrophilic. In SCP, Chl b molecules replace two Chl a molecules at positions 602 and 610 in spinach LHCII. The substituted Chl b molecules expand the Chl b cluster on the stromal side into a 610-608-609-601′-602′ macrocluster.

The high-resolution, 3D structure of SCP determined in this study provides the basis for the microscopic modeling of light (energy) absorption, excitation energy transfer, and quenching in SCP, which is required to explain spectroscopic observations. Furthermore, since SCP has somewhat different pigment than plant-type LHCII with a high-resolution structure, it will provide insight into the unresolved issues regarding Chl site energies and quenching sites in plant-type LHCII.

Author contributions

SS: Conceptualization; Sample preparation; Characterization; Modeling and refining; Writing-original draft; Writing-review and editing. TN: Conceptualization; Methodology; Writing-review and editing. PCH: Cryo-EM data collection. KS: Cryo-EM data collection. AK: Cryo-EM data processing; Modeling and refining; Writing-original draft. HT: Modeling and refining. PQ: Conceptualization; Cryo-EM data collection; Cryo-EM data processing; writing-review and editing. GK: Conceptualization; Methodology; Writing-review and editing. RF: Conceptualization; Methodology; Characterization; Writing-review and editing.

Declaration of Competing Interest

The authors declare no competing interests.

Data availability

• Data will be made available on request.