[Expired] PhD thesis in photosynthesis: Water oxidation mechanism of photosystem II probed in the far infrared range

with Rainer Hienerwadel in Marseille.


Photosystem II is the key enzyme for oxygenic photosynthesis in plants and cyanobacteria. The photochemical mechanisms at the heart of Photosystem II activity, which uses light energy to extract electrons from water by producing molecular oxygen, are a fundamental research model for the development of catalysts to generate fuels such as hydrogen from solar energy starting from an abundant and inexpensive substrate : water.

The particularity of the enzyme is that it is capable of breaking down this oxidation into four stages triggered by the sequential absorption of the energy of four photons, and to optimize the oxidation and proton transfer reactions at each stage, to greatly reduce the energies involved.

These reactions occur at a metal complex consisting of 4 manganese atoms and 1 calcium atom chelated by inorganic ligands and by amino acids of the protein. The three-dimensional structures recently obtained by X-ray diffraction gave a detailed image of the Mn4Ca complex (Figure 1 and ref included). Numerous spectroscopic techniques such as EXAFS, FTIR, EPR or mass spectrometry, and time-resolved UV-Vis or IR spectroscopy approaches, combined with the use of isotope labelling or chemical substitution of essential co-factors (Sr/Ca, Cl/Br,I) have played a decisive role in making it possible to specify, among other things, the role of different amino acids in the catalytic mechanisms, the redox state of Mn cations, the sequences for binding substrate water molecules and proton starters, etc…

However, the intrinsic mechanisms of water oxidation remain to be elucidated as well as the position of the reactive molecules or reaction intermediates. On the other hand, it is the use of techniques taken to their extremes (polarized EXAFS techniques, high frequency EPR, “flash” crystallography, etc…) that allow us to advance in the understanding of this complex system.

Figure 1: Structural scheme of the water oxidation catalytic site and currently proposed mechanism (hV corresponds to the absorption of one photon, the S states are the different charge states of the complex. Ref : a) H Nilsson, F Rappaport, A Boussac, J Messinger (2014) Nat. Commun. 5 4305-4311 ; b) N Cox, M Retegan, F Neese, D Pantazis, A Boussac, A., W Lubitz, (2014) Science 345, 804-808.

Objectives of the thesis

In this context, this thesis concerns the study of key steps of water oxidation by FTIR spectroscopy in a domain still little exploited: the far IR domain up to 100 cm-1, to probe the properties of the Mn4Ca complex, the oxo – hydroxo bonds, as well as the hydrogen bonds formed with the substrate water molecules.

This project will be supervised by Prof. R. Hienerwadel (AMU, LGBP) and Dr. C. Berthomieu (CEA, LIPM), specialists in FTIR spectroscopy, part of whose activity has concerned the study of photosystem II in the mid-IR domain [1-2 and ref. attached]. It involves collaboration with the team of Dr. A. Boussac (CNRS, CEA-Saclay), an internationally renowned specialist of Photosystem II and in particular in biochemistry and EPR spectroscopy approaches on Photosystem II [3-5 for recent publications] and the far infrared team Ailes of Synchrotron SOLEIL (Drs. J.B. Brubach and P. Roy) with whom we have been collaborating for several years to exploit the brightness of synchrotron radiation in the far IR domain [6-7]. We have done pioneering work in the use of FTIR difference spectroscopy in the far IR range to study in detail the properties of metallic sites and interactions with aqueous solvent [6-9].

Light-induced FTIR difference spectroscopy has become one of the main techniques for studying the oxidation mechanism of water [10,11], as it allows the identification of changes in chemical bond strength and structure, and thus allows the characterization of dynamic aspects of the reaction mechanisms, especially concerning the properties of water molecules or protonation/deprotonation reactions. In the mid-infrared range, many publications have investigated the role of amino acid ligands and residues located near the Mn4Ca cluster in catalysis [10,11]. More recently, polarized hydrogen bond networks and water molecules involved in weak hydrogen bonds with neighboring amino acids have been probed in the 2200-3700 cm-1 range [10,11].

Experimental data are scarce in the far infrared region, below 800 cm-1, where contributions are expected from the Mn4CaO5 cluster itself and from water molecules associated with this cluster [12-14]. Moreover, atomic resolution structure data were not available at that time and it remains to fully exploit this region and especially to extend the study to 100 cm-1, to probe not only the Mn ligand molecules but also the hydrogen bonds involving water molecules during the whole water oxidation cycle.

The thesis work will first of all concern the “S2-to-S3” transition by probing different states of the complex characterized by different EPR spin states, then the “S3-to-S0” transition which corresponds to the formation of the oxygen molecule. Model compounds will also be investigated in the Far-IR domain to support the assignments of protein bands.

For these studies, we will benefit from samples of Thermosynechoccocus elongatus photosystem II which will be prepared under different specific biochemical experimental conditions, chosen to favour the formation of the different forms of the S2 or S3 states. T. elongatus is a thermophilic cyanobacterium which has become one of the model organisms in photosynthesis research because its photosystem II is very robust, allowing enzymological studies without risk of enzymatic degradation. In particular, its photosystem II is always active under the dehydration conditions required for FTIR spectroscopic studies.


[1] Berthomieu C, Hienerwadel R (2009) Fourier Transform InfraRed (FTIR) spectroscopy. Photosynt.

Res. 101, 157-170 Review;

[2] Hienerwadel R, Diner BA, Berthomieu C (2008) Molecular origin of the pH dependence of tyrosine D kinetics and radical stability in photosystem II. Biochim. Biophys. Acta Bioenerg. 1777, 525-531.

[3]Rappaport F, Ishida N, Sugiura M, Boussac A (2011) Ca2+ determines the entropy changes associated with the formation of transition states during water oxidation by Photosystem II. Energ. Environ. Sci. 4, 2520-2524;

[4] Nilsson H., Rappaport F., Boussac A., Messinger J. (2014) Substrate-water exchange in Photosystem II is arrested prior to dioxygen formation. Nat. Commun. 5, 4305-4311

[5] Cox N, Retegan M, Neese F, Pantazis D A, Boussac A, Lubitz W (2014) Electronic structure of the oxygen evolving complex in Photosystem II prior to O-O bond formation Science 345, 804-808.

[6] Vita N, Brubach JB, Hienerwadel R, Bremond N, Berthomieu D, Roy P, Berthomieu C, (2013) Electrochemically induced far-infrared difference spectroscopy on metalloproteins using advanced synchrotron technology. Anal Chem. 85, 2891-8;

[7] Dalla Bernardina, S, Alabarse F,  Kalinko  A, Roy, P, Vita N,  Hienerwadel  R, Berthomieu C, Judein-stein P,  Zanotti J-M, Bantignies JL,  Haines J,  Catafesta  J, Creff G, Manceron L,  Brubach  JB (2014) New experimental set up for studing water trapping in condensed matter at AILES beamline of SO-LEIL.  Vibrational Spectroscopy 75 : 154–161

[8] Marboutin L, Petitjean H, Xerri B, Vita N, Dupeyrat F, Flament J-P, Berthomieu D & Berthomieu C

(2011) Profiling the active site of a cuproenzyme through its far-infrared fingerprint (680-50 cm-1).
Angew. Chem. Int. Ed. 50, 80623

[9] Motomura T, Zuccarello L, Sétif P, Boussac A, Umena Y, Lemaire D, Tripathy JN, Sugiur M, Hienerwadel R, Shen JR, Berthomieuet C (2019) An Alternative Plant-like Cyanobacterial Ferredoxin with Unprecedented Structural and Functional Properties.
Biochimica et Biophysica Acta (BBA) – Bioenergetics 1860 : 148084

[10] Debus RJ (2014) Biochim. Biophys. Acta, DOI: 10.1016/j.bbabio.2014.07.007 revue

[11] Noguchi T (2014) Biochim. Biophys Acta DOI: 10.1016/j.bbabio.2014.06.009 revue

[12] Chu HA, Sackett H, BabcockGT (2000) Biochemistry 39, 14371–14376; Chu HA, Hillier W, Law NA, Babcock GT (2001) Biochim. Biophys. Acta 1503, 69–82.

[13] Kimura Y, Ishii A, Yamanari T, Ono TA (2005) Biochemistry 44, 7613–7622; Kimura Y, Hasegawa K, Yamanari

T, Ono TA (2005) Photosynth. Res. 84, 245–250; Mizusawa N, Kimura Y, Ishii A, Yamanari T, Nakazawa S, Teramoto H, Ono TA (2004) J. Biol. Chem. 279, 29622–29627.

[14] Hou LH, Wu CM, Huang HH, Chu HA (2011) Biochemistry 50, 9248–9254.