Structure of Reconstituted Photosystem II Extrinsic Proteins Reveals a Cause of Reduced Oxygen Evolution Activity

【Research Highlights】

We determined the three-dimensional structure at a high resolution of 2.0 Å of the giant protein complex "Photosystem II (PSII)," which decomposes water and generates oxygen during photosynthesis, after its three extrinsic proteins were removed and then reattached.

It was found that the reattached three extrinsic proteins (PsbO, PsbV, and PsbU) returned to their correct positions on the main body of PSII.

On the other hand, slight changes were observed in the orientation of bicarbonate ions involved in the oxygen evolution reaction and the arrangement of water molecules.

Specifically, the arrangement of water molecules and the hydrogen bond network were disrupted in the "O1 channel," which is thought to be the pathway for water in the oxygen evolution reaction.

This research revealed that even if the extrinsic proteins return to their correct positions, the oxygen evolution activity of photosynthesis may decrease because the arrangement of water molecules and small molecules inside PSII is not completely restored.

【Research Overview】

Associate Professor Ryo Nagao of the Faculty of Agriculture, Shizuoka University, along with Assistant Professor Yoshiki Nakajima and Professor Jian-Ren Shen of the Institute for Fundamental Chemistry (Interdisciplinary Field), Okayama University, determined the molecular structure of Photosystem II (PSII) (Note 1) from the thermophilic cyanobacterium Thermosynechococcus vulcanus after removing and reattaching the three extrinsic proteins PsbO, PsbV, and PsbU (Note 2), using X-ray crystallography (Note 3).

The analysis revealed that the reattached PsbO, PsbV, and PsbU all bound to their correct positions on the main body of PSII. While the overall structure around the metal cluster involved in the oxygen evolution reaction was largely maintained, changes were observed in the orientation of bicarbonate ions and their interactions with surrounding water molecules. Furthermore, disruptions in the arrangement of water molecules and the hydrogen bond network were confirmed in the O1 channel, which is thought to be the pathway for water necessary for the oxygen evolution reaction. These results suggest that even if the extrinsic proteins return to their correct positions, the arrangement of water molecules and small molecules inside PSII may not be completely restored, potentially leading to a decrease in oxygen evolution activity.

This research was published in the international journal "ACS Catalysis" on June 25, 2026.

【Researcher Comments】

Ryo Nagao, Associate Professor, Faculty of Agriculture, Shizuoka University

PSII is an extremely important protein complex for life on Earth, decomposing water and generating oxygen. In this study, we structurally demonstrated that even when extrinsic proteins return to their correct positions, slight disruptions remain in the arrangement of water molecules and bicarbonate ions inside.

【Research Background】

Oxygenic photosynthesis (Note 4) is a fundamental metabolic process by which plants, algae, and cyanobacteria utilize sunlight to split water and produce oxygen. PSII, which plays a central role in this reaction, is a giant membrane protein complex containing chlorophylls, carotenoids, and metal clusters. By extracting electrons and protons from water using the energy of sunlight, it supports the oxygen environment on Earth.

The oxygen evolution reaction in PSII proceeds at the Mn4CaO5 cluster, composed of manganese, calcium, and oxygen. Water molecules and amino acid residues are precisely arranged around this metal cluster, and extrinsic proteins such as PsbO, PsbV, and PsbU bind to the outside of PSII to maintain a structural environment suitable for the oxygen evolution reaction.

Reconstitution experiments, where extrinsic proteins are removed from PSII and then reattached, have been conducted previously. While oxygen evolution activity is largely restored by reconstitution, it does not fully recover compared to native PSII, and the reasons for this have not been fully understood. Possible explanations include the possibility that the extrinsic proteins do not return to their original positions correctly, or that even if they do, subtle changes remain in the oxygen-evolving center and its surrounding molecular environment.

By determining the high-resolution structure of PSII with reattached PsbO, PsbV, and PsbU using X-ray crystallography, we have now clarified that these extrinsic proteins return to their correct positions on the main body of PSII. On the other hand, local disruptions were observed in the orientation of bicarbonate ions and their interactions with surrounding water molecules, as well as in the arrangement of water molecules and the hydrogen bond network in the O1 channel. This suggests that the incomplete recovery of PSII's oxygen evolution activity may be related to disruptions in the precise arrangement of water molecules and small molecules inside, rather than large structural changes in the entire protein.

【Research Achievements】

This study revealed the high-resolution three-dimensional structure of PSII with the reattached extrinsic proteins PsbO, PsbV, and PsbU. The main achievements are as follows (Figure 1):

Determination of the high-resolution structure of reconstituted PSII

In this study, we determined the three-dimensional structure at a resolution of 2.0 Å of PSII from the thermophilic cyanobacterium Thermosynechococcus vulcanus after the removal and reattachment of PsbO, PsbV, and PsbU. This provides a basis for comparing reconstituted PSII at the atomic level.

Confirmation of correct reattachment of extrinsic proteins

The analysis revealed that the reattached PsbO, PsbV, and PsbU all bound to their original positions on the main body of PSII. Furthermore, the overall structure of PSII was very similar to that of native PSII, indicating that the reattachment of extrinsic proteins did not cause significant disruption to the entire PSII structure.

Detection of local changes while maintaining the overall structure of the oxygen-evolving center

The overall structure of the Mn4CaO5 cluster, the center of the oxygen evolution reaction, was largely maintained in the reconstituted PSII. However, local structural changes were confirmed around the oxygen-evolving center, including a slight shortening of the Mn–O bond distance. This change is interpreted as a subtle change in the microenvironment of the oxygen-evolving center, rather than a major structural collapse.

Discovery of structural changes around bicarbonate ions

In the reconstituted PSII, the orientation of bicarbonate ions involved in electron transfer was altered. Furthermore, changes were observed in the interactions with surrounding amino acids, such as D1-Y246 and D1-E244, and water molecules. These changes may affect the environment for electron transfer within PSII and contribute to the decrease in oxygen evolution activity.

Confirmation of disruption in the water molecule network in the O1 channel

In the O1 channel, considered one of the pathways for water in the oxygen evolution reaction, some water molecules observed in native PSII were less clearly observed in reconstituted PSII. Changes were also confirmed in the hydrogen bond network around D1-N298. This suggests that the arrangement and mobility of water molecules have changed in the reconstituted PSII, potentially affecting water supply and the stability of the reaction site.

Confirmation that Cl-1 and O4 channels are largely unchanged

On the other hand, no significant structural changes were observed between native PSII and reconstituted PSII in the Cl-1 and O4 channels, which are thought to be other water/proton transport pathways within PSII. This indicates that the effects of reconstitution are not uniformly distributed throughout PSII but are relatively localized changes around the bicarbonate ions and in the O1 channel.

These findings indicate that even when extrinsic proteins are correctly reattached, the arrangement of water molecules and small molecules inside PSII is not completely restored. The incomplete recovery of PSII's oxygen evolution activity is thought to be related to disruptions in the precise arrangement of molecules around bicarbonate ions and in the O1 channel, rather than large structural changes in the entire protein. This is an important finding demonstrating that the water-splitting reaction in photosynthesis is supported by a precise molecular network, including individual water molecules.

Figure 1. Overview of this research.

【Publication Information】

Journal: ACS Catalysis

Paper Title: Structural basis for impaired oxygen evolution in extrinsic-protein-reconstituted photosystem II

Authors: Yoshiki Nakajima, Koji Kato, Jian-Ren Shen, Ryo Nagao

DOI: https://doi.org/10.1021/acscatal.6c03852

【Glossary】

Note 1: Photosystem II (PSII)

A protein complex in photosynthesis carried out by plants, algae, and cyanobacteria that decomposes water and generates oxygen. It uses the energy of sunlight to extract electrons from water, producing oxygen in the process.

Note 2: Extrinsic proteins

Proteins that bind to the outside of PSII and function to stabilize the oxygen evolution reaction. In this study, we focused on three types of proteins that bind to cyanobacterial PSII: PsbO, PsbV, and PsbU.

Note 3: X-ray crystallography

A technique used to determine the positions of atoms constituting a molecule by crystallizing molecules such as proteins and analyzing the diffraction patterns obtained by irradiating the crystals with X-rays. Because it can reveal the three-dimensional structure of proteins at the atomic level, it allows for detailed analysis of which molecules bind where and how water molecules and metal clusters are arranged. In this study, this technique was used to determine the structure of reconstituted PSII at a resolution of 2.0 Å.

Note 4: Oxygenic photosynthesis

There are two types of photosynthesis: oxygenic and anoxygenic. Oxygenic photosynthesis is driven by membrane protein complexes called Photosystem I, cytochrome b6f, Photosystem II, and ATP synthase, and uses light energy to synthesize organic matter and oxygen from water and carbon dioxide. It is thought that anoxygenic photosynthetic organisms evolved into oxygenic photosynthetic organisms.