Osaka Metropolitan University and the University of Osaka perform analysis on the triplet excited state of the green alga Codium fragile

In order to elucidate the mechanisms by which photosynthetic organisms efficiently use sunlight for chemical reactions, it is extremely important to accumulate precise structural and light response experimental data on the photosynthetic antenna LHCII, which consists of similar proteins that differ only in the structure and arrangement of pigments.

A research group led by Associate Professor Ritsuko Fujii of the Research Center for Artificial Photosynthesis at Osaka Metropolitan University, Specially Appointed Research Scientist (Full-time) Soichiro Seki of the Institute for Protein Research at the University of Osaka, and Associate Professor Alessandro Agostini of the University of Padova in Italy used EPR spectroscopy to analyze the photosynthetic antennae of spinach and the green alga Codium fragile, revealing that the quenching efficiency by carotenoids in the organism is extremely high. Furthermore, by utilizing multiple analytical methods, the researchers revealed that siphonein, which binds near carotenoids, has a mechanism to prevent excess energy by receiving and efficiently releasing excess excitation energy from chlorophyll a. In the future, the characteristics of the molecular structure of carotenoids that increase quenching efficiency are expected to become clearer. Ultimately, it is anticipated that molecular design of pigments to optimize photosynthetic antennae will become possible. The research was published in Cell Reports Physical Science.

In the photosynthetic antenna LHCII, an extremely large number of pigments (14 chlorophyll molecules and 4 carotenoid molecules per monomer) are densely packed in a confined space. When this is photoexcited, numerous excited states are generated simultaneously. Initially, a singlet excited state (S1) is generated, accumulating in chlorophyll a, which has the lowest energy among the pigments. From there, a triplet excited state is generated through intersystem crossing.

Carotenoids quench this triplet excited state of chlorophyll a, which is harmful to living organisms. The key to the quenching mechanism is how efficiently it can be deactivated. Therefore, EPR spectroscopy is necessary to specifically detect only the triplet excited state. Because EPR signals are weak, the research group has developed equipment capable of extremely sensitive detection by eliminating thermal noise through measurements at low temperatures and accumulating only synchronized signals using the lock-in amplifier method.

Using this equipment to measure time-resolved EPR, they revealed that while weak triplet excited states of chlorophyll were observed in spinach, they were not observed at all in C. fragile. In other words, the quenching efficiency by carotenoids is extremely high, completely eliminating the triplet state of chlorophyll. This "formation of carotenoid triplet states from the excited state of chlorophyll a" was clearly correlated by combining the T-S method.

Within the light-harvesting antenna of C. fragile, the carotenoids that are close enough to chlorophyll for this energy transfer to occur are siphonein at the L1 site and siphonaxanthin at the L2 site. Because the positional relationship between chlorophyll and carotenoids differs subtly at these two locations, the energy transfer efficiency should also differ. Therefore, based on the precise structure obtained by cryo-electron microscopy, simulations using the DFT (density functional theory) method were performed, revealing that this extremely high-efficiency quenching is due to siphonein at the L1 site.

Alternative technologies have not yet been obtained for the initial light-harvesting component in the photosynthetic mechanism that uses sunlight to cause photochemical reactions and leads to material production. Elucidating the light-harvesting mechanisms possessed by photosynthetic organisms is becoming important for the creation of a core theory for technology to use light at the level of ambient light for energy production, rather than special light with large amounts of energy that can cause burns. The results from this study clearly demonstrated that the special carotenoids possessed by marine photosynthetic organisms not only play a role in harvesting the blue-green light available in the ocean but also serve to improve biological protection functions by increasing quenching efficiency. This is currently only one example. By increasing the number of cases in the future, it is expected that the characteristics of the molecular structure of carotenoids that increase quenching efficiency become clearer and that ultimately molecular design of pigments to optimize photosynthetic antennae become possible.

Fujii commented: "The catalyst for starting this research was receiving an enthusiastic message from Dr. Agostini immediately after publishing a paper on the high-resolution structure of LHCII from the green alga Codium fragile in 2022. The mechanism for utilizing sunlight depends on energy exchange between pigments and can only be revealed by performing detection of precise structure and light response and quantum chemical calculations that connect these. This time, we were able to create a good team that shares values, and I truly felt that research has no borders."

Agostini commented: "Our research on Cf-LHCII led to many new discoveries by combining expertise in structural analysis, spectroscopy, and theory. There is still much to be clarified about the photosynthesis of lesser-known types of organisms. In this research, it was revealed that the antenna structure possessed by photosynthetic green algae has exceptional photoprotective properties."

Journal Information
Publication: Cell Reports Physical Science
Title: Siphonein enables an effective photoprotective triplet-quenching mechanism in green algal light-harvesting complexes
DOI: 10.1016/j.xcrp.2025.102873