The First State in the Catalytic Cycle of the Water-Oxidizing Enzyme: Identification of a Water-Derived μ-Hydroxo Bridge

Nature’s water-splitting catalyst, an oxygen-bridged tetramanganese calcium (Mn 4 O 5 Ca) complex, sequentially activates two substrate water molecules generating molecular O 2 . Its reaction cycle is composed of five intermediate (S i ) states, where the index i indicates the number of oxidizing equivalents stored by the cofactor. After formation of the S 4 state, the product dioxygen is released and the cofactor returns to its lowest oxidation state, S 0 . Membrane-inlet mass spectrometry measurements suggest that at least one substrate is bound throughout the catalytic cycle, as the rate of 18 O-labeled water incorporation into the product O 2 is slow, on a millisecond to second time scale depending on the S state. Here, we demonstrate that the Mn 4 O 5 Ca complex poised in the S 0 state contains an exchangeable hydroxo bridge. On the basis of a combination of magnetic multiresonance (EPR) spectroscopies, comparison to biochemical models and theoretical calculations we assign this bridge to O5, the same bridge identified in the S 2 state as an exchangeable fully deprotonated oxo bridge [Pérez Navarro, M.; et al. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 15561]. This oxygen species is the most probable candidate for the slowly exchanging substrate water in the S 0 state. Additional measurements provide new information on the Mn ions that constitute the catalyst. A structural model for the S 0 state is proposed that is consistent with available experimental data and explains the observed evolution of water exchange kinetics in the first three states of the catalytic cycle.