Architecture and functional regulation of a plant PSII-LHCII megacomplex

Jianyu Shan; Dariusz M Niedzwiedzki; Rupal S Tomar; Zhenfeng Liu; Haijun Liu

doi:10.1126/sciadv.adq9967

. 2024 Dec 13;10(50):eadq9967. doi: 10.1126/sciadv.adq9967

Architecture and functional regulation of a plant PSII-LHCII megacomplex

Jianyu Shan ^1,², Dariusz M Niedzwiedzki ^3,⁴, Rupal S Tomar ^5,^†, Zhenfeng Liu ^1,^2,^*, Haijun Liu ^5,^*

PMCID: PMC11640958 PMID: 39671473

Abstract

Photosystem II (PSII) splits water in oxygenic photosynthesis on Earth. The structure and function of the C₄S₄M₂-type PSII-LHCII (light-harvesting complex II) megacomplexes from the wild-type and PsbR-deletion mutant plants are studied through electron microscopy (EM), structural mass spectrometry, and ultrafast fluorescence spectroscopy [time-resolved fluorescence (TRF)]. The cryo-EM structure of a type I C₄S₄M₂ megacomplex demonstrates that the three domains of PsbR bind to the stromal side of D1, D2, and CP43; associate with the single transmembrane helix of the redox active Cyt b₅₅₉; and stabilize the luminal extrinsic PsbP, respectively. This megacomplex, with PsbR and PsbY centered around the narrow interface between two dimeric PSII cores, provides the supramolecular structural basis that regulates the plastoquinone occupancy in Q_B site, excitation energy transfer, and oxygen evolution. PSII-LHCII megacomplexes (types I and II) and LHC aggregation levels in Arabidopsis psbR mutant were also interrogated and compared to wild-type plants through EM and picosecond TRF.

Structural location of PsbR and PsbY in PSII megacomplex and their roles in higher-order structure of PSII in higher plants.

INTRODUCTION

In plant and algal cells, chloroplasts are specialized organelles responsible for carrying out a fundamental chemistry on the Earth known as oxygenic photosynthesis, by converting solar energy into chemical energy and releasing oxygen as a byproduct (1). Functioning at the early stage of oxygenic photosynthesis, photosystem II (PSII) is a multi-subunit supramolecular machinery present in the thylakoid membranes of cyanobacteria, algae, and higher plants (2). Plant and green algal PSII assembles with the light-harvesting complexes II (LHCII) to form the PSII-LHCII supercomplexes (3–5). Under diurnal and constantly changing natural light conditions, reorganization of the PSII-LHCII supercomplexes at different higher-order structural levels in the crowded thylakoid membrane is essentially required for adaptation, including numerous lateral assemblies known as the PSII-LHCII megacomplexes in diverse forms, a disordered (or random) organization and a semicrystalline array state (6–8).

Multiple types of plant PSII-LHCII megacomplexes have been observed through negative-stain electron microscopy (EM) image analysis (8–11). In plants, the most common and abundant ones are type I and type II megacomplexes consisting of two C₂S₂M or C₂S₂M₂ (C, PSII core; S, strongly associated LHCII; and M, moderately associated LHCII) PSII-LHCII supercomplexes assembled antiparallelly and through different interfacial contacts (3, 6, 9). The type I megacomplex exhibits an open structure with a central cavity surrounded by two adjacent PSII cores and two CP24 (a peripheral antenna) complexes, whereas the type II megacomplex forms a more closed structure with two CP24 complexes closely packed in the central region (3). In the semicrystalline arrays, the PSII-LHCII supercomplexes are organized in rows with a large (26.3 nm) or small (23 nm) spacing (6, 12). Both the PSII-LHCII megacomplex and the array architectures are likely induced under low-light conditions (<30 μmol photons m⁻² s⁻¹), whereas ordinary or high-light (300 or 1000 μmol photons m⁻² s⁻¹) treatment tends to trigger their rearrangements into the disordered state (10, 13). Notably, the PSII-LHCII megacomplexes and array complexes (with three units of PSII-LHCII supercomplexes) exhibit higher light-harvesting capability but lower thermal dissipation capacity than that of the monomeric supercomplexes (10).

Despite tremendous progress in structural biology studies on PSII complexes, the structural location and physiological functions of plant PsbR and PsbY (PSII subunits) remain not fully understood for decades. PsbR is a nuclear-encoded low molecular mass protein subunit found in PSII from plants and green algae (14–16) but absent in red algae and cyanobacteria (17, 18). Notably, the location and topology of PsbR subunit in thylakoid membranes are controversial. It was once considered as the fourth extrinsic protein located on the lumenal side of a plant PSII in addition to PsbO, PsbP, and PsbQ (14, 19, 20). On the other hand, a “hinge” structure positioned on the stromal surface of the stacked–PSII-LHCII supercomplexes was also suggested to belong to PsbR and connect two adjacent supercomplexes across the stromal gaps (21, 22). It might also be an integral membrane protein with a transmembrane helix (TMH) located nearby PsbE (a subunit of cytochrome b₅₅₉, Cyt b₅₅₉) (5). PsbR has a relatively short transit peptide in the N-terminal region, which is used to target PsbR to chloroplast stroma (23, 24). Functional studies on the PsbR deletion mutant of Arabidopsis thaliana (A. thaliana) and the transgenic potato with PsbR expression level reduced by antisense RNA revealed that the electron transfer from Q_A⁻ to Q_B became slower in the mutants, indicating defective PSII acceptor side (15, 25, 26) and contradicting its hypothetical lumenal location. Besides, the steady state oxygen evolution (OE) activity of PSII in the thylakoids of the psbR mutant was markedly decreased when compared to the wild type (WT) (27). The expression level of PsbP, PsbQ, and D2 subunit were also decreased in the psbR mutant in comparison to WT, especially under low light, suggesting that the PsbR subunit may have close interactions with PsbP, PsbQ, and D2 subunits and provides a docking site for the assembly of PsbP to PSII from the lumenal side (26–28). Moreover, PsbR from Chlamydomonas reinhardtii is required for binding of a photoprotective protein named LHC stress-related protein 3 (LhcSR3) to the PSII-LHCII supercomplex (16). Recent research on the PSII-LHCII supercomplex from a high light–resistant green alga, Chlorella ohadii, indicated that a PsbR homolog named Psb10 associates closely with PsbE and PsbY at a peripheral region (22).

PsbY is a 4.7-kDa protein with a single TMH located at the periphery of Cyt b₅₅₉ as shown in the structure of cyanobacterial PSII (29–32). Following the TMH, the C-terminal region of PsbY in cyanobacteria contains a short amphipathic α helix located on the stromal surface (31, 33). In plants, a single copy of psbY gene is translated into an ~20-kDa precursor polyprotein, and the polyprotein is posttranslationally processed through proteolytic cleavage into two short polypeptides, namely, PsbY-A1 and PsbY-A2 (34, 35). The exact location of plant PsbY-A1/A2 in PSII is elusive and remains to be resolved.

Here, we report the cryo-EM map and structural model of a C₄S₄M₂-type PSII-LHCII megacomplex from Spinacia (S.) oleracea at 3.22-Å resolution. The megacomplex contains two copies of PsbR and PsbY subunits each, located around the center of dimerization interface between two adjacent C₂S₂M supercomplexes. Cross-linking mass spectrometry (MS) confirmed the structural proximity of PsbR on the stromal side and provided the evidence of the PsbR-mediated higher-order organization of the stromal side–coupled PSII supercomplexes using the PSII membranes. Ultrafast time-resolved fluorescence (TRF) spectroscopy further probed the altered excitation energy transfer landscape in the PSII membrane lacking PsbR in A. thaliana. Our combined efforts provide crucial insights into the supramolecular bases for regulation of PSII light-harvesting and electron transport processes, resolving a long-standing enigma that have puzzled the community for four decades (14).

RESULTS

Overall assembly of the C₄S₄M₂ PSII-LHCII megacomplex

As shown in Fig. 1 (A and B), the C₄S₄M₂ PSII-LHCII megacomplex from S. oleracea has an overall shape matching with the type I megacomplex reported previously (3) and is composed of two antiparallel C₂S₂M supercomplexes (monomer1/monomer2) related by a C2 symmetry axis running through the center. Specific details about sample preparation and characterization, cryo-EM data collection and processing, model building, and structure refinement are summarized in figs. S1 to S3 and Tables 1 and 2. The megacomplex measures 320 Å by 290 Å by 112 Å in size and the overall molecular weight is about 2.7 MDa. Within the megacomplex, each C₂S₂M supercomplex is asymmetric and contains eight peripheral antenna complexes flanking on two sides of the PSII core dimer (C₂), including two CP29 (Lhcb4) monomers, two CP26 (Lhcb5) monomers, two strongly associated LHCII trimers (S-LHCII), one CP24 (Lhcb6) monomer, and one moderately associated LHCII trimer (M-LHCII). On the inner side facing the megacomplex center, the two C2-related PSII core monomers harbor the PsbR and PsbY subunits near the central region (Fig. 1, A and D), in addition to the other intrinsic and extrinsic subunits (Table 2). The assignment of PsbR and PsbY was made by careful fitting of the structural models with the corresponding cryo-EM densities and using the amino acid residues with bulky side chains (e.g., Trp, Tyr, or Phe) as characteristic landmarks (fig. S4). In comparison, the binding sites for PsbR and PsbY are vacant in the other two PSII core monomers located on the peripheral sides of the megacomplex (Fig. 1, A and C, and fig. S4, A and B). Putatively, the stable binding of PsbR and PsbY may rely on the formation of an enclosed local environment at the center of the megacomplex (Fig. 2A) so that their dissociation or movement is greatly restricted, plausibly free from the stripping effects of the detergents used in the membrane solubilization.

Table 1. Statistics of the cryo-EM data collection, processing, and refinement of spinach type I C₄S₄M₂ PSII-LHCII megacomplex structure.

Data collection and processing
Magnification	130,000
Voltage (kV)	300
Electron exposure (e⁻ Å⁻²)	60
Defocus range (μm)	from −1.5 to −2.0
Pixel size (Å)	1.35
Symmetry imposed	C2
Initial particle images (no.)	4,053,536
Final particle images (no.)	93,684
Map resolution (Å)	3.22
FSC threshold	0.143
Map resolution range (Å)	3.15–3.86
Refinement
Initial model used (PDB code)	3JCU, 5XNL
Model resolution (Å)	3.61
FSC threshold	0.5
Map sharpening B factor (Å)	−76.3
Model composition
Nonhydrogen atoms	340,147
Protein residues	17,264
Ligands	682
B factor (Å)
Protein	107.69
Ligand	103.11
R.m.s. deviations
Bond lengths (Å)	0.011
Bond angles (°)	1.169
Validation
MolProbity score	1.47
Clash score	3.62
Poor rotamers (%)	0.00
Ramachandran plot
Favored (%)	95.5
Allowed (%)	4.48
Outliers (%)	0.02

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Table 2. The protein and cofactor components in the final structural model of spinach type I C₄S₄M₂ PSII-LHCII megacomplex.

Subunit	Chain name	Range of amino acid residues traced	Chlorophylls	Carotenoids	Lipids	Others
PsbA	A, AA, Aa, a	10–344	16 Chl a	4 BCR	4 PG	4 OEX
			8 PHO		4 MGDG	4 FE2
					6 SQDG	8 CL
					2 DGDG	2 PL9
PsbB	B, BB, Bb, b	2–500	64 Chl a	16 BCR	10 MGDG
					2 DGDG
					8 SQDG
PsbC	C, CC, Cc, c	24–472	52 Chl a	16 BCR	2 PG
					2 MGDG
					12 DGDG
PsbD	D, DD, Dd, d	13–351	8 Chl a	4 BCR	14 PG	4 BCT
					14 PG	4 PL9
					4 MGDG	2 LMU
PsbE	E, EE, Ee, e	2–81			2 PG
PsbF	F, FF, Ff, f	12–45				4 HEM
PsbH	H, HH, Hh, h	14–72		4 BCR	4 DGDG
PsbI	I, II, Ii, i	1–35
PsbJ	J, JJ, Jj, j	4–40			2 PG
PsbK	K, KK, Kk, k	23–59
PsbL	L, LL, Ll, l	4–38			4 PG
PsbM	M, MM, Mm, m	1–33
PsbO	O, OO, Oo, o	88–330
PsbP	P, PP, Pp, p	83–269
PsbQ	Q, QQ, Qq, q	85–231
PsbR	R, RR, Rr, r	42–140			2 PG	2 LMU
PsbT	T, TT, Tt, t	1–30
PsbTn	U, UU, Uu, u	73–99
PsbW	W, WW, Ww, w	84–137			2 MGDG
PsbX	X, XX, Xx, x	72–117
PsbY	5, 55	2–41
PsbZ	Z, ZZ, Zz, z	2–65
CP24	4, 44	74–259	12 Chl a	2 LUT	2 PG
CP24	4, 44	74–259	10 Chl b	2 XAT	2 PG
CP26	S, SS, Ss, s	23–244	36 Chl a	6 LUT	2 PG
CP26	S, SS, Ss, s	23–244	16 Chl b	6 LUT	2 PG
CP29	R, RR, Rr, r	11–243	42 Chl a	4 LUT	4 PG	2 LMU
			12 Chl b	4 XAT
			12 Chl b	4 NEX
S-LHCII	G, GG, Gg, g	49–266	32 Chl a	8 LUT	4 PG
			24 Chl b	4 XAT
			24 Chl b	2 NEX
	N, NN, Nn, n	49–266	32 Chl a	8 LUT	4 PG
			24 Chl b	4 XAT
			24 Chl b	4 NEX
	Y, YY, Yy, y	49–266	32 Chl a	8 LUT	4 PG
			24 Chl b	4 XAT
			24 Chl b	4 NEX
M-LHCII	1, 11 (Lhcb1)	49–266	16 Chl a		2 PG
	1, 11 (Lhcb1)	49–266	10 Chl b		2 PG
	2, 22 (Lhcb1)	49–266	16 Chl a	2 LUT	2 PG
	2, 22 (Lhcb1)	49–266	10 Chl b	2 LUT	2 PG
	3, 33 (Lhcb3)	44–263	16 Chl a		2 PG
	3, 33 (Lhcb3)	44–263	10 Chl b		2 PG

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BCR, β-carotene; BCT, bicarbonate ion; Chl a, b, chlorophyll a, b; CL, Cl⁻ ion; DGDG, digalactosyl diacylglycerol; FE2, Fe²⁺ ion; LMU, dodecyl-α-d-maltoside; LUT, lutein; MGDG, monogalactosyl diacylglycerol; NEX, neoxanthin; OEX, Mn₄O₅Ca cluster; PHO, pheophytin a; PG, phosphatidylglycerol; SQDG, sulfoquinovosyl diacylglycerol; XAT, violaxanthin/zeaxanthin; PL9, plastoquinone 9.

Fig. 2. — (A) The central locations of PsbR and PsbY subunits in the C₄S₄M₂ PSII-LHCII megacomplex. The dash oval indicates the local regions around PsbR and PsbY subunits in the megacomplex. The dashed stocking-shaped rings indicate the vacant PsbY and PsbR sites at the peripheral regions, respectively. (B) The potential interaction network between PsbR, PsbY, and adjacent subunits. Differently colored nodes represent individual subunits with letter labels defined as follows: A, D1 (golden); C, CP43 (blue); D, D2 (tomato); E, PsbE (orange); F, PsbF (pale green); J, PsbJ (slate); P, PsbP (dark green); R, PsbR (cyan); X, PsbX (dark cyan); Y, PsbY (violet). Node sizes are approximately proportional to the chain surface areas. The solid lines indicate the major interactions of PsbR and PsbY with the adjacent subunits, whereas the dash lines suggest that they have weak interactions. (C) The cryo-EM densities and the corresponding structural models of PsbR and PsbY subunits. The densities are presented as transparent surfaces, while the structural models are shown as sticks. (D) The membrane topology of PsbR and PsbY subunits. The dash curve indicates the untraced irregular structure. (E) The interactions between PsbR and the adjacent D1, D2, and CP43 subunits. (F) The interactions between the TMH of PsbR and the TMH of PsbE. (G) The interactions of PsbR with PsbJ and PsbY. (H) The interaction between the C-terminal region of PsbR and the amino-proximal region of PsbP. All distances indicated in the panels are in angstrom.

Interactions of PsbR and PsbY with nearby PSII subunits

At the dimer interface of the type I C₄S₄M₂ megacomplex, two PsbR and two PsbY proteins fill their TMHs in a narrow central region between two adjacent C₂S₂M supercomplex (Fig. 2A). In the local region, PsbR and PsbY′ (′ indicates the protein from the adjacent supercomplex), PsbR and PsbR′, and PsbY and PsbY′ are separated by distances of 9 to 15 Å, 18 to 24 Å, and 11 to 19 Å, respectively. Evidently, the gaps are too wide for PsbR to form direct interactions with PsbY′ or PsbR′. There are, however, some weak unidentified electron densities in the gap region, presumably belonging to some lipid molecules, which may serve as bridges between PsbR and PsbY′ (or between PsbR′ and PsbY) and stabilize the assembly interface. To analyze the role of PsbR in mediating the assembly of PSII-LHCII megacomplexes, negative-stain EM analyses were carried out with the samples prepared from the psbR mutant and WT A. thaliana. In the absence of PsbR, the thylakoid membrane contains much less type I megacomplex (34.0% versus 43.9%) but more type II megacomplex (36.7% versus 13.3%) than that of the WT (fig. S5, A and B). Because of the absence of PsbR, the central cavity becomes larger and the gap between PsbR and PsbY′ in the type I megacomplex is expanded to PsbE on the side lacking PsbR and the width is increased from 14 to 25 Å, whereas the PsbY-PsbY′ gap is not much affected (fig. S5, C and D). Moreover, the distance between the two adjacent PSII dimer centers remains constant at 174 Å, indicating that the overall architectures of type I megacomplexes are similar in WT and psbR mutant (fig. S5C). On the other hand, the central gap of the type II megacomplex may become smaller in the psbR mutant as the PsbY-PsbY′ and CP24-CP24′ distances are decreased from 40 to 35 Å and 42 to 23 Å, respectively (fig. S5, E and F). Besides, the distance between the two adjacent PSII dimer centers becomes shorter, indicating that the type II megacomplex from the psbR mutant is more compact than the WT (fig. S5E). In the absence of PsbR, the two adjacent supercomplexes may form direct contacts between PsbE-PsbY and CP24′. For the type II megacomplex from the WT, PsbR instead of PsbE-PsbY directly interacts with CP24′ from the adjacent supercomplex. This is a strong indication that PsbR plays important roles in mediating the proper assembly of both type I and type II PSII-LHCII megacomplexes, either by filling in the central gap of type I megacomplex along with the other components (such as PsbY and lipid molecules) or by forming direct contacts with the adjacent subunits at the assembly interface of type II megacomplex. To further examine the role of PsbR on the organization of PSII in the grana membranes, we have performed the negative-stain EM imaging experiments using grana membranes from the leaves of the WT and psbR mutant of A. thaliana (fig. S6). Our analysis indicates that while most of the membranes have the PSII-like particles arranged in a random way (fig. S6, A and B), there are a few membrane fragments with the PSII semicrystalline arrays found in the samples from both WT and psbR mutant (fig. S6, C to E). The presence of PSII array complexes in the psbR mutant was further verified through the single-particle analysis on the negative-stain EM images of the partially purified sample from the detergent-solubilized grana membranes (fig. S6F). The grana membrane sample from the psbR mutant, however, exhibits less semicrystalline array membrane fragments than the one from the WT (fig. S6E). It suggests that formation of the PSII arrays in the psbR mutant is also affected, and PsbR may play a role in this process.

As recapitulated in Fig. 2B, the type I megacomplex has PsbR anchored to four PSII core proteins, namely, PsbA (D1), PsbD (D2), PsbC (CP43), and PsbE (the α subunit of Cyt b₅₅₉). Meanwhile, the other three subunits of PSII core, namely, PsbF (the β subunit of Cyt b₅₅₉), PsbJ, and PsbP, may also have relatively weak interactions with PsbR. Overall, the amino-proximal region of PsbR is located at the stromal side and has an extended hairpin-like domain (HPD) connected to the carboxy-proximal TMH through a short loop (Fig. 2, C and D). The HPD includes an N-terminal nine amino acid polypeptide segment (SGVKKIKVD) (24) clearly resolved in our structure. The HPD of PsbR associates with the stromal surface regions of PsbA, PsbD, and PsbC (Fig. 2E), while its TMH forms close interactions with PsbE (Fig. 2F). Besides, the conserved Asp¹⁰⁵ in the stromal loop region of PsbR likely forms a salt bridge with Arg7_PsbJ, and the van der Waals contact between Trp117_PsbR and Trp23_PsbY connects PsbR with PsbY (Fig. 2G). As a result, PsbR, PsbY, and PsbJ encircle the Cyt b₅₅₉ to shield it from the bulk lipids in thylakoid membrane. Numerous lipid molecules are trapped in the PQ-PQH₂ exchange cavity on the side with PsbR associated, whereas the cavity on the peripheral side without PsbR associated appears to be vacant (fig. S7). On the lumenal side, the C-terminal tail of PsbR clamps an amino-proximal segment (Val⁹¹–Phe⁹²) of PsbP against the C-terminal region of PsbF (Fig. 2H), enhancing the stability of PsbP-binding site on the lumenal surface. Consistently, it was observed that lack of PsbR led to drastic reduction of PsbP and PsbQ levels, and the light-saturated rate of OE declines dramatically under low-light condition (27).

PsbY has a TMH extending from the lumenal side to the stromal side. The amino-proximal region of PsbY contains a short flexible irregular structure extending toward a carboxy-proximal amphipathic α helix of PsbE on the lumenal side and interacts with the amino-proximal region of PsbP (fig. S8A). Besides, PsbE carboxyl-proximal Arg⁸¹ may interact with the PsbY amino-proximal Glu². In comparison, the shortened PsbY in PSII from cyanobacteria [Protein Data Bank (PDB): 7N8O] and unicellular algae (PDB: 8BD3) do not contain the extended N-terminal region (see alignment in fig. S8D). The functionally active cyanobacterial PSII does not contain PsbP (36). On the stromal side, the carboxy-proximal region of PsbY forms a short amphipathic α helix extending toward PsbX (fig. S8B). Flanking on one side of Cyt b₅₅₉, PsbY simultaneously forms close contacts with PsbE and PsbF (Fig. 2B). The side chain of Leu²¹, Val²⁴, and Ile²⁸ residues from PsbY may form van der Waals interactions with the heme group of Cyt b₅₅₉, potentially involved in modulating the redox potential of Cyt b₅₅₉ (fig. S8C). In psbY knockout plants, Cyt b₅₅₉ is only present in its low-potential oxidized form, and the mutants are more susceptible to photoinhibition (37). Therefore, the close interactions between PsbY and Cyt b₅₅₉ may be crucial for its redox control and photoprotection.

Arrangement of peripheral antenna complexes in the megacomplex

At the interface between two adjacent C₂S₂M supercomplexes, M-LHCII and CP24 from one C₂S₂M supercomplex make close contacts with CP26 and PsbZ from the adjacent supercomplex (CP26′ and PsbZ′), respectively (Fig. 3, A to C). Thereby, M-LHCII, CP24, CP26′, and PsbZ′ serve as bridges connecting two C₂S₂M supercomplexes so that they can assemble into one C₄S₄M₂ megacomplex. In Pisum (P.) sativum and A. thaliana, each PSII core dimer can bind up to two M-LHCII and two CP24 in addition to two S-LHCII and two CP29, forming a C₂S₂M₂ PSII-LHCII supercomplex (38, 39). When the C₂S₂M₂ PSII-LHCII supercomplexes from P. sativum and A. thaliana are superimposed on the corresponding part in the type I C₄S₄M₂ megacomplex (with their D1 and D2 proteins aligned) from S. oleracea, it is apparent that the M-LHCII of the C₄S₄M₂ megacomplex is rotated by 19° or 17° counterclockwise in the membrane plane relative to the M-LHCII trimers in the C₂S₂M₂ supercomplex from A. thaliana or P. sativum, respectively (fig. S9, A and B). Besides, CP24 also shows an obvious orientation change in the spinach C₄S₄M₂ megacomplex, namely, 9° or 13° clockwise rotation in the membrane plane relative to the position of CP24 in A. thaliana or P. sativum C₂S₂M₂ supercomplex (fig. S9, A and B). Moreover, the vertical positions of M-LHCII and CP24 in respect to the membrane normal are also slightly different from their counterparts in the A. thaliana or P. sativum C₂S₂M₂ supercomplex (fig. S9, C and D).

Fig. 3. — (A) The locations of M-LHCII, CP24, CP26, and PsbZ at the interface between two adjacent supercomplexes of the C₄S₄M₂ megacomplex. Color codes: blue green, M-LHCII; purple, CP24; pink, CP26′; blue, CP43′; gray, PsbZ′ (the ′ symbol labels the complexes/protein from the adjacent supercomplex). The red dash lines show the interfaces between the peripheral antenna complexes from two adjacent supercomplexes. (B) Interactions between M-LHCII and CP26′ from the adjacent supercomplex. (C) Interactions between CP24 and PsbZ′ from the adjacent supercomplex. (D and E) Potential energy transfer pathway between M-LHCII and CP26′ on the stromal side (D) and lumenal side (E). (F) The role of PsbZ′ in connecting CP24 with CP26′-CP43′. (G and H) Overall arrangement of Chls molecules (shown as ball-and-stick models) and potential energy transfer pathways in the stromal (G) and lumenal layers (H) within the type I PSII-LHCII megacomplex. The dashed black line defines the approximate interfaces between two adjacent C₂S₂M supercomplexes of the megacomplex. The red arrow inside the dashed red ovals indicates the potential energy transfer between two adjacent supercomplexes of the megacomplex. The black arrows indicate the potential energy transfer pathways within the individual supercomplexes.

Close contacts between M-LHCII from one C₂S₂M supercomplex and CP26′ from the adjacent C₂S₂M supercomplex may establish probable excitation energy transfer pathways between them. In the stromal layer, two chlorophylls Chl a611 and Chl b601 from monomer 2 of M-LHCII of one C₂S₂M supercomplex are likely connected with Chl a609_CP26´ from the adjacent C₂S₂M supercomplex at Mg-Mg distances of ~15 Å (Fig. 3D). At the lumenal layer, Chl b606_CP26′ and Chl b607_CP26′ are connected to Chl a614_M-LHCII at Mg-Mg distances of 15 and 17 Å, respectively (Fig. 3E). While Chls b may serve as an excitation energy donor for the receiving Chl a, they do not function as efficient bridges in mediating excitation energy transfer between adjacent antenna complexes (40). Meanwhile, the Chls from CP24 are separated by PsbZ from their counterparts in CP26′ and CP43′ of the adjacent supercomplex. Those are too distant (Mg-Mg distance of 27 to 30 Å) to enable efficient excitation energy transfer (Fig. 3F). Therefore, the Chl a611_M-LHCII–Chl a609_CP26′ connection is the only pair of the interfacial Chl a molecules potentially functioning in mediating excitation energy transfer between two adjacent C₂S₂M supercomplexes (Fig. 3, G and H).

Supramolecular basis for electron transport regulation

As shown in Fig. 4 (A to C), the two Q_B sites in the C₂S₂M supercomplex of the type I C₄S₄M₂ megacomplex exhibited evident differences as they have distinct local environments. The Q_B site in the CSM monomer is in an enclosed local environment surrounded by PsbY, PsbR, PsbX, PsbJ, CP29-CP24 heterodimer, and the C′S′M′ monomer from the adjacent supercomplex. A well-resolved PQ molecule density is present at the Q_B site of the CSM monomer of the C₂S₂M supercomplex (Fig. 4C). The PQ head group is surrounded by the side chains of Leu²¹⁸, Leu²⁷¹, and Phe²⁵⁵ from D1 and forms van der Waals contacts with them. His²¹⁵ and Ser²⁴⁶ of D1 protein form hydrogen bonds with the head group of PQ molecule (Fig. 4D). The phytyl tail extends to the interface between two adjacent supercomplexes through a side portal between PsbF and the first TMH of D2 protein. A lipid molecule (PG2632) with its head group attached to the stromal domain of PsbR forms hydrophobic interactions with the tail of PQ molecule at the Q_B site, stabilizing it in the position (Fig. 4E). It appears that the stromal domain of PsbR caps over the PQ/PQH₂ exchange cavity and forms close interactions with the lipid molecules inside the cavity. In comparison, the Q_B site in the CS monomer is located on the other side exposed to detergent (or lipid) environment. There is neither PQ-like density observed at the Q_B site of CS monomer nor those of PsbR residues (Fig. 4B).

As for the Q_A site in the CSM and CS-PSII monomers, these are both occupied by PQ molecules and the local environments are overall very similar (fig. S10). While the amino acid residues (Trp253_D2, Thr217_D2, and His214_D2) interacting with the head group of the PQ molecule exhibit similar positions at the two Q_A sites, Phe261_D2 near the Q_A site in the CSM monomer shows slight orientation change and becomes closer to the head group of PQ than Phe261_D2 in the CS monomer. Met246_D2 in the CSM monomer becomes slightly more distanced from the head group of PQ than the one in the CS monomer (fig. S10). Such differences suggest that the Q_A site is likely flexible and may slightly adjust its position according to the status of PQ molecule trapped inside the pocket.

Previously, it was demonstrated that both PQ and the semiquinone radical (PQ^•−) bind to the Q_B site more favorably than the product (PQH₂), the release of which, however, is more thermodynamically favorable (41). Therefore, the Q_B sites in the CSM monomers of the C₄S₄M₂ megacomplex are most likely occupied by PQ and/or PQ^•−. Normally, after reduction of PQ to PQH₂ at Q_B site, it diffuses to PQ pool in a microdomain surrounded by PSII, LHCII, Cyt b₆f complexes, and lipids, where exchange of PQH₂ with the oxidized PQ from the PQ pool occurs (42). Nevertheless, our data indicate that on the CSM side, at the interface between the two adjacent supercomplexes, PsbR, PsbY, PsbZ, antenna complexes (CP24 and CP29), and the two PSII cores from two adjacent supercomplexes in the type I megacomplex, collectively form a closed membrane island (Fig. 4A). In the local area, the diffusion and exchange of PQH₂ with PQ might be greatly hindered, unless the abovementioned island opens up along the dimer interface (Fig. 1A, black dash line) to allow in and out of the oxidized and reduced PQs.

How is the oxygen-evolution activity of PSII in the megacomplex compared to that of the supercomplex sample where both Q_B sites are open for PQ/PQH₂ exchange? The oxygen-evolution activities of the megacomplex are much lower than those of PSII-LHCII supercomplex. The maximal oxygen-evolution activity of the PSII-LHCII megacomplex [163 ± 11 μmol O₂ (mg Chl)⁻¹ hour⁻¹] is evidently lower than that of the PSII-LHCII supercomplex [327 ± 14 μmol O₂ (mg Chl)⁻¹ hour⁻¹] (Fig. 4F and Table 3). The result suggests that about half of the PSII core in the megacomplex sample is inactive, probably because of the restricted access of 2,6-dichloro-1,4-benzoquinone (DCBQ), an artificial electron acceptor, to the Q_B site in the interface membrane island (Fig. 4A). Moreover, the megacomplex exhibits lower F_v/F_m value than that of the supercomplex sample, suggesting that a portion of PSII reaction centers in the megacomplex fails to perform charge separation and is photoinhibited (43) (Table 3), presumably because of the altered Q_B sites around the megacomplex center.

Table 3. Functional characterization of the PSII-LHCII megacomplex (PSII-MC) and supercomplex (PSII-SC) samples.

	PSII-SC	PSII-MC
OE_max [μmol O₂ (mg Chl)⁻¹ hour⁻¹]	327 ± 14*	163 ± 11
F_v/F_m	0.70 ± 0.03	0.58 ± 0.03

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*Mean ± SEM (n = 5 for PSII-MC or 6 for PSII-SC)

The flash-induced fluorescence relaxation kinetics of the PSII-LHCII megacomplex differs from that of the PSII-LHCII supercomplex (Fig. 4G and Table 4). The decay kinetic curves of both samples could be fitted with a similar set of three exponentially decaying phases that reflect the functional status of the electron acceptor and donor sides of PSII (44, 45). Evidently, the lifetimes and amplitudes of the three phases are different between the two samples. The fast decay phase, corresponding to the forward electron transfer from Q_A⁻ to oxidized Q_B, for the PSII-LHCII megacomplex has shorter lifetime but higher amplitude (τ_fast = 0.35 ms, A_fast = 60.5%) than the one for the supercomplex (0.40 ms, A_fast = 56.1%). The middle decay component corresponds to the forward electron transfer from Q_A⁻ limited by the binding of a plastoquinone to the Q_B site when the Q_B site is unoccupied. While the PSII-LHCII megacomplex sample exhibits a lifetime of the middle exponential decay component (τ_fast = 6.53 ms) slightly longer than that of the supercomplex sample (6.03 ms), its amplitude (A_middle = 14.0%) is lower than that of the PSII-LHCII supercomplex (20.2%). For the slow decay phase corresponding to a charge recombination reaction of Q_A⁻ with the S₂ states of the O₂-evolving complex, the lifetime and amplitude for the PSII-LHCII megacomplex (τ_slow = 101 ms, A_slow = 13.8%) are longer and lower than those of the PSII-LHCII supercomplex (τ_slow = 79.7 ms, A_slow = 19.5%) respectively. Notably, the forward electron transfer from Q_A⁻ to oxidized Q_B is the major component of the decay kinetics of the PSII-LHCII megacomplex, and it occurs faster and at higher amplitude than the one of the PSII-LHCII supercomplex (Fig. 4G and Table 4). Besides, the residual fraction of the fluorescence yield (11.7%) for the megacomplex is larger than that of the supercomplex (4.2%), suggesting that the equilibrium between Q_A⁻ and Q_B is more pronounced in the megacomplex sample. The result is consistent with the cryo-EM observation that half of the Q_B sites in the PSII-LHCII megacomplex are occupied by PQ (or PQ^•−).

Table 4. Flash-induced fluorescence relaxation kinetics of the PSII-LHCII megacomplex (PSII-MC) and supercomplex (PSII-SC) samples.

	A_fast(%)	τ_fast(ms)	A_middle(%)	τ_middle (ms)	A_slow (%)	τ_slow	A_Residual (%)
PSII-SC	56.1 ± 1.1*	0.40 ± 0.01	20.2 ± 0.4	6.03 ± 0.19	19.5 ± 0.1	79.7 ± 2.1	4.2 ± 1.6
PSII-MC	60.5 ± 2.6	0.35 ± 0.02	14.0 ± 0.7	6.53 ± 0.60	13.8 ± 0.3	101 ± 11	11.7 ± 3.6

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*Mean ± SEM (n = 3)

Flexible stromal domain of PsbR as probed with cross-linking MS

The irregular secondary structures in the stromal HPD domain of PsbR (Fig. 2, C and D) prompted us to perform bioinformatic analysis on the protein. It revealed that PsbR has an overall high content of disorder-promoting amino acid residues, namely Gly (>18%) and Ser (>7%) (fig. S11), a signature of intrinsically disordered proteins/regions (IDP/Rs) (46). An IDP/R is defined as a protein that lacks a unique fold, either entirely or in parts when isolated in solution, typically in the absence of its macromolecular interaction partners, such as other proteins or RNA. Besides, IDP/Rs exist as dynamic ensembles of rapidly interconverting conformers in equilibrium (47, 48). To test whether the PsbR structure observed in the PSII-LHCII megacomplex (Fig. 1A) represents a unique or universal entity associated with various PSII complexes, we performed the chemical cross-linking reactions (isotopically encoded BS₃-H12/D12) using the BBY particles [from Berthod, Babcock, and Yocum—names of the authors of the original preparation protocol that has led to many major discoveries of PSII (49)] and interrogated the cross-linked products using liquid chromatography tandem MS (LC-MS/MS). The PSII-enriched grana membranes (BBY particles), if isolated, are routinely used to further separate various PSII supercomplexes with different antenna size and functionality (50), simply owing to its heterogenous nature. In higher plant thylakoids, the heterogenous distribution of photosynthetic protein complexes is a determinant for the formation of grana, stacks of membrane discs that are densely populated with PSII and LHCII (21). Numerous biochemical and structural research works support several levels of PSII heterogeneity, such as antenna size heterogeneity and reducing side heterogeneity (6, 51–53).

Chemical cross-linkers covalently link amino acid pairs found in proximity to each other in a protein or a protein complex. It is particularly useful to capture protein ensemble structures that contain large loop regions that are recalcitrant for x-ray crystallography and cryo-EM studies (54). Table S1 tabulates the detailed MS1 (precursor ion) information of the identified cross-links and loop-links. In short, three cross-links and three loop-links were identified with high confident isotopically encoded features from the cross-linker (table S2). Figures S12 and S13 show the representative MS1 and MS2 quality spectra of two cross-links. Firstly, Lys51_PsbR and Lys75_PsbR were found cross-linked to the N terminus of Ser² of PsbE, which is located on the stromal side of PSII, indicating that a large portion of the N-terminal domain (NTD) of PsbR is located on the stromal side, consistent with the cryo-EM structure (Fig. 1A). The distance between two primary amines from Lys51_PsbR and the N-terminal Ser2_PsbE in cryo-EM structure is ~11.1 Å (fig. S14), matching with the cross-linking chemistry of BS₃ cross-linker (spacer arm length of 11.4 Å). However, the distance between Lys75_PsbR and Ser2_PsbE is ~30 Å, much larger than the spacer arm of the cross-linker (fig. S14), and the apparent spatial conflicts seem to preclude the cross-linking chemistry from occurring. Note that these measurements were based on our cryo-EM structure of the PSII-LHCII megacomplex, while the cross-linking MS was indeed based on BBY PSII particle, which contains more heterogeneous PSII. The apparent contradictive results of our cross-link Lys75_PsbR and Ser2_PsbE versus the cryo-EM data suggest that there might be alternative models of PsbR in the BBY particles in addition to the one that has been isolated and resolved using the cryo-EM method (Fig. 1). On the basis of the cryo-EM structure and guided by our MS data, we propose a stromal side–coupled PSII supercomplex organization model (Fig. 5). In this model, the stromal domain of PsbR comprises the major contact region for two PSII supercomplexes from the oppositely stacked thylakoid membrane (Fig. 5A). The distance of the solvent accessible Lys75_PsbR and Ser2_PsbE is 16.5 Å; from two PSIIs that are located across the stromal gap, respectively, no apparent spatial conflicts are observed, agreeing with the cross-linking chemistry (Fig. 5B). This dimeric PsbR orientation model is also consistent with the observed 22 kDa cross-linked species reported in a recent study of spinach PSII BBY particles (20). The orientation of two oppressed PSII from adjacent layers may vary, and the interactions between such PSIIs from the two stacked membranes may not be very specific (12). However, the cross-link chemistry can lock such interactions and allow us to interrogate the spatial relationships of the binding partners using LC-MS/MS. Other groups have used nonisotopically encoded chemical cross-linker to interrogate the trans-partition structure of PSII-LHCII supercomplex. A model was subsequently proposed and supported by their cross-linking data (55).

Fig. 5. — (A) Side view of the C₂S₂M megacomplex stacked with another PSII from the opposite thylakoid membrane. (B) The close distance, between K75_PsbR from top PSII and S2_PsbE from lower PSI in an enlarged view (16.5 Å), is consistent with the experimental data (XL-MS) (fig. S13). Color code: PsbR (pink and cyan from top and bottom, respectively, in the stacked membranes), PsbE (orange), all other PSII components (wheat and lime, respectively).

Loop-links are cross-linked products connecting two close amino acid residues within one single peptide (56, 57). These are less important for probing nearest neighbor analysis between two proteins/subunits; however, they provide constrain or proximity information of different domains of one protein. We noticed that two identified loop-links of PsbR (i.e., Lys⁴⁸–Lys⁵¹, Lys⁷¹–Lys⁷⁵; tables S1 and S2) are consistent with the cryo-EM data (fig. S15). Identification of Lys⁵¹–Lys⁶⁰ indicates structural proximity between these two amino acids. The distance between them in PsbR in the cryo-EM structure is ~20.8 Å with serious spatial conflicts (fig. S15B). The discrepancy indicates that, in addition to the structure of PsbR observed in the megacomplex, PsbR may adopt a different conformation in the other PSII units in the BBY particles in which Lys⁵¹ and Lys⁶⁰ are in proximity that enabled the cross-linking chemistry to occur and be identified using our MS system. These experimentally observed constraints are consistent with some AlphaFold2-generated models (e.g., fig. S15C). The structural repertoire of PsbR in PSII-enriched BBY particles may seem more complex and awaits future research effort.

Evaluation of functional roles of PsbR in PSII membrane via static and time-resolved optical spectroscopies using PsbR deletion mutant and WT

As shown in figs. S5 and S6, deletion of PsbR subunit has a meaningful effect on heterogeneity of the PSII-LHCII megacomplexes, particularly affecting stoichiometry of type I and II megacomplexes, morphological property of the individual megacomplex type due to spatial rearrangement of LHCs and core complexes, and formation of semicrystalline PSII arrays as well (fig. S6). Type I megacomplex from the psbR mutant shows weaker spatial coupling of two supercomplexes (fig. S5D) but, on the other hand, type II shows slightly stronger coupling between supercomplexes and better alignment between peripheral LHCs (fig. S5, E and F). The latter one should lead to more efficient excitation energy transfer through the LHC network toward core complexes. The question that we asked is whether these changes could also be spectroscopically investigated by some means as changes brought by mutation, especially in spatial arrangement of LHCs, likely influence the excited state properties of Chls. These aspects were investigated with application of absorption and fluorescence spectroscopies at 77 K. To maintain the native nature of the samples, the experiments were carried out on BBY particles. Because of the heterogenous nature of the BBY particles, it is expected to observe sort of average effect from all mutation-related changes (type I/II stoichiometry, changes in the morphologies of the same megacomplex type, and other possible but unaccounted for effects). Therefore, some rebalance in fluorescence emission primarily from LHCs should be seen because of spatial reorganization of LHCs in the megacomplexes in the mutant BBY particles. Simultaneously, changes in the sample’s absorption due to altered aggregation states of LHCs may be detectable. However, because of competing partial contributions from both types of megacomplexes, it is difficult to predict a priori if the mutant BBY shows higher or lower aggregation state.

Condensed overview of spectroscopic studies is shown in Fig. 6. The data reveal that absence of PsbR is associated with a decreased level of complexes aggregation. This is confirmed by our negative-stain EM imaging analysis using PSII membranes (fig. S6). The WT-minus-ΔPsbR absorption difference spectrum (Fig. 6A) shows two distinct positive bands at 505 and 684 nm, spectral features characteristic of higher aggregation level of LHC complexes in the membrane, as previously demonstrated for model studies of LHCII (58, 59). The pseudo-color map of TRF decay of Chls a (Fig. 6B) and the subsequent global spectro-kinetic analysis (Fig. 6, C and D) demonstrated rebalancing in the spectro-temporal components indicative of better energetic coupling between LHCs (more efficient excitation energy transfer) and/or partial de-oligomerization of LHC in the ΔPsbR BBY particles (60) (for details, refer to figs. S16 and S17 and the associated supplementary description).

DISCUSSION

In the type I C₄S₄M₂ PSII-LHCII megacomplex from spinach, multiple pairs of closely connected Chl molecules have been found at the interface between M-LHCII from one supercomplex and CP26′ from the adjacent supercomplex (Fig. 3, G and H). Among them, Chl a611_M-LHCII–Chl a609_CP26′ may serve to mediate excitation energy transfer between two adjacent supercomplexes. Previous studies indicated that low light might induce formation of semicrystalline PSII arrays to overcome potential PQ diffusion problems in the crowded membrane (13), and the C₂S₂M₂ supercomplexes are packed in the semicrystalline arrays more tightly in low-light plants than in normal-light or high-light plants to facilitate energy transfer between supercomplexes (61). As a fraction of the semicrystalline PSII arrays, the PSII-LHCII megacomplexes mainly exist and function under low-light and normal-light conditions (10, 61). While the semicrystalline arrays of C₂S₂M₂ and C₂S₂-type supercomplexes were also observed in the grana membranes of high-light plants, they are less abundant than those found in the grana membranes of low-light and normal-light plants (61). The formation of megacomplexes and further assembly into the semicrystalline array may dramatically increase the light harvesting cross section for each PSII so that it can harvest photon energy more efficiently under low-light conditions. Despite that the peripheral antenna system for PSII are expanded, a fraction of PSII reaction centers may still be idle most of the time when the photon flux is low and due to the rate-limiting steps of photon absorption, excitation energy transfer, and trapping processes (6). In this case, the electron transport process in the PSII-LHCII megacomplexes may be regulated accordingly to avoid random distribution of energy. The excitation energy might be directionally transferred to the PSII reaction centers in the outer region (edge) of the megacomplex where the exchange of PQH₂/PQ with Cyt b₆f is not spatially/physically hindered, as in the case of the CS-PSII monomer (Fig. 4A, the exposed Q_B site in the blue dashed oval in the silver region versus the blocked Q_B site in the red dashed oval in the green region).

Our TRF data clearly indicate that the PSII and LHCII aggregation state is altered in the absence of PsbR (reduced PsbY as well). We propose that this effect could be more related to the fact that presence of (a pair of) PsbR (and PsbY as well) aligns two adjacent PSII dimers so that the space between them allows M-LHCII–CP24 from one supercomplex and CP26′-PsbZ′ from the adjacent supercomplex in the megacomplex to bind (Fig. 1A). Absence of PsbR, however, may disrupt or weaken the integrity of the interface between two PSII dimers (fig. S5, C to F); alter the spatial accommodation of CP26-PsbZ, M-LHCII–CP24, and their symmetry-related counterparts along the axis region (Fig. 1A, the dark dash line); affect the formation of semicrystalline PSII arrays (fig. S6, A to E); and subsequently destabilize the overall interaction network in the grana membrane.

Previous EM and biochemical studies demonstrated that the protein density of the intact grana thylakoid membranes has been maintained at a specific value (62) so as to achieve efficient light harvesting for PSII photochemistry. Reduction of the protein packing density below a critical point improves the light-harvesting functionality of PSII, while further dilution to such value leads to functional disconnection of LHCII from PSII (63). Diffusion of proteins (e.g., PSII, LHCII, and Cyt b₆f) and mobile electron carriers (e.g., PQ) is fairly restricted in such a crowded membrane environment (62). It was proposed that PSII, LHCII, and Cyt b₆f complexes will likely form a closed diffusion space known as the microdomains for diffusion of PQ (42, 62, 64). For the linear electron transport process, the slowed reoxidation of PQ by Cyt b₆f complex is the major limiting step in the electron-shuttling process (42). Under low-light conditions, PSIIs tend to rearrange from the monomeric supercomplexes into the megacomplexes and semicrystalline arrays (13). The PSII cores on the inner side near the assembly interface are likely arrested at an inhibited or closed state because the diffusion of the PQ/PQH₂ is dramatically hindered by the occluded environments around the Q_B sites. From the point of view on light harvesting, however, these PSII cores, together with its associated LHCs, may behave as one light-harvesting unit to capture and transfer the excitation energy to the PSII cores located in the peripheral region of the PSII membrane region (Fig. 4, A and B, blue dash oval) where photochemistry takes place sustainably, since the exchange of PQH₂/PQ with the PQ diffusion microdomain is likely not limited with Cyt b₆f complex in vicinity. This adaptive low-light scheme strategy enables the linear electron transport to occur at the interface between PSII and Cyt b₆f under limited light conditions at the expense of shutting down half of the PSII cores of the megacomplexes (or a portion of the PSII enriched membrane). The number of the active PSII cores available for the linear electron transport process, however, is thereby down-regulated under low-light conditions.

CP24 has a crucial role in mediating the assembly of type I and type II PSII-LHCII megacomplexes (Fig. 3A and fig. S5, C to F). In high-light plants, the contents of CP24 and Lhcb3 (a component of M-LHCII involved in binding CP24 and CP29) are reduced to ~50% of those in the normal-light plants, and semicrystalline PSII arrays composed of the C₂S₂-type supercomplexes besides those of C₂S₂M₂ supercomplexes were observed with low frequency (61). In the CP24-knockout (koCP24) plants, the C₂S₂M₂ supercomplexes are absent and the C₂S₂ supercomplexes form ordered arrays in the grana membranes (65, 66). A Chl b (Chl b614) molecule at the C-terminal region of CP29 is crucial for the formation of the C₂S₂M₂ supercomplex by interacting with CP24, and the mutant plants lacking Chl b614 in CP29 (CP29-H242L) also contain semicrystalline PSII arrays of the C₂S₂-type supercomplexes in the grana membranes as in the koCP24 plants (67). Both the koCP24 and CP29-H242L mutant plants exhibit decreased electron transport rates and restricted electron transport from Q_A to Q_B, likely due to the limited PQ diffusion to the Q_B site in the ordered PSII arrays (65, 67). The mechanism underlying the PQ diffusion restriction in the C₂S₂ arrays remains unclear and needs to be investigated further in future works.

Like other intrinsic membrane subunits of PSII, PsbR contains three domains, namely, the stromal N-terminal domain (NTD) (large portion), a transmembrane α helix, and a small lumenal C-terminal domain (CTD). PsbR-CTD, although short, indeed provides docking sites for the lumenal domain of PsbE and PsbP (Fig. 2G). The absence of PsbR (CTD) apparently abolishes such stabilization effects, which eventually provide a reasonable explanation for the dramatically decreased level of PsbP and PsbQ in the literature (26–28). The increased amount of dark oxidized Cyt b559 in a previous report (25) can also be interpreted by the absence of the PsbR transmembrane α helix that interacts with and stabilize the TMH of PsbE. Note that both PsbR and PsbY are closely associated with the TMH of PsbE (Fig. 2G). The absence of PsbY also results in the elevated oxidized form of Cyt b559 and in the decreased expression level of PsbR as well (37). PsbR-NTD, accounting for ~80% of the PsbR total length, putatively plays important roles in optimizing the electron transfer from Q_A to Q_B by capping the Q_B cavity from the stromal side through interactions with D1, D2, PsbE, and CP43, providing the structural basis for several previous research (15, 26, 27).

Under low-light conditions, PSIIs may rearrange from the monomeric supercomplexes to the megacomplexes, which could block the exchange of PQH₂ with the free PQ pool for oxidation. While the danger of charge recombination within PSII, concomitant production of singlet oxygen and photoinhibition increases, Cyt b559 protects PSII from photoinhibition by functioning as a PQH₂ oxidase (68, 69). Notably, the increased level of oxidized Cyt b559 was observed in the absence of PsbR/PsbY in both PsbR and PsbY mutants (25, 37). It was found that plant Cyt b559 in the native PSII membranes exhibits variable redox potentials including the high-, intermediate-, and low-potential forms (70), whereas cyanobacterial Cyt b559 is mainly present as a low-potential form in the thylakoid membrane and lacks the high-potential form found in higher plants (71). The formation of PSII megacomplexes in plants generates a specific local environment around Cyt b559, and it is potentially related to the shift of its redox potential. For future studies, it will be interesting to characterize the redox level of Cyt b559 that are located at the position where PsbR/PsbY is present or absent respectively in the PSII-LHCII megacomplex when it becomes technically feasible. On the basis of current observation, we propose that the Cyt b559–mediated photoprotective activity in the different PSII units of the PSII-LHCII megacomplex could be different because of the absence/presence of PsbR/PsbY.

The PsbR protein in the type I C₄S₄M₂ PSII-LHCII megacomplex from spinach contains an intrinsically disordered region (IDR) at the amino-proximal region followed by a TMH and the short carboxy-proximal domain. Despite a large portion of irregular secondary structures observed in PsbR, they may serve versatile functionalities (72). For instance, a functional IDP protein that exists at a multitude of various conformational states allows it to interact promiscuously with many different partners. Studies have shown that proteins containing IDRs hold central roles in protein interaction networks, specifically acting as a hub protein within the nucleus and enabling molecular communication via protein-protein interactions. For the versatile irregular secondary structures in PsbR, it indeed forms a cap on the stromal surface above the PQ/PQH₂ exchange cavity and may be involved in stabilizing the lipid molecules within the cavity so that the exchange of PQH₂ in Q_B site with external PQ is inhibited or slowed down. Moreover, our cross-linking MS results strongly support that the irregular secondary structures in the IDR of PsbR may mediate the stacking of two PSII from two appressed thylakoid membrane region. Such conformational variability might be crucial for mediating the dynamic assembly of the two closely stacked PSII complexes in the life cycle of grana thylakoid membranes.

METHODS

Thylakoid membrane preparation and purification of PSII-LHCII megacomplexes

Fresh spinach leaves were obtained from a local market and stored at 4°C in a dim room before use to reduce the starch content and acclimate the leaves under low-light conditions. Intact thylakoid membranes were prepared as described in a previous report (73). The thylakoid membranes were suspended in a storage buffer solution containing 50 mM MES-NaOH (pH 6.5) and 0.2 M sucrose. The samples were frozen in liquid nitrogen and stored at −80°C until next use. For further preparation, the thylakoid samples were thawed and washed once by centrifuging it at 21,100g at 4°C for 10 min (Legend Micro 21R Centrifuge, Thermo Fisher Scientific, Waltham, MA), and the pellets were resuspended in a resuspension buffer with 50 mM MES-NaOH (pH 6.5) for further experiments. For purification of the PSII-LHCII megacomplexes, the thylakoid membrane sample at 1 mg Chl ml⁻¹ concentration was solubilized with dodecyl-α-d-maltoside (α-DDM, 2.5% final concentration) on ice in a dark room for 5 min and then centrifuged at 21,100g at 4°C for 5 min to remove the insoluble components. The pellets were discarded, and the supernatant was collected for further application in the GraFix procedure through an ultracentrifugation step with the sample centrifuged into a gradient with increasing concentration of a chemical fixation reagent for stabilization of the complexes (74). For the GraFix experiment, the sucrose density gradient (SDG) solution contain 10 to 40% sucrose, 50 mM MES-NaOH (pH 6.5), 1 M betaine, 0.02% α-DDM, and 0 to 2 mM glutaraldehyde was used. After the solubilized thylakoid samples were applied on the top of SDG tubes, they were ultracentrifuged at 256,000g at 4°C for 18 hours (Beckman Optima XPN-100 Ultracentrifuge, SW40 rotor). PSII particles (BBY particles) were prepared as previously described (49).

WT and psbR mutant plants of A. thaliana were grown at 24°C with a light-dark period of 8-hour light (100 μmol photons m⁻² s⁻¹) and 16-hour dark. After growing for 6 weeks in soil, the leaves were harvested for preparation of the megacomplex samples. About 10 g of leaves per batch were ground in liquid nitrogen, thawed, and suspended in 100 ml of lysis buffer [20 mM tricine-KOH (pH 7.8), 400 mM NaCl, 5 mM MgCl₂, 0.2 mM benzamidine, and 1 mM ε-aminocaproic acid]. The thylakoid membranes were collected as pellets by centrifuging the suspension at 1400g for 10 min, resuspended in the storage buffer, frozen in liquid nitrogen, and stored at −80°C. The subsequent protocols for solubilizing the thylakoid membranes and purification of the PSII-LHCII megacomplex are the same as described above.

The PSII-enriched membrane (BBY particle) samples from WT and psbR mutant of A. thaliana were prepared as previously described (61). The leaves were adapted under dark overnight before being used for sample preparation. The intact thylakoids obtained as described above were homogenized in a resuspension buffer [25 mM Hepes-NaOH (pH 7.5), 15 mM NaCl, and 5 mM MgCl₂] in advance. The PSII-enriched membrane was purified from the intact spinach thylakoid sample by solubilizing it with 0.4% α-DDM (per 1 mg Chl ml⁻¹). After being incubated at 4°C for 20 min, the mixture was then centrifuged at 3500g at 4°C for 5 min to remove the pellets. The supernatant was collected and further centrifuged at 21,100g at 4°C for 3 min. Afterward, the pellets were collected and resuspended in the resuspension buffer for further preparation of the negative-stain EM samples.

Electrophoresis and immunoblotting

After the SDG ultracentrifugation, the relative amount of each individual fractions in SDG were extracted and quantified through the piston gradient fractionation system (Biocomp Instruments, Fredericton, Canada). The components in the B6 and B7 fractions at a Chl concentration of 5 μg ml⁻¹ were analyzed through the SDS–polyacrylamide gel electrophoresis (SDS-PAGE) (8 to 16% w/v tris-tricine, SolarBio) and blue native–PAGE (10% w/v acrylamide) methods (fig. S1, A to D). For the Western blot analysis, the anti-PsbR and anti-PsbY antibodies were obtained from Agrisera (Vännäs, Sweden).

Mass spectrometry

Protein identification

The protein bands of interest from the SDS-PAGE gel stained by the Coomassie brilliant blue were excised, destained, and applied for in-gel trypsin digestion procedure. The products were analyzed by a Ultra Performance Liquid Chromatography (UPLC) system (UltrafleXtreme, Bruker, Billerica, MA) coupled to an Orbitrap Elite mass spectrometer (Bruker, Billerica, MA) (75).

Chemical cross-linking MS and data analysis

PSII particles (BBY particles) were dissolved in the resuspension buffer [total chlorophyll concentration of 0.2 mg ml⁻¹ in 400 mM sucrose, 15 mM NaCl, 5 mM MgCl₂, and 20 mM MES-NaOH (pH 6.3)] in the immediate presence of the isotopic-coded BS3 cross-linker mixture (BS3-H₁₂/D₁₂, Creative Molecule Inc.) incubated together for 10 min in the dark at 25°C, where the cross-linker was in 10-, 50-, and 100-fold excess with respect to the PSII content. A Dionex Ultimate high-performance liquid chromatography (HPLC) is connected to a Nanospray Flex source coupled with a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA). See reference (76) for technical details. For cross-linked peptide identification, MS and MS/MS data were imported into pLink software (77). Searching parameters were as follows: Enzyme was trypsin (up to three missed cleavages) with 20 parts per million (ppm) of precursor tolerance and 60 ppm of fragment tolerance. Variable modifications were as follows: oxidation of M and deamidation of N, Q, and N terminus. The minimum number of peptide length is 6 with peptide mass of 600 Da, and the maximum number is 60 with peptide mass of 6000 Da. The false discovery rate was equal to or smaller than 5% at spectral level with a 10-ppm filter tolerance. Isotopic pairs were examined manually in raw files to confirm cross-link identification. The theoretical product-ion mass list was calculated in Protein Prospector. Manual validation of the fragments was further performed as a comparison to the pLink assignment.

Negative-staining EM analysis

The concentration of the purified spinach PSII-LHCII megacomplex and supercomplex samples was adjusted to 50 μg Chl ml⁻¹ by diluting it in a buffer with 50 mM MES-NaOH (pH 6.5) and 0.02% α-DDM in advance. Subsequently, 5 μl of the diluted samples was applied onto a Cu 230-mesh holey grid covered with a common carbon support film (Zhongjingkeyi/EMCN, Beijing, China), which was pretreated with H₂/O₂ glow discharge in a plasma ion bombarder (PIB-10, Vacuum Device) for 1 min. After the excess sample was removed by filter paper, 5 μl of 2% uranyl acetate was applied on the grid to stain the sample. The excess stain was removed by filter paper until a thin liquid film covers the grid surface and the grid was dried under room temperature. The prepared grids of the samples were used for image collection on a 120-kV transmission electron microscope (Tecnai Spirit 120 kV) equipped with an UltraScan 1000 (2 k by 2 k) charge-coupled device (CCD) camera. The data were obtained at 98,000 magnification and recorded with a pixel size of 5.78 Å. For data processing of the spinach PSII-LHCII megacomplex, 500 particles were picked from 50 images by using the manual-picker module in cryoSPARC (78). They were used as the templates for the template-based autopicking procedures, and, as a result, 13,717 and 15,754 particles were extracted from the images of PSII-LHCII megacomplex and supercomplex samples, respectively. Subsequently, all particles of the two samples were applied to two-dimensional (2D) classification and divided into 20 classes. The 2D-class images corresponding to the PSII-LHCII supercomplexes and megacomplexes were identified on the basis of the overall shape of the particles (fig. S1, E and F). For the PSII-LHCII megacomplexes from WT and psbR A. thaliana, 24,823 and 24,662 particles were selected from 296 and 274 images, respectively (fig. S5B), through multiple rounds of 2D classification processes by using the cryoSPARC software (78).

The negative-staining EM samples of the grana membranes from the WT and psbR mutant plants were prepared through the same methods mentioned above. The images were recorded on the Tecnai Spirit 120-kV microscope through an objective and unbiased approach.

Absorption spectra measurement

Absorption spectra of B6 and B7 fractions from the SDG ultracentrifugation at Chl concentration of 5 μg ml⁻¹ (fig. S2A) were recorded on a U-3900 spectrophotometer (Hitachi, Japan) at room temperature at a spectral range between 350 and 750 nm.

Pigment analysis through the HPLC method

To analyze the pigment compositions, the HPLC analysis was carried out on the extracts of the PSII-LHCII supercomplex and PSII-LHCII megacomplex samples as described previously and with some minor alteration (79). The pigments of 1 μl of the supercomplex/megacomplex sample (10 μg Chl μl⁻¹) were extracted in 200 μl 80% (v/v) acetone. After being vortexed, the preparation was centrifuged at 13,000g at 4°C for 15 min. After centrifugation, 20 μl of the supernatant (0.5 mg Chl ml⁻¹) was injected into the LiChrospher 100 RP-18 column (Alltech Allsphere ODS-2) on a Hitachi L-8900 separation system equipped with a Hitachi L2450 diode array detector. Two mobile phases were used for gradient elution of pigments with different hydrophobicity, namely buffer A with two organic phases and one aqueous phase [acetonitrile:methanol:0.1 M tris-HCl (pH 8.0) = 87:10:3, v:v:v] and buffer B with two organic phases (methanol:hexane = 4:1, v:v). A flow rate of 2 ml/min was used in the entire separation process. For the first 9 min, buffer A was applied to separate pigments like neoxanthin (Neo) and lutein (Lut); in the next 3.5 min, the gradient of 0 to 100% buffer B was applied and then 100% buffer B was maintained for 5.5 min to elute and separate chlorophylls. Last, a reverse gradient of 100 to 0% buffer B was applied in 1 min and then 100% buffer A was maintained for 5 min to elute β-carotene. For comparison, the HPLC profiles with absorption at 440 nm versus the retention time of the pigment fractions of the two samples are superposed and shown in fig. S2B.

77 K chlorophyll fluorescence emission measurement

To prepare the sample, an aliquot (20 μg Chl) of the B6 or B7 fractions was pipetted into 250 μl of dilution buffer [50 mM MES-NaOH (pH 6.5), 5 mM CaCl₂, and 0.02% α-DDM] and then mixed well with an equal volume of 60% glycerol (w/v) for the measurement of chlorophyll fluorescence under 77 K. The whole measuring process is carried out in a sample pool environment filled with liquid nitrogen by using Micro-FluorCam FC 2000 (Photon Systems Instruments, Czechia). The fluorescence emission spectra were recorded at three different excitation wavelengths at 436 nm (Chl a), 473 nm (Chl b), and 500 nm (carotenoids) by using the FL solutions-F-7000 FL spectrophotometer (Thermo Fisher Scientific, Waltham, MA) (fig. S2, C to D).

Measurement of the steady state OE activity

The light-saturated steady-state rates of OE for the PSII-LHCII supercomplex and megacomplex samples were measured at 25°C by using the Chlorolab-2 oxygen electrode system (Hansatech Instruments Ltd., Norfolk, UK). To sustain the reaction during measurement, DCBQ was used as an artificial electron acceptor. Before starting the measurement, the zero-oxygen line was achieved by complete consumption of oxygen in the oxygen-saturated water by sodium dithionite. The PSII-LHCII supercomplex or megacomplex sample with 20 μg Chl was diluted into 2 ml of the measurement buffer containing 25 mM MES-NaOH (pH 6.5), 5 mM CaCl₂, 0.02% α-DDM, 1 mM potassium ferricyanide [K₃Fe(CN)₆], and 0.25 mM DCBQ. During the measurement, a gradient of light with intensity increasing gradually from 0 to saturating intensity (2000 μmol photons m⁻² s⁻¹) was applied. After continuous irradiation under a light condition of specific intensity for 1 min, the light intensity is ramped up to the next level for a new measurement. The experimental data of the OE (μmol mg⁻¹ min⁻¹) were measured by using the gas-phase oxygen electrode chamber (Hansatech Instruments Ltd., Norfolk, UK) and recorded once every second at room temperature. The steady-state rate of oxygen release was calculated by dividing the increment of oxygen concentration under a specific light intensity with the duration of time [μmol OE (mg Chl)⁻¹ hour⁻¹]. To quantify and compare the amount of PSII reaction center in the two samples, the protein components were separated by SDS-PAGE, and the relative contents of D1 protein bands were quantified by the gray scale values measured through the ImageJ program (v1.8.0). When the gray scale value of the D1 band of the supercomplex sample was normalized at 1 with an arbitrary unit (a.u.), the amount of D1 protein of megacomplex was estimated to be 0.89 a.u. (relative to that of supercomplex). The final steady-state rate of oxygen release in the PSII-LHCII megacomplex was calibrated by dividing the OE value with the amount of arbitrary unit of D1 protein [μmol OE per D1_AU (mg Chl)⁻¹ hour⁻¹] (Fig. 4F). For curve fitting, the steady-state approximation model, expressed by a hyperbolic function OE^th = OE^th_max I/(L_1/2+I), was applied to the photon flux density–dependent OE data as described in a previous work (80). OE_th(max) represents the theoretical OE maximum observed at very high irradiance when the maximal possible PSII reaction centers are all open. I is the light intensity (μmol photons m⁻² s⁻¹), and L_1/2 is the irradiance giving OE^th_max/2.

Flash-induced fluorescence relaxation kinetics

The fluorescence relaxation kinetics of Chl a in the purified spinach PSII-LHCII supercomplex and megacomplex samples were measured with Dual-PAM-100 (WALZ, Germany), and the F_V/F_M were obtained according to the protocol reported in a previous work (81). The monitoring light pulses from a light-emitting diode were provided at 650-nm wavelength and about 0.016–μmol photon m⁻² s⁻¹ intensity with 10-μs duration at a frequency of 1.6 kHz. Single saturating actinic flashes of approximately 7.5-μs width at the half maximum were provided by a saturating single turnover flash lamp. Each sample contains 40 μg ml⁻¹ Chl and was dark-adapted for 5 min before the fluorescence detection procedure was initiated (Fig. 4G).

Cryo-EM data collection, processing, classification, and reconstruction

After the GraFix SDG ultracentrifugation, the target bands were carefully collected by using syringes with thin needles. The sample was concentrated in a 100-kDa cutoff Amicon ultracentrifugal filters (MilliporeSigma, Burlington, MA) through centrifugation at 2,500g at 4°C for 5 min each time until no more volume can be reduced. Then, 10 times sample volume of dilution buffer with 50 mM MES-NaOH (pH 6.5) and 0.02% α-DDM was added to the sample and mixed well with the sample. The diluted sample was concentrated, and the process was repeated twice to minimize the concentration of sucrose and betaine in the sample. Subsequently, 3.5 μl of the concentrated samples at a Chl concentration of 6 mg ml⁻¹ was applied to a Cu 200 mesh Holey Carbon Films grid (Quantifoil R 1.2/1.3). The grid with target sample was blotted for 3 s with a blotting force of level 2 and waiting time of 4 s at 100% humidity and 4°C and then plunged into liquid ethane on the FEI vitrobot IV (Thermo Fisher Scientific, Waltham, MA). The frozen sample in liquid ethane was further transferred rapidly to liquid nitrogen for storage and data collection.

For data collection, a total of 17,310 movies were recorded on a 300 kV Titan Krios microscope (FEI) equipped with a K3 camera (Gatan Inc., Pleasanton, CA) in the super-resolution mode. The defocus value is at the range of −1.5 to −2.0 μm. The physical pixel size of 1.35 Å, and a total dose of 60 e⁻ Å⁻² was used. The beam-induced motion in each movie with 32 frames were aligned and corrected by MotionCor2 (82). Subsequently, the contrast transfer function (CTF) parameters were estimated by using CTFFIND (v4.1) (83). About 1000 particles were selected manually from 200 images in cryoSPARC (78) and sorted into 2D classes, which were used as templates for subsequent autopicking procedure. After autopicking, a total of 4,053,536 particles were used for multiple rounds of 2D classification iterations. Among them, 285,475 particles showing good contrast and detailed features in 2D class images were selected for further ab initio 3D reconstruction and classification procedure. Two of the 10 3D classes were identified as particles of the PSII-LHCII megacomplexes and combined for multiple rounds of 3D heterogeneous refinement. Subsequently, 104,040 particles with good contrast were selected for further nonuniform refinement with C2 symmetry applied, local and global CTF refinement, and local motion correction. As a result, a cryo-EM density map at a medium resolution of 4.5 Å was obtained. The particles were further combined with the other set of lower-resolution PSII-LHCII megacomplex particles from 3D heterogeneous refinement and divided into five equal particle stacks. Subsequently, reference volumes of three resolution gradients (4, 10, and 30 Å) were used for further 3D classification by using five parallel particle stacks. Last, 93,684 particles merged from five subsets of 3D classification results are used for nonuniform (NU)-refinement with C2 symmetry applied after the duplicates are removed. The overall resolution of the cryo-EM map reached 3.7 Å at this stage. Then, the symmetry expansion procedure was applied to further improve the map of C₂S₂M region (one-half of the C₄S₄M₂ megacomplex). The particle subtraction and local refinement procedures were carried out to further improve the local map quality around PSII core, CP26-CP29-LHCII_S-type, and CP24-LHCII_M-type regions individually, and the resolutions of these three local regions were estimated to be 3.22, 3.30, and 4.26 Å, respectively. By using the combine_focus_map procedure in Phenix program (v1.20.1) (84), the local maps were merged to generate a composite map. The resolution of final composite map was estimated by using the gold standard Fourier shell correlation (FSC) with a cutoff at 0.143.

Model building and refinement

To build the structural model of C₄S₄M₂ PSII-LHCII megacomplex, the structures of S. oleracea C₂S₂ PSII-LHCII supercomplex (PDB code: 3JCU) and P. sativum C₂S₂M₂ PSII-LHCII supercomplex (PDB code: 5XNL) were used as the initial models. The spinach C₂S₂ PSII-LHCII supercomplex model and pea M-LHCII–CP24 model were docked into the 3.22-Å composite map by using the UCSF Chimera program (85), respectively. Then, the models were refined through the function of Rigid Body Fit Zone in COOT (86) to achieve improved matching of the models with the corresponding densities of the cryo-EM map. Subsequently, the amino acid residues in the initial model from P. sativum supercomplex were corrected and registered by referring to the sequences of the corresponding proteins from S. oleracea. The models of PsbR and PsbY-A1 subunit were built manually through a de novo model building process by using the amino acid sequence and the cryo-EM map as references. The precursor of plant PsbY was found to be processed into two mature proteins, namely, PsbY-A1 and PsbY-A2 (35). As PsbY-A1 and PsbY-A2 share high sequence identity (73%) and similarity (87%) and due to limited resolution, it is not possible to distinguish them unambiguously according to the local map features. Hence, the PsbY-A1 model was built in the map tentatively, and it fits well with the density (fig. S4, D and F). For the small molecules, two plastoquinone molecules and some lipid molecules were found in PQ/PQH₂ exchange cavity in the CSM monomer on the inner side of the megacomplex. After model building, real-space refinements were performed by using Phenix program (v. 1.20.1) (84), and the geometric restraints for the cofactors and the Chl Mg-ligand coordination bond were supplied during the refinement. After real-space refinement, manual adjustment and correction was carried out iteratively in COOT. The geometries of the structural model were assessed by using Phenix (84), and the detailed model information was summarized in Table 1.

Bioinformatic analysis of higher plant PsbR

More than 800 homologs of PsbR protein sequences were downloaded manually from National Center for Biotechnology Information (NCBI) using reference list. Among them, 704 homolog sequences producing notable alignments were manually trimmed to remove large indels in some sequences. The trimmed file was then used for NCBI COBALT analysis, which computes a multiple protein sequence alignment using conserved domain and local sequence similarity information. The .aln file was downloaded and used for visualization with Weblogo 3.7.4. The structures were analyzed by using PyMOL (The PyMOL Molecular Graphics System, version 2.5.0, Schrödinger LLC).

Growth of A. thaliana plants

The ΔPsbR A. thaliana T-DNA insertion mutant (SALK_114469C) was obtained from the Arabidopsis Biological Resource Center, Ohio State University, Columbus, USA. The seeds of WT and ΔPsbR were kept for germination after cold treatment for 48 hours at 4°C and grown in a growth chamber with control conditions at 22° ± 1°C under 50 to 80 μmol photons m⁻² s⁻¹ of white light with a 16 hour-light and 8-hour dark photoperiod. Fully expanded rosette leaves of 4- to 6-week-old plants were harvested to isolate the PSII particles.

Isolation and characterization of PSII particles (BBY)

The PSII particles were isolated from WT and ΔPsbR A. thaliana plants (49). The leaves were ground in a glass homogenizer with a chloroplast isolation buffer [100 mM sucrose and 50 mM sodium potassium phosphate (pH 7.4)], the homogenate was then passed through two layers of Miracloth (Calbiochem, USA), and the chloroplast thylakoids were pelleted by centrifugation at 2000g for 5 min. After being washed with resuspension buffer RB [400 mM sucrose, 15 mM NaCl, 5 mM MgCl₂, and 20 mM MES-NaOH (pH 6.3)], the thylakoids were resuspended in a small amount of the same buffer to bring Chl concentration to 2 to 3 mg Chl ml⁻¹ concentration determined by the method of Arnon (87). Triton X-100 (20%, Sigma-Aldrich, St. Louis, USA) was then added to a final detergent to Chl ratio of 20:1, followed by 25-min incubation on ice. Samples were centrifuged at 7000g for 5 min at 4°C, and the supernatant was centrifuged again at 38,000g for 25 min at 4°C. The pellets were resuspended again in resuspension buffer and centrifuged with the same speed. Typical preparations had a Chl a/b ratio of 1.7 to 2.0. SDS-PAGE was performed on 18 to 24% polyacrylamide gradient gels (88) with 15 μg of Chl loaded per lane, and the gel was stained by 0.1% Coomassie brilliant blue for visualization of the protein bands.

Basic spectroscopic characterization of BBY from WT and ΔPsbR A. thaliana

Steady state absorption spectra of the samples were recorded using UV-Vis 1800 spectrophotometer from Shimadzu. Cryogenic measurements of the absorption spectra were performed in the liquid nitrogen vapor-based VNF-100 cryostat (Janis, USA) at 77 K. The samples were dissolved in a mixture of glycerol/20 mM MES–NaOH (pH 6.3) (v/v at 60/40) that forms transparent glass upon freezing to 77 K.

Time-resolved fluorescence

TRF measurements were was performed using C5680 from Hamamatsu streak camera consisting of a cooled N51716-04 streak tube, a digital CCD camera (Orca2), a synchroscan unit M5675, a synchronous delay generator C6878, and an A6365-01 spectrograph from Bruker (Bruker Corporation, USA). Excitation pulses at 410 nm were generated by an ultrafast optical parametric oscillator Inspire100 (Spectra-Physics, USA) and pumped using a Mai-Tai (Spectra-Physics, USA) ultrafast Ti:Sapphire laser generating ~90-fs laser pulses at 820 nm with a frequency of 80 MHz. The repetition rate of the excitation laser was set to 8 MHz using pulse picker (Spectra-Physics, USA). The excitation beam was depolarized before sample and focused on the sample to a circular spot of ~1 mm and adjusted to a very low photon flux of ~10¹⁰ photons cm⁻² to minimize singlet-singlet annihilation processes that could be easily triggered between excited Chls in the PSII-LHC assemblies in the BBY particles. Chl a fluorescence emission was measured at a right angle from the excitation beam, and a long-pass 610-nm filter was placed at the entrance slit of the spectrograph. For TRF measurements, the samples were dispersed in the RB buffer.

Global analysis of TRF datasets

The TRF decay datasets were fitted with application of target analysis. In general, TRF decay signals at any time delay and wavelength, Fl(t, λ) can be decomposed to a superposition of n^th C_i(t)⋅SAFS_i(λ), (Species Associated Fluorescence Spectra) products (89)

Fl (t, λ) = \sum_{i = 1}^{n} C_{i} (t) SAF S_{i} (λ)

(1)

where C_i(t) are time-dependent SAFS concentration, defined by a kinetic model intended to mimic the true excitation decay pathway and typically reflects complex interactions between mutually dependent molecular species. This global (or whole data) modeling is typically referred to as target analysis. The modeling includes anticipated microscopic rates between interacting species and unlike typical unbiased global fitting producing macroscopic (observed) transfer rates, focuses rather on correct decomposition of datasets to well-defined spectral components (so-called molecular species). Because 410-nm excitation essentially excites all spectral forms of fluorescing Chls a, it assumed that all C_i(t) were initially convoluted by the excitation pulse represented by the instrument response function, having the full width at half maximum of ~0.35 ns (this is temporal resolution of streak camera system in 5-ns time delay window used for experiments). Subsequently, it was assumed that fluorescence decay follows irreversible sequential decay pathway from the fastest to the slowest SAFS. This could be mathematically described as

\frac{d C_{i - 1} (t)}{dt} = k_{i - 1} C_{i - 1} (t) - k_{i} C_{i - 1} (t), i \neq 1, k_{i - 1} > k_{i}

(2)

Reversed transfers were ignored considering a very low thermal energy available at 77 K that would allow us to repopulate molecular species of higher energy. To make qualitative comparison between SAFS, the “raw” SAFS were multiplied (corrected) by the maximum value of their time-dependent concentration C(t) (SAFS × C_max). This product more adequately represents amplitude of fluorescence associated with each molecular species of Chl a. Target analysis of TRF images was performed using CarpetView software (Light Conversion, Lithuania).

Acknowledgments

We thank X.-J. Huang, B.-L. Zhu, X.-J. Li, L.-H, Chen, and other staff members for support in cryo-EM data collection at the Center for Biological Imaging (CBI), Core Facilities for Protein Science at the Institute of Biophysics, Chinese Academy of Sciences (IBP, CAS). We are grateful to X.-B, Liang for assistance in sample preparation and data storage. We also thank A.-J. Li, Q. Zhou, Y.-P. Zhu, and X.-Y. Liu for technical support with sample preparation, biochemical experiments, and data processing.

Funding: The project is funded by the National Natural Science Foundation of China (31925024 to Z.L.), the Chinese Academy of Sciences Project for Young Scientists in Basic Research (YSBR-015 to Z.L.), and the Strategic Priority Research Program of CAS (XDB37020101 to Z.L.). D.M.N. acknowledges the Center for Solar Energy and Energy Storage at McKelvey School of Engineering at Washington University in Saint Louis for financial support. This research was also supported by the Danforth Seed Grant of the Department of Biology at Washington University in Saint Louis (to H.L.). R.S.T. was supported by the US Department of Energy (DOE), Office of Basic Energy Sciences, Photosynthetic Systems (PS) Program (grant DE-FG02-07ER15902 to H.L.).

Author contributions: J.S. purified the protein complex samples for biochemical characterization and cryo-EM study, carried out negative stain EM and cryo-EM data collection, processed the data, and prepared the figures. D.M.N. designed and performed the ultrafast time-resolved fluorescence analysis and prepared the figures. R.S.T. prepared the samples for biochemical and time-resolved fluorescence analysis. Z.L. and J.S. built and refined the atomic model for the cryo-EM map and analyzed the structure. H.L. prepared samples for and performed the mass spectrometry analysis and built the structural model of stromal side–coupled PSII supercomplexes. J.S., D.M.N., Z.L., and H.L. wrote the paper. All authors participated in the discussion. Z.L. and H.L. were in charge of conceptualization, project administration, and funding acquisition.

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: The atomic coordinate of the type I PSII-LHCII megacomplex from spinach has been deposited in the Protein Data Bank with accession code 8Z9D. The cryo-EM map has been deposited in the Electron Microscopy Data Bank with accession code of EMD-39860. All other data needed to evaluate the conclusions in this paper are present in the paper and/or the Supplementary Materials.

Supplementary Materials

This PDF file includes:

Figs. S1 to S17

Tables S1 and S2

References

sciadv.adq9967_sm.pdf^{(6.9MB, pdf)}

REFERENCES AND NOTES

1.Barber J., Photosystem II: An enzyme of global significance. Biochem. Soc. Trans. 34, 619–631 (2006). [DOI] [PubMed] [Google Scholar]
2.Boekema E. J., Hankamer B., Bald D., Kruip J., Nield J., Boonstra A. F., Barber J., Rogner M., Supramolecular structure of the photosystem II complex from green plants and cyanobacteria. Proc. Natl. Acad. Sci. U.S.A. 92, 175–179 (1995). [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Boekema E. J., van Roon H., Calkoen F., Bassi R., Dekker J. P., Multiple types of association of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes. Biochemistry 38, 2233–2239 (1999). [DOI] [PubMed] [Google Scholar]
4.Wei X., Su X., Cao P., Liu X., Chang W., Li M., Zhang X., Liu Z., Structure of spinach photosystem II–LHCII supercomplex at 3.2 Å resolution. Nature 534, 69–74 (2016). [DOI] [PubMed] [Google Scholar]
5.Sheng X., Watanabe A., Li A. J., Kim E., Song C. H., Murata K., Song D. F., Minagawa J., Liu Z. F., Structural insight into light harvesting for photosystem II in green algae. Nat. Plants 5, 1320–1330 (2019). [DOI] [PubMed] [Google Scholar]
6.Dekker J. P., Boekema E. J., Supramolecular organization of thylakoid membrane proteins in green plants. Biochim. Biophys. Acta. 1706, 12–39 (2005). [DOI] [PubMed] [Google Scholar]
7.Kouřil R., Dekker J. P., Boekema E. J., Supramolecular organization of photosystem II in green plants. Biochim. Biophys. Acta. 1817, 2–12 (2012). [DOI] [PubMed] [Google Scholar]
8.Nosek L., Semchonok D., Boekema E. J., Ilík P., Kouřil R., Structural variability of plant photosystem II megacomplexes in thylakoid membranes. Plant J. 89, 104–111 (2017). [DOI] [PubMed] [Google Scholar]
9.Boekema E. J., Van Roon H., Van Breemen J. F., Dekker J. P., Supramolecular organization of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes. Eur. J. Biochem. 266, 444–452 (1999). [DOI] [PubMed] [Google Scholar]
10.Kim E., Watanabe A., Duffy C. D. P., Ruban A. V., Minagawa J., Multimeric and monomeric photosystem II supercomplexes represent structural adaptations to low- and high-light conditions. J. Biol. Chem. 295, 14537–14545 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Kouřil R., Nosek L., Opatíková M., Arshad R., Semchonok D. A., Chamrád I., Lenobel R., Boekema E. J., Ilík P., Unique organization of photosystem II supercomplexes and megacomplexes in Norway spruce. Plant J. 104, 215–225 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Boekema E. J., van Breemen J. F., van Roon H., Dekker J. P., Arrangement of photosystem II supercomplexes in crystalline macrodomains within the thylakoid membrane of green plant chloroplasts. J. Mol. Biol. 301, 1123–1133 (2000). [DOI] [PubMed] [Google Scholar]
13.Kirchhoff H., Haase W., Wegner S., Danielsson R., Ackermann R., Albertsson P.-A., Low-light-induced formation of semicrystalline photosystem II arrays in higher plant chloroplasts. Biochemistry 46, 11169–11176 (2007). [DOI] [PubMed] [Google Scholar]
14.Ljungberg U., Åkerlund H.-E., Andersson B., The release of a 10-kDa polypeptide from everted photosysthem II thylakoid membranes by alkaline tris. FEBS Lett. 175, 255–258 (1984). [Google Scholar]
15.Stockhaus J., Höfer M., Renger G., Westhoff P., Wydrzynski T., Willmitzer L., Anti-sense RNA efficiently inhibits formation of the 10 kd polypeptide of photosystem II in transgenic potato plants: Analysis of the role of the 10 kd protein. EMBO J. 9, 3013–3021 (1990). [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Xue H., Tokutsu R., Bergner S. V., Scholz M., Minagawa J., Hippler M., Photosystem II subunit R is required for efficient binding of light-harvesting complex stress-related protein3 to photosystem II-light-harvesting supercomplexes in Chlamydomonas reinhardtii. Plant Physiol. 167, 1566–1578 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Ferreira K. N., Iverson T. M., Maghlaoui K., Barber J., Iwata S., Architecture of the photosynthetic oxygen-evolving center. Science 303, 1831–1838 (2004). [DOI] [PubMed] [Google Scholar]
18.Allen J. F., de Paula W. B. M., Puthiyaveetil S., Nield J., A structural phylogenetic map for chloroplast photosynthesis. Trends Plant Sci. 16, 645–655 (2011). [DOI] [PubMed] [Google Scholar]
19.Ljungberg U., Akerlund H. E., Andersson B., Isolation and characterization of the 10-kDa and 22-kDa polypeptides of higher-plant photosystem 2. Eur. J. Biochem. 158, 477–482 (1986). [DOI] [PubMed] [Google Scholar]
20.Ido K., Nield J., Fukao Y., Nishimura T., Sato F., Ifuku K., Cross-linking evidence for multiple interactions of the PsbP and PsbQ proteins in a higher plant photosystem II supercomplex. J. Biol. Chem. 289, 20150–20157 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Albanese P., Melero R., Engel B. D., Grinzato A., Berto P., Manfredi M., Chiodoni A., Vargas J., Sorzano C. Ó. S., Marengo E., Pea PSII-LHCII supercomplexes form pairs by making connections across the stromal gap. Sci. Rep. 7, 10067 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Fadeeva M., Klaiman D., Caspy I., Nelson N., Structure of Chlorella ohadii photosystem II reveals protective mechanisms against environmental stress. Cells 12, 1971 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Karlin-Neumann G. A., Tobin E. M., Transit peptides of nuclear-encoded chloroplast proteins share a common amino acid framework. EMBO J. 5, 9–13 (1986). [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Webber A. N., Packman L. C., Gray J. C., A 10 kDa polypeptide associated with the oxygen-evolving complex of photosystem II has a putative C-terminal non-cleavable thylakoid transfer domain. FEBS Lett. 242, 435–438 (1989). [DOI] [PubMed] [Google Scholar]
25.Allahverdiyeva Y., Mamedov F., Suorsa M., Styring S., Vass I., Aro E.-M., Insights into the function of PsbR protein in Arabidopsis thaliana. Biochim. Biophys. Acta. 1767, 677–685 (2007). [DOI] [PubMed] [Google Scholar]
26.Liu H., Frankel L. K., Bricker T. M., Characterization and complementation of a psbR mutant in Arabidopsis thaliana. Arch. Biochem. Biophys. 489, 34–40 (2009). [DOI] [PubMed] [Google Scholar]
27.Suorsa M., Sirpio S., Allahverdiyeva Y., Paakkarinen V., Mamedov F., Styring S., Aro E. M., PsbR, a missing link in the assembly of the oxygen-evolving complex of plant photosystem II. J. Biol. Chem. 281, 145–150 (2006). [DOI] [PubMed] [Google Scholar]
28.Allahverdiyeva Y., Suorsa M., Rossi F., Pavesi A., Kater M. M., Antonacci A., Tadini L., Pribil M., Schneider A., Wanner G., Leister D., Aro E.-M., Barbato R., Pesaresi P., Arabidopsis plants lacking PsbQ and PsbR subunits of the oxygen-evolving complex show altered PSII super-complex organization and short-term adaptive mechanisms. Plant J. 75, 671–684 (2013). [DOI] [PubMed] [Google Scholar]
29.Loll B., Kern J., Saenger W., Zouni A., Biesiadka J., Towards complete cofactor arrangement in the 3.0 Å resolution structure of photosystem II. Nature 438, 1040–1044 (2005). [DOI] [PubMed] [Google Scholar]
30.Kato K., Miyazaki N., Hamaguchi T., Nakajima Y., Akita F., Yonekura K., Shen J. R., High-resolution cryo-EM structure of photosystem II reveals damage from high-dose electron beams. Commun. Biol. 4, 382 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Kawakami K., Iwai M., Ikeuchi M., Kamiya N., Shen J.-R., Location of PsbY in oxygen-evolving photosystem II revealed by mutagenesis and x-ray crystallography. FEBS Lett. 581, 4983–4987 (2007). [DOI] [PubMed] [Google Scholar]
32.Gisriel C. J., Wang J., Liu J., Flesher D. A., Reiss K. M., Huang H. L., Yang K. R., Armstrong W. H., Gunner M. R., Batista V. S., Debus R. J., Brudvig G. W., High-resolution cryo-electron microscopy structure of photosystem II from the mesophilic cyanobacterium, Synechocystis sp. PCC 6803. Proc. Natl. Acad. Sci. U.S.A. 119, e2116765118 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
33.L. E. Thornton, J. L. Roose, H. B. Pakrasi, M. Ikeuchi, Photosystem II: The light-driven water: Plastoquinone oxidoreductase, T. J. Wydrzynski, K. Satoh, J. A. Freeman, Eds. (Springer, 2005), vol. 22, pp. 121–137. [Google Scholar]
34.Gau A. E., Thole H. H., Pistorius E. K., Isolation and partial characterization of a manganese requiring ʟ-arginine metabolizing enzyme being present in photosystem II complexes of spinach and tobacco. Z. Naturforsch. C 50, 638–651 (1995). [Google Scholar]
35.Gau A. E., Thole H. H., Sokolenko A., Altschmied L., Hermann R. G., Pistorius E. K., PsbY, a novel manganese-binding, low-molecular-mass protein associated with photosystem II. Mol. Gen. Genet. 260, 56–68 (1998). [DOI] [PubMed] [Google Scholar]
36.Shen J. R., The structure of photosystem II and the mechanism of water oxidation in photosynthesis. Annu. Rev. Plant Biol. 66, 23–48 (2015). [DOI] [PubMed] [Google Scholar]
37.von Sydow L., Schwenkert S., Meurer J., Funk C., Mamedov F., Schröder W. P., The PsbY protein of Arabidopsis photosystem II is important for the redox control of cytochrome b₅₅₉. Biochim. Biophys. Acta. 1857, 1524–1533 (2016). [DOI] [PubMed] [Google Scholar]
38.Su X., Ma J., Wei X., Cao P., Zhu D., Chang W., Liu Z., Zhang X., Li M., Structure and assembly mechanism of plant C₂S₂M₂-type PSII-LHCII supercomplex. Science 357, 815–820 (2017). [DOI] [PubMed] [Google Scholar]
39.Graca A. T., Hall M., Persson K., Schroder W. P., High-resolution model of Arabidopsis photosystem II reveals the structural consequences of digitonin-extraction. Sci. Rep. 11, 15534 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Croce R., van Amerongen H., Light harvesting in oxygenic photosynthesis: Structural biology meets spectroscopy. Science 369, eaay2058 (2020). [DOI] [PubMed] [Google Scholar]
41.De Causmaecker S., Douglass J. S., Fantuzzi A., Nitschke W., Rutherford A. W., Energetics of the exchangeable quinone, Q_B, in photosystem II. Proc. Natl. Acad. Sci. U.S.A. 116, 19458–19463 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Kirchhoff H., Horstmann S., Weis E., Control of the photosynthetic electron transport by PQ diffusion microdomains in thylakoids of higher plants. Biochim. Biophys. Acta. 1459, 148–168 (2000). [DOI] [PubMed] [Google Scholar]
43.Maxwell K., Johnson G. N., Chlorophyll fluorescence—A practical guide. J. Exp. Bot. 51, 659–668 (2000). [DOI] [PubMed] [Google Scholar]
44.Bowes J. M., Crofts A. R., Binary oscillations in the rate of reoxidation of the primary acceptor of photosystem II. Biochim. Biophys. Acta 590, 373–384 (1980). [DOI] [PubMed] [Google Scholar]
45.Reifarth F., Christen G., Seeliger A. G., Dormann P., Benning C., Renger G., Modification of the water oxidizing complex in leaves of the dgd1 mutant of Arabidopsis thaliana deficient in the galactolipid digalactosyldiacylglycerol. Biochemistry 36, 11769–11776 (1997). [DOI] [PubMed] [Google Scholar]
46.Uversky V. N., Intrinsically disordered proteins and their “mysterious” (meta)physics. Front. Phys. 7, 10 (2019). [Google Scholar]
47.Marsh J. A., Forman-Kay J. D., Ensemble modeling of protein disordered states: Experimental restraint contributions and validation. Proteins 80, 556–572 (2012). [DOI] [PubMed] [Google Scholar]
48.Fisher C. K., Stultz C. M., Constructing ensembles for intrinsically disordered proteins. Curr. Opin. Struct. Biol. 21, 426–431 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Berthold D. A., Babcock G. T., Yocum C. F., A highly resolved, oxygen-evolving photosystem-II preparation from spinach thylakoid membranes - EPR and electron-transport properties. FEBS Lett. 134, 231–234 (1981). [Google Scholar]
50.Crepin A., Santabarbara S., Caffarri S., Biochemical and spectroscopic characterization of highly stable photosystem II supercomplexes from Arabidopsis. J. Biol. Chem. 291, 19157–19171 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Anderson J. M., Melis A., Localization of different photosystems in separate regions of chloroplast membranes. Proc. Natl. Acad. Sci. U.S.A. 80, 745–749 (1983). [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Govindjee G., Photosystem II heterogeneity: The acceptor side. Photosynth. Res. 25, 151–160 (1990). [DOI] [PubMed] [Google Scholar]
53.Veerman J., McConnell M. D., Vasil'ev S., Mamedov F., Styring S., Bruce D., Functional heterogeneity of photosystem II in domain specific regions of the thylakoid membrane of spinach (Spinacia oleracea L.). Biochemistry 46, 3443–3453 (2007). [DOI] [PubMed] [Google Scholar]
54.Mummadisetti M., Su X., Lui H., An approach to nearest neighbor analysis of pigment-protein complexes using chemical cross-linking in combination with mass spectrometry. Methods Enzymol. 680, 139–162 (2022). [DOI] [PubMed] [Google Scholar]
55.Albanese P., Tamara S., Saracco G., Scheltema R. A., Pagliano C., How paired PSII-LHCII supercomplexes mediate the stacking of plant thylakoid membranes unveiled by structural mass-spectrometry. Nat. Commun. 11, 1361 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Sinz A., Cross-linking/mass spectrometry for studying protein structures and protein-protein interactions: Where are we now and where should we go from here? Angew. Chem. Int. Ed. Engl. 57, 6390–6396 (2018). [DOI] [PubMed] [Google Scholar]
57.Leitner A., Walzthoeni T., Kahraman A., Herzog F., Rinner O., Beck M., Aebersold R., Probing native protein structures by chemical cross-linking, mass spectrometry, and bioinformatics. Mol. Cell. Proteomics 9, 1634–1649 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Shukla M. K., Watanabe A., Wilson S., Giovagnetti V., Moustafa E. I., Minagawa J., Ruban A. V., A novel method produces native light-harvesting complex II aggregates from the photosynthetic membrane revealing their role in nonphotochemical quenching. J. Biol. Chem. 295, 17816–17826 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Kirchhoff H., Hinz H. J., Rosgen J., Aggregation and fluorescence quenching of chlorophyll a of the light-harvesting complex II from spinach in vitro. Biochim. Biophys. Acta. 1606, 105–116 (2003). [DOI] [PubMed] [Google Scholar]
60.Natali A., Gruber J. M., Dietzel L., Stuart M. C., van Grondelle R., Croce R., Light-harvesting complexes (LHCs) cluster spontaneously in membrane environment leading to shortening of their excited state lifetimes. J. Biol. Chem. 291, 16730–16739 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Kouril R., Wientjes E., Bultema J. B., Croce R., Boekema E. J., High-light vs. low-light: Effect of light acclimation on photosystem II composition and organization in Arabidopsis thaliana. Biochim. Biophys. Acta. 1827, 411–419 (2013). [DOI] [PubMed] [Google Scholar]
62.Kirchhoff H., Diffusion of molecules and macromolecules in thylakoid membranes. Biochim. Biophys. Acta. 1837, 495–502 (2014). [DOI] [PubMed] [Google Scholar]
63.Haferkamp S., Haase W., Pascal A. A., van Amerongen H., Kirchhoff H., Efficient light harvesting by photosystem II requires an optimized protein packing density in grana thylakoids. J. Biol. Chem. 285, 17020–17028 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
64.Joliot P., Lavergne J., Béal D., Plastoquinone compartmentation in chloroplasts. I. Evidence for domains with different rates of photo-reduction. Biochim. Biophys. Acta. 1101, 1–12 (1992). [Google Scholar]
65.De Bianchi S., Dall'Osto L., Tognon G., Morosinotto T., Bassi R., Minor antenna proteins CP24 and CP26 affect the interactions between photosystem II subunits and the electron transport rate in grana membranes of Arabidopsis. Plant Cell 20, 1012–1028 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]
66.Kovács L., Damkjær J., Kereïche S., Ilioaia C., Ruban A. V., Boekema E. J., Jansson S., Horton P., Lack of the light-harvesting complex CP24 affects the structure and function of the grana membranes of higher plant chloroplasts. Plant Cell 18, 3106–3120 (2006). [DOI] [PMC free article] [PubMed] [Google Scholar]
67.Guardini Z., Gomez R. L., Caferri R., Dall'Osto L., Bassi R., Loss of a single chlorophyll in CP29 triggers re-organization of the photosystem II supramolecular assembly. Biochim. Biophys. Acta. 1863, 148555 (2022). [DOI] [PubMed] [Google Scholar]
68.Thompson L. K., Brudvig G. W., Cytochrome b-559 may function to protect photosystem II from photoinhibition. Biochemistry 27, 6653–6658 (1988). [DOI] [PubMed] [Google Scholar]
69.Bondarava N., Gross C. M., Mubarakshina M., Golecki J. R., Johnson G. N., Krieger-Liszkay A., Putative function of cytochrome b559 as a plastoquinol oxidase. Physiol. Plant. 138, 463–473 (2010). [DOI] [PubMed] [Google Scholar]
70.Stewart D. H., Brudvig G. W., Cytochrome b₅₅₉ of photosystem II. BBA-Bioenergetics 1367, 63–87 (1998). [DOI] [PubMed] [Google Scholar]
71.Ortega J. M., Hervás M., De La Rosa M. A., Losada M., Redox properties of cytochrome b559 in photosynthetic membranes from the Cyanobacterium Synechocystis sp. PCC 6803. J. Plant Physiol. 144, 454–461 (1994). [Google Scholar]
72.Cumberworth A., Lamour G., Babu M. M., Gsponer J., Promiscuity as a functional trait: Intrinsically disordered regions as central players of interactomes. Biochem. J. 454, 361–369 (2013). [DOI] [PubMed] [Google Scholar]
73.Caffarri S., Kouril R., Kereïche S., Boekema E. J., Croce R., Functional architecture of higher plant photosystem II supercomplexes. EMBO J. 28, 3052–3063 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
74.Kastner B., Fischer N., Golas M. M., Sander B., Dube P., Boehringer D., Hartmuth K., Deckert J., Hauer F., Wolf E., Uchtenhagen H., Urlaub H., Herzog F., Peters J. M., Poerschke D., Lührmann R., Stark H., GraFix: Sample preparation for single-particle electron cryomicroscopy. Nat. Methods 5, 53–55 (2008). [DOI] [PubMed] [Google Scholar]
75.Shevchenko A., Tomas H., Havli J., Olsen J. V., Mann M., In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006). [DOI] [PubMed] [Google Scholar]
76.Liu H., Cyanobacterial phycobilisome allostery as revealed by quantitative mass spectrometry. Biochemistry 62, 1307–1320 (2023). [DOI] [PubMed] [Google Scholar]
77.Chen Z. L., Meng J. M., Cao Y., Yin J. L., Fang R. Q., Fan S. B., Liu C., Zeng W. F., Ding Y. H., Tan D., Wu L., Zhou W. J., Chi H., Sun R. X., Dong M. Q., He S. M., A high-speed search engine pLink 2 with systematic evaluation for proteome-scale identification of cross-linked peptides. Nat. Commun. 10, 3404 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
78.Punjani A., Rubinstein J. L., Fleet D. J., Brubaker M. A., cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017). [DOI] [PubMed] [Google Scholar]
79.Farber A., Young A. J., Ruban A. V., Horton P., Jahns P., Dynamics of xanthophyll-cycle activity in different antenna subcomplexes in the photosynthetic membranes of higher plants (The relationship between zeaxanthin conversion and nonphotochemical fluorescence quenching). Plant Physiol. 115, 1609–1618 (1997). [DOI] [PMC free article] [PubMed] [Google Scholar]
80.Fragata M., Dudekula S., Nonlinear enhancement of oxygen evolution in thylakoid membranes: Modeling the effect of light intensity and beta-cyclodextrin concentration. J. Phys. Chem. B 109, 14707–14714 (2005). [DOI] [PubMed] [Google Scholar]
81.Pesaresi P., Hertle A., Pribil M., Kleine T., Wagner R., Strissel H., Ihnatowicz A., Bonardi V., Scharfenberg M., Schneider A., Pfannschmidt T., Leister D., Arabidopsis STN7 kinase provides a link between short- and long-term photosynthetic acclimation. Plant Cell 21, 2402–2423 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
82.Zheng S. Q., Palovcak E., Armache J.-P., Verba K. A., Cheng Y., Agard D. A., MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
83.Rohou A., Grigorieff N., CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
84.Afonine P. V., Poon B. K., Read R. J., Sobolev O. V., Terwilliger T. C., Urzhumtsev A., Adams P. D., Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol. 74, 531–544 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
85.Pettersen E. F., Goddard T. D., Huang C. C., Couch G. S., Greenblatt D. M., Meng E. C., Ferrin T. E., UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). [DOI] [PubMed] [Google Scholar]
86.Emsley P., Lohkamp B., Scott W. G., Cowtan K., Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
87.Arnon D. I., Copper enzymes in isolated chloroplasts. polyphenoloxidase in Beta Vulgaris. Plant Physiol. 24, 1–15 (1949). [DOI] [PMC free article] [PubMed] [Google Scholar]
88.Kashino Y., Koike H., Satoh K., An improved sodium dodecyl sulfate-polyacrylamide gel electrophoresis system for the analysis of membrane protein complexes. Electrophoresis 22, 1004–1007 (2001). [DOI] [PubMed] [Google Scholar]
89.van Stokkum I. H. M., Larsen D. S., van Grondelle R., Global and target analysis of time-resolved spectra. Biochim. Biophys. Acta. 1657, 82–104 (2004). [DOI] [PubMed] [Google Scholar]
90.Huyer J., Eckert H.-J., Irrgang K.-D., Miao J., Eichler H.-J., Renger G., Fluorescence decay kinetics of solubilized pigment protein complexes from the distal, proximal, and core antenna of photosystem II in the range of 10–277 K and absence or presence of sucrose. J. Phys. Chem. B 108, 3326–3334 (2004). [Google Scholar]
91.Wilson S., Li D. H., Ruban A. V., The structural and spectral features of light-harvesting complex II proteoliposomes mimic those of native thylakoid membranes. J. Phys. Chem. Lett. 13, 5683–5691 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
92.Weisz D. A., Johnson V. M., Niedzwiedzki D. M., Shinn M. K., Liu H., Klitzke C. F., Gross M. L., Blankenship R. E., Lohman T. M., Pakrasi H. B., A novel chlorophyll protein complex in the repair cycle of photosystem II. Proc. Natl. Acad. Sci. U.S.A. 116, 21907–21913 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figs. S1 to S17

Tables S1 and S2

References

sciadv.adq9967_sm.pdf^{(6.9MB, pdf)}

[R1] 1.Barber J., Photosystem II: An enzyme of global significance. Biochem. Soc. Trans. 34, 619–631 (2006). [DOI] [PubMed] [Google Scholar]

[R2] 2.Boekema E. J., Hankamer B., Bald D., Kruip J., Nield J., Boonstra A. F., Barber J., Rogner M., Supramolecular structure of the photosystem II complex from green plants and cyanobacteria. Proc. Natl. Acad. Sci. U.S.A. 92, 175–179 (1995). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R3] 3.Boekema E. J., van Roon H., Calkoen F., Bassi R., Dekker J. P., Multiple types of association of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes. Biochemistry 38, 2233–2239 (1999). [DOI] [PubMed] [Google Scholar]

[R4] 4.Wei X., Su X., Cao P., Liu X., Chang W., Li M., Zhang X., Liu Z., Structure of spinach photosystem II–LHCII supercomplex at 3.2 Å resolution. Nature 534, 69–74 (2016). [DOI] [PubMed] [Google Scholar]

[R5] 5.Sheng X., Watanabe A., Li A. J., Kim E., Song C. H., Murata K., Song D. F., Minagawa J., Liu Z. F., Structural insight into light harvesting for photosystem II in green algae. Nat. Plants 5, 1320–1330 (2019). [DOI] [PubMed] [Google Scholar]

[R6] 6.Dekker J. P., Boekema E. J., Supramolecular organization of thylakoid membrane proteins in green plants. Biochim. Biophys. Acta. 1706, 12–39 (2005). [DOI] [PubMed] [Google Scholar]

[R7] 7.Kouřil R., Dekker J. P., Boekema E. J., Supramolecular organization of photosystem II in green plants. Biochim. Biophys. Acta. 1817, 2–12 (2012). [DOI] [PubMed] [Google Scholar]

[R8] 8.Nosek L., Semchonok D., Boekema E. J., Ilík P., Kouřil R., Structural variability of plant photosystem II megacomplexes in thylakoid membranes. Plant J. 89, 104–111 (2017). [DOI] [PubMed] [Google Scholar]

[R9] 9.Boekema E. J., Van Roon H., Van Breemen J. F., Dekker J. P., Supramolecular organization of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes. Eur. J. Biochem. 266, 444–452 (1999). [DOI] [PubMed] [Google Scholar]

[R10] 10.Kim E., Watanabe A., Duffy C. D. P., Ruban A. V., Minagawa J., Multimeric and monomeric photosystem II supercomplexes represent structural adaptations to low- and high-light conditions. J. Biol. Chem. 295, 14537–14545 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] 11.Kouřil R., Nosek L., Opatíková M., Arshad R., Semchonok D. A., Chamrád I., Lenobel R., Boekema E. J., Ilík P., Unique organization of photosystem II supercomplexes and megacomplexes in Norway spruce. Plant J. 104, 215–225 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R12] 12.Boekema E. J., van Breemen J. F., van Roon H., Dekker J. P., Arrangement of photosystem II supercomplexes in crystalline macrodomains within the thylakoid membrane of green plant chloroplasts. J. Mol. Biol. 301, 1123–1133 (2000). [DOI] [PubMed] [Google Scholar]

[R13] 13.Kirchhoff H., Haase W., Wegner S., Danielsson R., Ackermann R., Albertsson P.-A., Low-light-induced formation of semicrystalline photosystem II arrays in higher plant chloroplasts. Biochemistry 46, 11169–11176 (2007). [DOI] [PubMed] [Google Scholar]

[R14] 14.Ljungberg U., Åkerlund H.-E., Andersson B., The release of a 10-kDa polypeptide from everted photosysthem II thylakoid membranes by alkaline tris. FEBS Lett. 175, 255–258 (1984). [Google Scholar]

[R15] 15.Stockhaus J., Höfer M., Renger G., Westhoff P., Wydrzynski T., Willmitzer L., Anti-sense RNA efficiently inhibits formation of the 10 kd polypeptide of photosystem II in transgenic potato plants: Analysis of the role of the 10 kd protein. EMBO J. 9, 3013–3021 (1990). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] 16.Xue H., Tokutsu R., Bergner S. V., Scholz M., Minagawa J., Hippler M., Photosystem II subunit R is required for efficient binding of light-harvesting complex stress-related protein3 to photosystem II-light-harvesting supercomplexes in Chlamydomonas reinhardtii. Plant Physiol. 167, 1566–1578 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R17] 17.Ferreira K. N., Iverson T. M., Maghlaoui K., Barber J., Iwata S., Architecture of the photosynthetic oxygen-evolving center. Science 303, 1831–1838 (2004). [DOI] [PubMed] [Google Scholar]

[R18] 18.Allen J. F., de Paula W. B. M., Puthiyaveetil S., Nield J., A structural phylogenetic map for chloroplast photosynthesis. Trends Plant Sci. 16, 645–655 (2011). [DOI] [PubMed] [Google Scholar]

[R19] 19.Ljungberg U., Akerlund H. E., Andersson B., Isolation and characterization of the 10-kDa and 22-kDa polypeptides of higher-plant photosystem 2. Eur. J. Biochem. 158, 477–482 (1986). [DOI] [PubMed] [Google Scholar]

[R20] 20.Ido K., Nield J., Fukao Y., Nishimura T., Sato F., Ifuku K., Cross-linking evidence for multiple interactions of the PsbP and PsbQ proteins in a higher plant photosystem II supercomplex. J. Biol. Chem. 289, 20150–20157 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] 21.Albanese P., Melero R., Engel B. D., Grinzato A., Berto P., Manfredi M., Chiodoni A., Vargas J., Sorzano C. Ó. S., Marengo E., Pea PSII-LHCII supercomplexes form pairs by making connections across the stromal gap. Sci. Rep. 7, 10067 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.Fadeeva M., Klaiman D., Caspy I., Nelson N., Structure of Chlorella ohadii photosystem II reveals protective mechanisms against environmental stress. Cells 12, 1971 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] 23.Karlin-Neumann G. A., Tobin E. M., Transit peptides of nuclear-encoded chloroplast proteins share a common amino acid framework. EMBO J. 5, 9–13 (1986). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] 24.Webber A. N., Packman L. C., Gray J. C., A 10 kDa polypeptide associated with the oxygen-evolving complex of photosystem II has a putative C-terminal non-cleavable thylakoid transfer domain. FEBS Lett. 242, 435–438 (1989). [DOI] [PubMed] [Google Scholar]

[R25] 25.Allahverdiyeva Y., Mamedov F., Suorsa M., Styring S., Vass I., Aro E.-M., Insights into the function of PsbR protein in Arabidopsis thaliana. Biochim. Biophys. Acta. 1767, 677–685 (2007). [DOI] [PubMed] [Google Scholar]

[R26] 26.Liu H., Frankel L. K., Bricker T. M., Characterization and complementation of a psbR mutant in Arabidopsis thaliana. Arch. Biochem. Biophys. 489, 34–40 (2009). [DOI] [PubMed] [Google Scholar]

[R27] 27.Suorsa M., Sirpio S., Allahverdiyeva Y., Paakkarinen V., Mamedov F., Styring S., Aro E. M., PsbR, a missing link in the assembly of the oxygen-evolving complex of plant photosystem II. J. Biol. Chem. 281, 145–150 (2006). [DOI] [PubMed] [Google Scholar]

[R28] 28.Allahverdiyeva Y., Suorsa M., Rossi F., Pavesi A., Kater M. M., Antonacci A., Tadini L., Pribil M., Schneider A., Wanner G., Leister D., Aro E.-M., Barbato R., Pesaresi P., Arabidopsis plants lacking PsbQ and PsbR subunits of the oxygen-evolving complex show altered PSII super-complex organization and short-term adaptive mechanisms. Plant J. 75, 671–684 (2013). [DOI] [PubMed] [Google Scholar]

[R29] 29.Loll B., Kern J., Saenger W., Zouni A., Biesiadka J., Towards complete cofactor arrangement in the 3.0 Å resolution structure of photosystem II. Nature 438, 1040–1044 (2005). [DOI] [PubMed] [Google Scholar]

[R30] 30.Kato K., Miyazaki N., Hamaguchi T., Nakajima Y., Akita F., Yonekura K., Shen J. R., High-resolution cryo-EM structure of photosystem II reveals damage from high-dose electron beams. Commun. Biol. 4, 382 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] 31.Kawakami K., Iwai M., Ikeuchi M., Kamiya N., Shen J.-R., Location of PsbY in oxygen-evolving photosystem II revealed by mutagenesis and x-ray crystallography. FEBS Lett. 581, 4983–4987 (2007). [DOI] [PubMed] [Google Scholar]

[R32] 32.Gisriel C. J., Wang J., Liu J., Flesher D. A., Reiss K. M., Huang H. L., Yang K. R., Armstrong W. H., Gunner M. R., Batista V. S., Debus R. J., Brudvig G. W., High-resolution cryo-electron microscopy structure of photosystem II from the mesophilic cyanobacterium, Synechocystis sp. PCC 6803. Proc. Natl. Acad. Sci. U.S.A. 119, e2116765118 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R33] 33.L. E. Thornton, J. L. Roose, H. B. Pakrasi, M. Ikeuchi, Photosystem II: The light-driven water: Plastoquinone oxidoreductase, T. J. Wydrzynski, K. Satoh, J. A. Freeman, Eds. (Springer, 2005), vol. 22, pp. 121–137. [Google Scholar]

[R34] 34.Gau A. E., Thole H. H., Pistorius E. K., Isolation and partial characterization of a manganese requiring ʟ-arginine metabolizing enzyme being present in photosystem II complexes of spinach and tobacco. Z. Naturforsch. C 50, 638–651 (1995). [Google Scholar]

[R35] 35.Gau A. E., Thole H. H., Sokolenko A., Altschmied L., Hermann R. G., Pistorius E. K., PsbY, a novel manganese-binding, low-molecular-mass protein associated with photosystem II. Mol. Gen. Genet. 260, 56–68 (1998). [DOI] [PubMed] [Google Scholar]

[R36] 36.Shen J. R., The structure of photosystem II and the mechanism of water oxidation in photosynthesis. Annu. Rev. Plant Biol. 66, 23–48 (2015). [DOI] [PubMed] [Google Scholar]

[R37] 37.von Sydow L., Schwenkert S., Meurer J., Funk C., Mamedov F., Schröder W. P., The PsbY protein of Arabidopsis photosystem II is important for the redox control of cytochrome b₅₅₉. Biochim. Biophys. Acta. 1857, 1524–1533 (2016). [DOI] [PubMed] [Google Scholar]

[R38] 38.Su X., Ma J., Wei X., Cao P., Zhu D., Chang W., Liu Z., Zhang X., Li M., Structure and assembly mechanism of plant C₂S₂M₂-type PSII-LHCII supercomplex. Science 357, 815–820 (2017). [DOI] [PubMed] [Google Scholar]

[R39] 39.Graca A. T., Hall M., Persson K., Schroder W. P., High-resolution model of Arabidopsis photosystem II reveals the structural consequences of digitonin-extraction. Sci. Rep. 11, 15534 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R40] 40.Croce R., van Amerongen H., Light harvesting in oxygenic photosynthesis: Structural biology meets spectroscopy. Science 369, eaay2058 (2020). [DOI] [PubMed] [Google Scholar]

[R41] 41.De Causmaecker S., Douglass J. S., Fantuzzi A., Nitschke W., Rutherford A. W., Energetics of the exchangeable quinone, Q_B, in photosystem II. Proc. Natl. Acad. Sci. U.S.A. 116, 19458–19463 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R42] 42.Kirchhoff H., Horstmann S., Weis E., Control of the photosynthetic electron transport by PQ diffusion microdomains in thylakoids of higher plants. Biochim. Biophys. Acta. 1459, 148–168 (2000). [DOI] [PubMed] [Google Scholar]

[R43] 43.Maxwell K., Johnson G. N., Chlorophyll fluorescence—A practical guide. J. Exp. Bot. 51, 659–668 (2000). [DOI] [PubMed] [Google Scholar]

[R44] 44.Bowes J. M., Crofts A. R., Binary oscillations in the rate of reoxidation of the primary acceptor of photosystem II. Biochim. Biophys. Acta 590, 373–384 (1980). [DOI] [PubMed] [Google Scholar]

[R45] 45.Reifarth F., Christen G., Seeliger A. G., Dormann P., Benning C., Renger G., Modification of the water oxidizing complex in leaves of the dgd1 mutant of Arabidopsis thaliana deficient in the galactolipid digalactosyldiacylglycerol. Biochemistry 36, 11769–11776 (1997). [DOI] [PubMed] [Google Scholar]

[R46] 46.Uversky V. N., Intrinsically disordered proteins and their “mysterious” (meta)physics. Front. Phys. 7, 10 (2019). [Google Scholar]

[R47] 47.Marsh J. A., Forman-Kay J. D., Ensemble modeling of protein disordered states: Experimental restraint contributions and validation. Proteins 80, 556–572 (2012). [DOI] [PubMed] [Google Scholar]

[R48] 48.Fisher C. K., Stultz C. M., Constructing ensembles for intrinsically disordered proteins. Curr. Opin. Struct. Biol. 21, 426–431 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R49] 49.Berthold D. A., Babcock G. T., Yocum C. F., A highly resolved, oxygen-evolving photosystem-II preparation from spinach thylakoid membranes - EPR and electron-transport properties. FEBS Lett. 134, 231–234 (1981). [Google Scholar]

[R50] 50.Crepin A., Santabarbara S., Caffarri S., Biochemical and spectroscopic characterization of highly stable photosystem II supercomplexes from Arabidopsis. J. Biol. Chem. 291, 19157–19171 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R51] 51.Anderson J. M., Melis A., Localization of different photosystems in separate regions of chloroplast membranes. Proc. Natl. Acad. Sci. U.S.A. 80, 745–749 (1983). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R52] 52.Govindjee G., Photosystem II heterogeneity: The acceptor side. Photosynth. Res. 25, 151–160 (1990). [DOI] [PubMed] [Google Scholar]

[R53] 53.Veerman J., McConnell M. D., Vasil'ev S., Mamedov F., Styring S., Bruce D., Functional heterogeneity of photosystem II in domain specific regions of the thylakoid membrane of spinach (Spinacia oleracea L.). Biochemistry 46, 3443–3453 (2007). [DOI] [PubMed] [Google Scholar]

[R54] 54.Mummadisetti M., Su X., Lui H., An approach to nearest neighbor analysis of pigment-protein complexes using chemical cross-linking in combination with mass spectrometry. Methods Enzymol. 680, 139–162 (2022). [DOI] [PubMed] [Google Scholar]

[R55] 55.Albanese P., Tamara S., Saracco G., Scheltema R. A., Pagliano C., How paired PSII-LHCII supercomplexes mediate the stacking of plant thylakoid membranes unveiled by structural mass-spectrometry. Nat. Commun. 11, 1361 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R56] 56.Sinz A., Cross-linking/mass spectrometry for studying protein structures and protein-protein interactions: Where are we now and where should we go from here? Angew. Chem. Int. Ed. Engl. 57, 6390–6396 (2018). [DOI] [PubMed] [Google Scholar]

[R57] 57.Leitner A., Walzthoeni T., Kahraman A., Herzog F., Rinner O., Beck M., Aebersold R., Probing native protein structures by chemical cross-linking, mass spectrometry, and bioinformatics. Mol. Cell. Proteomics 9, 1634–1649 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R58] 58.Shukla M. K., Watanabe A., Wilson S., Giovagnetti V., Moustafa E. I., Minagawa J., Ruban A. V., A novel method produces native light-harvesting complex II aggregates from the photosynthetic membrane revealing their role in nonphotochemical quenching. J. Biol. Chem. 295, 17816–17826 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R59] 59.Kirchhoff H., Hinz H. J., Rosgen J., Aggregation and fluorescence quenching of chlorophyll a of the light-harvesting complex II from spinach in vitro. Biochim. Biophys. Acta. 1606, 105–116 (2003). [DOI] [PubMed] [Google Scholar]

[R60] 60.Natali A., Gruber J. M., Dietzel L., Stuart M. C., van Grondelle R., Croce R., Light-harvesting complexes (LHCs) cluster spontaneously in membrane environment leading to shortening of their excited state lifetimes. J. Biol. Chem. 291, 16730–16739 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R61] 61.Kouril R., Wientjes E., Bultema J. B., Croce R., Boekema E. J., High-light vs. low-light: Effect of light acclimation on photosystem II composition and organization in Arabidopsis thaliana. Biochim. Biophys. Acta. 1827, 411–419 (2013). [DOI] [PubMed] [Google Scholar]

[R62] 62.Kirchhoff H., Diffusion of molecules and macromolecules in thylakoid membranes. Biochim. Biophys. Acta. 1837, 495–502 (2014). [DOI] [PubMed] [Google Scholar]

[R63] 63.Haferkamp S., Haase W., Pascal A. A., van Amerongen H., Kirchhoff H., Efficient light harvesting by photosystem II requires an optimized protein packing density in grana thylakoids. J. Biol. Chem. 285, 17020–17028 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R64] 64.Joliot P., Lavergne J., Béal D., Plastoquinone compartmentation in chloroplasts. I. Evidence for domains with different rates of photo-reduction. Biochim. Biophys. Acta. 1101, 1–12 (1992). [Google Scholar]

[R65] 65.De Bianchi S., Dall'Osto L., Tognon G., Morosinotto T., Bassi R., Minor antenna proteins CP24 and CP26 affect the interactions between photosystem II subunits and the electron transport rate in grana membranes of Arabidopsis. Plant Cell 20, 1012–1028 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R66] 66.Kovács L., Damkjær J., Kereïche S., Ilioaia C., Ruban A. V., Boekema E. J., Jansson S., Horton P., Lack of the light-harvesting complex CP24 affects the structure and function of the grana membranes of higher plant chloroplasts. Plant Cell 18, 3106–3120 (2006). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R67] 67.Guardini Z., Gomez R. L., Caferri R., Dall'Osto L., Bassi R., Loss of a single chlorophyll in CP29 triggers re-organization of the photosystem II supramolecular assembly. Biochim. Biophys. Acta. 1863, 148555 (2022). [DOI] [PubMed] [Google Scholar]

[R68] 68.Thompson L. K., Brudvig G. W., Cytochrome b-559 may function to protect photosystem II from photoinhibition. Biochemistry 27, 6653–6658 (1988). [DOI] [PubMed] [Google Scholar]

[R69] 69.Bondarava N., Gross C. M., Mubarakshina M., Golecki J. R., Johnson G. N., Krieger-Liszkay A., Putative function of cytochrome b559 as a plastoquinol oxidase. Physiol. Plant. 138, 463–473 (2010). [DOI] [PubMed] [Google Scholar]

[R70] 70.Stewart D. H., Brudvig G. W., Cytochrome b₅₅₉ of photosystem II. BBA-Bioenergetics 1367, 63–87 (1998). [DOI] [PubMed] [Google Scholar]

[R71] 71.Ortega J. M., Hervás M., De La Rosa M. A., Losada M., Redox properties of cytochrome b559 in photosynthetic membranes from the Cyanobacterium Synechocystis sp. PCC 6803. J. Plant Physiol. 144, 454–461 (1994). [Google Scholar]

[R72] 72.Cumberworth A., Lamour G., Babu M. M., Gsponer J., Promiscuity as a functional trait: Intrinsically disordered regions as central players of interactomes. Biochem. J. 454, 361–369 (2013). [DOI] [PubMed] [Google Scholar]

[R73] 73.Caffarri S., Kouril R., Kereïche S., Boekema E. J., Croce R., Functional architecture of higher plant photosystem II supercomplexes. EMBO J. 28, 3052–3063 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R74] 74.Kastner B., Fischer N., Golas M. M., Sander B., Dube P., Boehringer D., Hartmuth K., Deckert J., Hauer F., Wolf E., Uchtenhagen H., Urlaub H., Herzog F., Peters J. M., Poerschke D., Lührmann R., Stark H., GraFix: Sample preparation for single-particle electron cryomicroscopy. Nat. Methods 5, 53–55 (2008). [DOI] [PubMed] [Google Scholar]

[R75] 75.Shevchenko A., Tomas H., Havli J., Olsen J. V., Mann M., In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006). [DOI] [PubMed] [Google Scholar]

[R76] 76.Liu H., Cyanobacterial phycobilisome allostery as revealed by quantitative mass spectrometry. Biochemistry 62, 1307–1320 (2023). [DOI] [PubMed] [Google Scholar]

[R77] 77.Chen Z. L., Meng J. M., Cao Y., Yin J. L., Fang R. Q., Fan S. B., Liu C., Zeng W. F., Ding Y. H., Tan D., Wu L., Zhou W. J., Chi H., Sun R. X., Dong M. Q., He S. M., A high-speed search engine pLink 2 with systematic evaluation for proteome-scale identification of cross-linked peptides. Nat. Commun. 10, 3404 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R78] 78.Punjani A., Rubinstein J. L., Fleet D. J., Brubaker M. A., cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017). [DOI] [PubMed] [Google Scholar]

[R79] 79.Farber A., Young A. J., Ruban A. V., Horton P., Jahns P., Dynamics of xanthophyll-cycle activity in different antenna subcomplexes in the photosynthetic membranes of higher plants (The relationship between zeaxanthin conversion and nonphotochemical fluorescence quenching). Plant Physiol. 115, 1609–1618 (1997). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R80] 80.Fragata M., Dudekula S., Nonlinear enhancement of oxygen evolution in thylakoid membranes: Modeling the effect of light intensity and beta-cyclodextrin concentration. J. Phys. Chem. B 109, 14707–14714 (2005). [DOI] [PubMed] [Google Scholar]

[R81] 81.Pesaresi P., Hertle A., Pribil M., Kleine T., Wagner R., Strissel H., Ihnatowicz A., Bonardi V., Scharfenberg M., Schneider A., Pfannschmidt T., Leister D., Arabidopsis STN7 kinase provides a link between short- and long-term photosynthetic acclimation. Plant Cell 21, 2402–2423 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R82] 82.Zheng S. Q., Palovcak E., Armache J.-P., Verba K. A., Cheng Y., Agard D. A., MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R83] 83.Rohou A., Grigorieff N., CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R84] 84.Afonine P. V., Poon B. K., Read R. J., Sobolev O. V., Terwilliger T. C., Urzhumtsev A., Adams P. D., Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol. 74, 531–544 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R85] 85.Pettersen E. F., Goddard T. D., Huang C. C., Couch G. S., Greenblatt D. M., Meng E. C., Ferrin T. E., UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). [DOI] [PubMed] [Google Scholar]

[R86] 86.Emsley P., Lohkamp B., Scott W. G., Cowtan K., Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R87] 87.Arnon D. I., Copper enzymes in isolated chloroplasts. polyphenoloxidase in Beta Vulgaris. Plant Physiol. 24, 1–15 (1949). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R88] 88.Kashino Y., Koike H., Satoh K., An improved sodium dodecyl sulfate-polyacrylamide gel electrophoresis system for the analysis of membrane protein complexes. Electrophoresis 22, 1004–1007 (2001). [DOI] [PubMed] [Google Scholar]

[R89] 89.van Stokkum I. H. M., Larsen D. S., van Grondelle R., Global and target analysis of time-resolved spectra. Biochim. Biophys. Acta. 1657, 82–104 (2004). [DOI] [PubMed] [Google Scholar]

[R90] 90.Huyer J., Eckert H.-J., Irrgang K.-D., Miao J., Eichler H.-J., Renger G., Fluorescence decay kinetics of solubilized pigment protein complexes from the distal, proximal, and core antenna of photosystem II in the range of 10–277 K and absence or presence of sucrose. J. Phys. Chem. B 108, 3326–3334 (2004). [Google Scholar]

[R91] 91.Wilson S., Li D. H., Ruban A. V., The structural and spectral features of light-harvesting complex II proteoliposomes mimic those of native thylakoid membranes. J. Phys. Chem. Lett. 13, 5683–5691 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R92] 92.Weisz D. A., Johnson V. M., Niedzwiedzki D. M., Shinn M. K., Liu H., Klitzke C. F., Gross M. L., Blankenship R. E., Lohman T. M., Pakrasi H. B., A novel chlorophyll protein complex in the repair cycle of photosystem II. Proc. Natl. Acad. Sci. U.S.A. 116, 21907–21913 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Architecture and functional regulation of a plant PSII-LHCII megacomplex

Jianyu Shan

Dariusz M Niedzwiedzki

Rupal S Tomar

Zhenfeng Liu

Haijun Liu

Roles

Abstract

INTRODUCTION

RESULTS

Overall assembly of the C4S4M2 PSII-LHCII megacomplex

Fig. 1. Cryo-EM density of the C4S4M2 PSII-LHCII megacomplex from spinach.

Table 1. Statistics of the cryo-EM data collection, processing, and refinement of spinach type I C4S4M2 PSII-LHCII megacomplex structure.

Table 2. The protein and cofactor components in the final structural model of spinach type I C4S4M2 PSII-LHCII megacomplex.

Fig. 2. The location and structural characteristics of PsbR and PsbY.

Interactions of PsbR and PsbY with nearby PSII subunits

Arrangement of peripheral antenna complexes in the megacomplex

Fig. 3. The roles of peripheral antenna complexes in establishing potential energy transfer pathways in the C4S4M2 megacomplex.

Supramolecular basis for electron transport regulation

Fig. 4. The local environments of the QB sites and functional characteristics of the megacomplex.

Table 3. Functional characterization of the PSII-LHCII megacomplex (PSII-MC) and supercomplex (PSII-SC) samples.

Table 4. Flash-induced fluorescence relaxation kinetics of the PSII-LHCII megacomplex (PSII-MC) and supercomplex (PSII-SC) samples.

Flexible stromal domain of PsbR as probed with cross-linking MS

Fig. 5. Stromal side–coupled PSII supercomplex organization model mediated by PsbR.

Evaluation of functional roles of PsbR in PSII membrane via static and time-resolved optical spectroscopies using PsbR deletion mutant and WT

Fig. 6. Changes in PSII-LHCII aggregation in BBY particles from WT and ΔPsbR A. thaliana investigated with absorption and TRF at 77 K.

DISCUSSION

METHODS

Thylakoid membrane preparation and purification of PSII-LHCII megacomplexes

Electrophoresis and immunoblotting

Mass spectrometry

Protein identification

Chemical cross-linking MS and data analysis

Negative-staining EM analysis

Absorption spectra measurement

Pigment analysis through the HPLC method

77 K chlorophyll fluorescence emission measurement

Measurement of the steady state OE activity

Flash-induced fluorescence relaxation kinetics

Cryo-EM data collection, processing, classification, and reconstruction

Model building and refinement

Bioinformatic analysis of higher plant PsbR

Growth of A. thaliana plants

Isolation and characterization of PSII particles (BBY)

Basic spectroscopic characterization of BBY from WT and ΔPsbR A. thaliana

Time-resolved fluorescence

Global analysis of TRF datasets

Acknowledgments

Supplementary Materials

This PDF file includes:

REFERENCES AND NOTES

Associated Data

Supplementary Materials

ACTIONS

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Overall assembly of the C₄S₄M₂ PSII-LHCII megacomplex

Fig. 1. Cryo-EM density of the C₄S₄M₂ PSII-LHCII megacomplex from spinach.

Table 1. Statistics of the cryo-EM data collection, processing, and refinement of spinach type I C₄S₄M₂ PSII-LHCII megacomplex structure.

Table 2. The protein and cofactor components in the final structural model of spinach type I C₄S₄M₂ PSII-LHCII megacomplex.

Fig. 3. The roles of peripheral antenna complexes in establishing potential energy transfer pathways in the C₄S₄M₂ megacomplex.

Fig. 4. The local environments of the Q_B sites and functional characteristics of the megacomplex.