Effects of high light and elevated temperature on the structure of Photosystem II core complexes isolated from Arabidopsis thaliana

Technical Summary: Effects of High Light and Elevated Temperature on the Structure of Photosystem II Core Complexes

Problem Statement
Photosystem II (PS II) is the membrane-bound pigment-protein complex responsible for water oxidation and oxygen evolution in photosynthesis. While light drives these reactions, excessive light intensity and elevated temperatures cause photodamage, leading to the degradation of the D1 subunit and the loss of oxygen-evolving activity. Although the repair cycle involving D1 degradation and replacement is well-characterized, the specific structural consequences of high light and heat stress on the intact PS II core complex, prior to proteolytic digestion, remain incompletely understood. Previous studies have relied on biochemical assays or in vivo observations, which often mask the initial structural states of damaged complexes due to rapid repair mechanisms.

Methodology
This study employed an in vitro exploratory approach using Arabidopsis thaliana PS II core complexes. The researchers isolated solubilized PS II complexes and subjected them to extreme stress conditions: 2000 µmol photons m⁻² s⁻¹ (high light) for 30 minutes, resulting in a temperature increase to 29°C. This treatment was designed to maximize damage while retaining sufficient intact complexes for structural analysis.

Structural determination was performed using single-particle cryo-electron microscopy (cryo-EM). Approximately 57,560 high-quality particles were processed to generate a Coulomb potential map with a global resolution of 2.74 Å. This "high light" structure was compared against a previously published control structure (PDB: 9I4T) of intact Arabidopsis PS II vitrified under dim light conditions (2.44 Å resolution). The control map was low-pass filtered to 2.74 Å to ensure a fair comparison. Model building involved fitting the control structure into the high-light map, deleting subunits lacking density, and manually refining residues using COOT and Phenix. Additional analyses included occupancy estimation (OccuPy), channel calculations (CAVER), and oxygen evolution assays to correlate structural changes with functional loss.

Key Results

1. Loss of Peripheral Subunits: The high-light treatment resulted in the complete loss of density for the strongly bound LHC II trimer antenna, as well as the CP26 and CP29 linker subunits. Furthermore, small transmembrane subunits (PsbJ, PsbW, PsbZ) and extrinsic subunits (PsbP, PsbQ) were absent. Notably, the Mn-cluster remained largely intact, with occupancy comparable to the control, although the D1:Glu189 residue, a ligand to the Mn-cluster, shifted position, moving out of ligation distance with the Mn1 atom.
2. Acceptor Side Damage and Loop Variability: Significant loss of electron density was observed in specific hydrophilic loops of the D1 and D2 subunits (D1:226-232, D1:242-245, D2:226-230, D2:233-240). This suggests damage-induced structural variability or mobility in these regions.
3. D1:His252 Movement and Protease Triad Formation: A critical structural change was observed in the D1:260-264 loop near the QB-binding pocket. The D1:His252 residue rotated to face inward toward the empty QB-binding site. This movement positions His252 within 4.2 Å of D1:Asn266 and 3.8 Å of D1:Ser264 (from the control structure). The authors propose this arrangement forms an atypical serine-protease triad (Ser-His-Asn) that may trigger the auto-proteolysis of the D1 subunit, a key step in the PS II repair cycle.
4. Bicarbonate Dissociation: The study confirmed the dissociation of the bicarbonate ligand from the non-heme iron (NHI) on the acceptor side. No density for bicarbonate was visible at a 3.0 RMSD contour level. At a lower contour (2.3 RMSD), a weak density appeared, which fit a monodentate bicarbonate or potentially an oxygen/water complex, but not the standard bidentate configuration. This supports the hypothesis that bicarbonate dissociation acts as a photoprotective "fuse" under high light.
5. Reaction Center Stability: Surprisingly, no obvious structural damage or resolution loss was detected around the P680 chlorophylls or pheophytin, suggesting that the core reaction center pigments remain structurally stable despite the loss of surrounding subunits and acceptor-side damage.

Significance and Claims
The authors claim this study provides the first high-resolution structural view of the average state of PS II complexes immediately following high light and elevated temperature stress, before proteolytic degradation occurs. The work offers direct structural evidence for:

• The passive loss of antenna and extrinsic subunits due to increased membrane fluidity in solubilized complexes.
• The specific dissociation of the bicarbonate ligand from the non-heme iron, validating biophysical models of photoprotection.
• A structural mechanism for D1 auto-cleavage, where the movement of D1:His252 creates a putative serine-protease triad in the absence of QB, explaining how D1 degradation can occur in isolated particles without accessory proteases.

The paper maintains a modest tone, acknowledging that the cryo-EM map represents an average of damaged complexes and that the proposed protease triad is a hypothesis based on structural proximity. The study establishes a foundation for future investigations into specific light/temperature conditions and exposure times, offering a new pathway to observe structural consequences of photoinhibition that are difficult to capture in vivo.