THESE DE DOCTORAT DE L ’ UNIVERSITE PARIS SUD - 11 Ecole

Photosystem II (PSII) uses light energy to oxidise water and reduce quinone. The water oxidation site is a Mn4Ca cluster located on the luminal side of the membrane protein complex, while the quinone reduction site is made up of two quinones (QA and QB) and a nonheme Fe located on the stromal side of the membrane protein. In this thesis I worked on both oxidation and reduction functions of the enzyme. QA •and QB •are magnetically couple to the Fe giving weak and complex EPR signals. The distorted octahedral Fe has four histidines ligands and an exchangeable (bi)carbonate ligand. Formate can displace the exchangeable (bi)carbonate ligand, slowing electron transfer out of the PSII reaction centre. Here I report the formate-modified QB •Fe EPR signal, and this shows marked spectral changes and has a greatly enhanced intensity. I also discovered a second new EPR signal from formate-treated PSII that is attributed to formate-modified QA •Fe in the presence of a 2-electron reduced form of QB. In addition, I found that the native QA •Fe and QB •Fe EPR signals have a strong feature that had been previously missed because of overlapping signals (mainly the stable tyrosyl radical TyrD). These previously unreported EPR signals should allow for the redox potential of this cofactor to be directly determined for the first time. I also observed that when QB Fe was formed; it was able to oxidise the iron slowly in the dark. This occurred in samples pumped to remove O2. This observation implies that at least in some centres, the QB /QBH2 couple has a higher potential then is often assumed and thus that the protein-bound semiquinone is thermodynamically less stable expected. It has yet to be determined if this represents a situation occurring in the majority of centres. Treatment of the system with dithionite generated a modified form of QA Fe state and a change in the association of the proteins on gels. This indicates a redox induced modification of the protein, possibly structurally important cysteine bridge in PSII. On the water oxidation side of the enzyme, I studied the first step in the assembly of the Mn4Ca cluster looking at Mn 2+ oxidation using kinetic EPR and high field EPR. Conditions were found for stabilising the first oxidised state and some discrepancies with the literature were observed. I also found that dithionite could be used to reduce the Mn4Ca, forming states that are formally equivalent to those that exist during the assembly of the enzyme.