DS0206 - Efficacité énergétique des procédés et des systèmes

Solar-driven water-splitting enzyme photosystem II probed by Far-IR spectroscopy – PS2FIR

Photolysis of water by photosynthetic organisms

The oxidation mechanism of water to molecular oxygen at the manganese and calcium complex (Mn4CaO5) of Photosystem II was studied by infrared spectroscopy in the far IR domain. The internal structure of the complex and its interactions with water molecules were probed.

Mechanism for photolysis of water into molecular oxygen during photosynthesis, studied by infrared spectroscopy in the far IR domain

The use of solar energy to produce energy and non-polluting substances is a major challenge for the future. Plants and cyanobacteria have developed an enzyme, Photosystem II (PSII), capable of splitting water molecules into molecular oxygen, protons and energy-rich electrons using solar energy. The PS2FIR project aims at better understanding this water photolysis mechanism, which is an excellent model for the development of non-polluting photocatalysts that can use water molecules as a substrate and produce oxygen as the only «waste« product.<br />The active site of PSII is an Mn4CaO5 complex where water oxidation occurs after a succession of four photochemical reactions. Remarkably, the mechanism minimizes the energy required for water oxidation by combining proton transfer reactions with electron transfer reactions. Crystallography data and theoretical chemistry calculations revealed the structure of a relaxed state of the complex and allowed mechanistic predictions. However, many questions are open and experimental approaches to answer them are rare and require very specific developments. This project proposes to use the far IR domain to probe the properties of water molecules and the insertion of oxygen into the complex, as well as Mn spin state changes and proton movements as the process progresses.

The properties of the PSII Mn4CaO5 complex and its interactions with its environment can be studied by its vibrational modes that absorb in the far infrared domain. The frequency and intensity of these modes are sensitive to the structure, oxidation state of the complex and interactions with its ligands. This project allowed us to buy and optimize a commercial infrared spectrometer to access this spectral domain and record the difference spectra between two redox states of the Mn4CaO5 complex, induced by photochemistry using a nanosecond pulse laser. We have optimized a sample wheel to measure multiple samples and accurately control the temperature and humidity of the samples, and automated the accumulation of spectra during experiments lasting about 12 hours. Part of the experiments required the brightness of synchrotron light. The team of the AILES beam line of the French synchrotron SOLEIL has developed a cryogenic system to study the spin transition of the complex induced by irradiation at 780 nm of the sample, as well as a sample wheel that can be thermostated between -10°C and +30°C. These new devices are available to all users of the beam line. . During the 4 years of the project, 8 projects using these two experimental set-ups (1 week of beamtime each) were accepted by the SOLEIL program committee. In addition, 15 weeks of in house beamtime using the internal source of the spectrometer and also the synchrotron beam, were devoted to this project.

These devices have allowed us to obtain very good quality spectra despite the very small size of the bands. The infrared spectra characteristic of the photo-oxidation of the PSII Mn4CaO5 complex from the dark stable state S1 to the oxidized state S2 revealed vibrations of distinct elements of the complex through isotopic water labelling (2H2O, H218O, Illustration) and replacement of calcium by strontium at the site. The transition S2 Low Spin (S=1/2) to S2 High Spin (S=5/2), involved in the enzyme cycle, was probed by the use of a mutant and by infrared photochemistry at cryogenic temperature. The identification of spectral signatures of an oxygen incorporated in the complex after one catalytic cycle and of a water molecule allow us to discuss the theoretical models proposed for the oxidation reaction of water.

The work carried out demonstrates the interest of the far IR domain in deciphering the catalytic cycle of PSII. This work is a basis for exploring the different stages of the catalytic cycle and the evolution of the key signals identified. In addition, we started a collaboration to exploit our experimental results with a theoretical analysis of the normal modes of the complex, to clarify the interpretation of the results and to better understand the catalytic mechanism. Ultimately, the understanding of the mechanism will serve as a model for bio-inspired systems.

This project has resulted in 4 publications and 3 others are in preparation, notably concerning photochemistry results between PSII S1 and S2 states and IR photochemistry. Teams 2&3 studied the effects of a V185T point mutation on PSII Mn4CaO5 spin states: Biochemistry and Biophysica Acta (BBA) - Bioenergetics (2018), 1859, 1259-1273. Team 2 published two articles on the spin transition in S2 Boussac et al (2018) BBA Bioenerg. 1859, 1259-1273 and the transition temperatures S2HS to S3 and the mechanical implications of Boussac A. (2019) BBA Bioenerg. 1860,508-518. During the optimization of the FTIR spectrometer, Team 1 performed a vibrational study of a ferredoxin purified by Team 2, which was found to have remarkable properties in the transfer of photosynthetic electrons Motomura et al (2019) BBA Bioenerg. 148084. doi: 10.1016/d.bbabio.

Solar-driven hydrogen production from the abundant and cheap electron source water is a promising way to produce renewable energy. Plants and cyanobacteria have developed a water splitting enzyme which is able to oxidize water into molecular oxygen, protons and electrons using visible light energy within the membrane protein photosystem II. The heart of the enzyme is a Mn4CaO5 cluster at which water oxidation takes place following four sequential light-induced steps. Reactions at the Mn4CaO5 cluster consist of concerted electron and proton transfer, and form intermediate states that minimize the activation energy necessary for the water oxidation process. Photosystem II is thus a paradigm for engineering bio-inspired solar energy converting applications.

A recent high-resolution three-dimensional structure of photosystem II gave a precise arrangement of the Mn-Ca cluster necessary for water oxidation. In addition, the combination of theoretical catalytic models with experimental data from numerous state-of-the-art spectroscopic techniques have given a possible view of Mn oxidation states during water oxidation, of water fixation steps, have revealed the importance of a set of amino acids in the catalytic mechanism, and given hints on proton transfer reactions involving extended hydrogen bonding networks. Despite these remarkable progresses in recent years, key questions remain opened. They concern the position of reactive molecules, the formation mechanisms of the oxygen molecule itself, and relaxation processes at the Mn4CaO5 cluster involving spin-state transitions and concerted electron and proton transfer.

The PS2FIR project will contribute to answer these questions. We will gather the complementary expertise of three research teams: Team 1, R. Hienerwadel & C. Berthomieu, UMR 7265; Team 2, A. Boussac UMR 9198; and Team 3, J.B. Brubach & P. Roy, Synchrotron SOLEIL, to probe light- and near infared (NIR)- induced transitions and spin conversions at the Mn4CaO5 cluster, using state-of-the-art far-infrared FTIR difference spectroscopy. Vibrational modes in the far-infrared down to 10 cm-1 will allow probing the valence state of the Mn ions, cluster conformation, and Mn-O/Ca-O interactions. Of particular interest will be the identification of libration and connectivity modes of water molecules associated to the cluster below 300 cm-1 during the reaction cycle. To overcome the challenge of exploiting small-bands in the Far-infrared domain, setups will be optimized to probe different samples in parallel and to optimize NIR-induced spin-state transitions by controlled temperature jumps. We will also benefit from the brilliance of the synchrotron AILES beamline at SOLEIL. Highly resistant photosystem II from Thermosynechoccocus elongatus prepared to precisely select within heterogeneous oxidation or spin states of photosystem II will allow to decipher the molecular origin of different Mn4CaO5 cluster conformations, and ultimately to contribute to our understanding of water oxidation and O-O bond formation.

Project coordination

Rainer Hienerwadel (Biologie Végétale et Microbiologie Environnementales)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


UMR9198 CEA / DSV / Institut de Biologie et Technologies de Saclay (iBiTec-S)
UMR7265 (CNRS-CEA-Aix-Marseille Université) Biologie Végétale et Microbiologie Environnementales

Help of the ANR 461,556 euros
Beginning and duration of the scientific project: September 2015 - 36 Months

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