Version française

Sunday 20 September 2009

ISHIDA-BLANC Naoko

Title : Mechanism of the water-splitting enzyme.


University : Paris XI Orsay

Jury :

  • Président : Ally Aukauloo
  • Rapporteurs : Elodie Anxolabehere-Mallart, Catherine Berthomieu, Philippe Champeil
  • Examinateurs :
  • Directeur de thèse : Alain Boussac


Summary :

Photosystem II uses solar energy to drive water oxidation and atmospheric O2 production. Its catalytic center comprises a cluster of four manganese and one calcium ions. The Mn4Ca cluster goes through five sequential oxidation states (S0 to S4) before O2 is evolved during the S4 to S0 transition. Although the Ca2+ ion is detected in the actual crystallographic structures, its role still needs to be clarified. A second cofactor is involved: the chloride, whose localization and role in the O2 evolution are unclear. To study both cofactors, we replaced biosynthetically the Ca2+ and the Cl- by Sr2+ and Br- in the thermophilic cyanobacteria Thermosynechococcus elongatus. In the substituted PSII, the O2 evolution under continuous illumination is decreased. It varies in the following order: Ca/Cl>Ca/Br>Sr/Cl>Sr/Br. In chapter III, we explain this activity decrease by a kinetic limitation of some of the electron transfer steps, in particular the S3 to S0 transition during which a lag phase is put in evidence. We demonstrate that this lag phase corresponds to a modification of the electrostatic environment of the chlorophyll P680, implying very likely proton movements. The significant slow down of the kinetics goes with a decrease of the free energy level of the S3 state and consequently with a decrease of the redox potential of the S3/S2 couple. In chapter IV, we study the exchangeability of the halide, and chloride is biochemically substituted with iodide. We observe a decrease of the catalytic activity due to the slowing down of the Mn4Ca cluster oxidation kinetics. The thermodynamic properties of PSII are also modified by the iodide, but the question still arises whether this is due to the reduction of the Mn4Ca cluster by I-, or to the increase of the redox potential of the S3/S2 couple. In chapter V, we focus on the structural aspects of the biosynthetically modified PSII samples. On one hand, we highlight the direct implication of Ca2+ in the binding of the substrate water molecules, and protonation/deprotonation phenomena during the S-states cycle. On the other hand, we show that our results support the existence of two halide binding sites. PDF

 

Keywords :

photosystem II – cofactors – calcium – chloride – electron transfer – exchange – cyanobacteria