Substrate water binding to the oxygen-evolving complex in photosystem II

Sammanfattning: Oxygenic photosynthesis in plants, algae and cyanobacteria converts sunlight into chemical energy. In this process electrons are transferred from water molecules to CO2 leading to the assembly of carbohydrates, the building blocks of life. A cluster of four manganese ions and one calcium ion, linked together by five oxygen bridges, constitutes the catalyst for water oxidation in photosystem II (Mn4CaO5 cluster). This cluster stores up to four oxidizing equivalents (S0,..,S4 states), which are then used in a concerted reaction to convert two substrate water molecules into molecular oxygen. The reaction mechanism of this four-electron four-proton reaction is not settled yet and several hypotheses have been put forward. The work presented in this thesis aims at clarifying several aspects of the water oxidation reaction by analyzing the mode of substrate water binding to the Mn4CaO5 cluster.Time-resolved membrane-inlet mass spectrometric detection of flash-induced O2 production after fast H218O labelling was employed to study the exchange rates between substrate waters bound to the Mn4CaO5 cluster and the surrounding bulk water. By employing this approach to dimeric photosystem II core complexes of the red alga Cyanidoschyzon merolae it was demonstrated that both substrate water molecules are already bound in the S2 state of the Mn4CaO5 cluster. This was confirmed with samples from the thermophilic cyanobacterium Thermosynechococcus elongatus. Addition of the water analogue ammonia, that is shown to bind to the Mn4CaO5 cluster by replacing the crystallographic water W1, did not significantly affect the exchange rates of the two substrate waters. Thus, these experiments exclude that W1 is a substrate water molecule.The mechanism of O-O bond formation was studied by characterizing the substrate exchange in the S3YZ? state. For this the half-life time of this transient state into S0 was extended from 1.1 ms to 45 ms by replacing the native cofactors Ca2+ and Cl- by Sr2+ and I-. The data show that both substrate waters exchange significantly slower in the S3YZ? state than in the S3 state. A detailed discussion of this finding lead to the conclusions that (i) the calcium ion in the Mn4CaO5 cluster is not a substrate binding site and (ii) O-O bond formation occurs via the direct coupling between two Mn-bound water-derived oxygens, which were assigned to be the terminal water/hydroxy ligand W2 and the central oxo-bridging O5.The driving force for the O2 producing S4?S0 transition was studied by comparing the effects of N2 and O2 pressures of about 20 bar on the flash-induced O2 production of photosystem II samples containing either the native cofactors Ca2+ and Cl- or the surrogates Sr2+ and Br-. While for the Ca/Cl-PSII samples no product inhibition was observed, a kinetic limitation of O2 production was found for the Sr/Br-PSII samples under O2 pressure. This was tentatively assigned to a significant slowdown of the O2 release in the Sr/Br-PSII samples. In addition, the equilibrium between the S0 state and the early intermediates of the S4 state family was studied under 18O2 atmosphere in photosystem II centers devoid of tyrosine YD. Water-exchange in the transiently formed early S4 states would have led to 16,18O2 release, but none was observed during a three day incubation time. Both experiments thus indicate that the S4?S0 transition has a large driving force. Thus, photosynthesis is not limited by the O2 partial pressure in the atmosphere.