Pulse EPR and related double resonance techniques are versatile tools for studying enzymatic processes involving transition metal ions as active sites. In the past such investigations have often been impeded by fast magnetic relaxation and sensitivity problems of the methods used. Recently, these limitations have been overcome by using higher microwave frequencies (95 GHz, W-band) and by the development of ELDOR-detected NMR (EDNMR), a double resonance method with high intrinsic sensitivity (1). As an example the catalytic water oxidation reaction in photosystem II (PSII) of oxygenic photosynthesis is presented. In PSII water oxidation takes place at an oxygen-bridged tetranuclear manganese calcium cluster (Mn4OxCa) located in the D1 protein subunit (2). The reaction cycle is comprised of 5 distinct redox intermediates Sn, where the subscript indicates the number of stored oxidizing equivalents in the manganese cluster (n = 0 - 4) required to split two water molecule and release one O2. The S-states are trapped by laser flash/freeze techniques; their electronic structure is studied by advanced EPR (ENDOR, ESEEM/HYSCORE, EDNMR). The data are corroborated by DFT calculations. The data analyses yield the spin and oxidation states of the manganese ions, the spin coupling in the cluster, the function of Ca2+, effect of the amino acid surrounding, the binding, location and reaction dynamics of the 2 substrate water molecules, the O-O bond formation and the release of O2 (3,4). A water oxidation mechanism is derived (5). The importance for the design of bioinspired catalysts for water oxidation is emphasized.

Multifrequency pulse EPR studies of the water oxidizing complex in photosynthesis

253rd National Meeting (2017)

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