Light-driven water oxidation is a fundamental reaction in the biosphere. The MnCa cluster of photosystem II cycles through five redox states termed S-S, after which oxygen is evolved. Critically, the timing of O-O bond formation within the Kok cycle remains unknown. By combining recent crystallographic, spectroscopic, and DFT results, we demonstrate an atomistic S state model with the possibility of a low barrier to O-O bond formation prior to the final oxidation step. Furthermore, the associated one electron oxidized S state does not provide more advantages in terms of spin alignment or the energy of O-O bond formation. We propose that a high energy peroxide isoform of the S state can preferentially be oxidized by Tyr in the course of final electron transfer leading to O evolution. Such a mechanism may explain the peculiar kinetic behavior of O evolution as well as serve as an evolutionary adaptation to avoid release of the harmful peroxides.

The realization of artificial photosynthesis carries the promise of cheap and abundant energy, however, significant advances in the rational design of water oxidation catalysts are required. Detailed information on the structure of the catalyst under reaction conditions and mechanisms of O-O bond formation should be obtained. Here, we used a combination of electron paramagnetic resonance (EPR), stopped flow freeze quench on a millisecond-second time scale, X-ray absorption (XAS), resonance Raman (RR) spectroscopy, and density functional theory (DFT) to follow the dynamics of the Ru-based single site catalyst, [Ru(NPM)(4-pic)(HO)] (NPM = 4-t-butyl-2,6-di(1',8'-naphthyrid-2'-yl)pyridine, pic = 4-picoline), under the water oxidation conditions. We report a unique EPR signal with g-tensor, g = 2.30, g = 2.18, and g = 1.83 which allowed us to observe fast dynamics of oxygen atom transfer from the Ru═O oxo species to the uncoordinated nitrogen of the NPM ligand. In few seconds, the NPM ligand modification results in [Ru(NPM-NO)(4-pic)(HO)] and [Ru(NPM-NO,NO)(4-pic)] complexes. A proposed [Ru(NPM)(4-pic)═O] intermediate was not detected under the tested conditions. We demonstrate that while the proximal base might be beneficial in O-O bond formation via nucleophilic water attack on an oxo species as shown by DFT, the noncoordinating nitrogen is impractical as a base in water oxidation catalysts due to its facile conversion to the N-O group. This study opens new horizons for understanding the real structure of Ru catalysts under water oxidation conditions and points toward the need to further investigate the role of the N-O ligand in promoting water oxidation catalysis.

Photosynthetic water oxidation is a fundamental process that sustains the biosphere. A Mn4Ca cluster embedded in the photosystem II protein environment is responsible for the production of atmospheric oxygen. Here, time-resolved x-ray emission spectroscopy (XES) was used to observe the process of oxygen formation in real time. These experiments reveal that the oxygen evolution step, initiated by three sequential laser flashes, is accompanied by rapid (within 50 μs) changes to the Mn Kβ XES spectrum. However, no oxidation of the Mn4Ca core above the all MnIV state was detected to precede O−O bond formation, and the observed changes were therefore assigned to O−O bond formation dynamics. We propose that O−O bond formation occurs prior to the transfer of the final (4th) electron from the Mn4Ca cluster to the oxidized tyrosine YZ residue. This model resolves the kinetic limitations associated with O−O bond formation, and suggests an evolutionary adaptation to avoid releasing of harmful peroxide species.

The biological generation of oxygen by the oxygen-evolving complex (OEC) in photosystem II (PS II) is one of nature's most important reactions. The OEC is a Mn 4 Ca cluster that has multiple Mn−O−Mn and Mn−O−Ca bridges and binds four water molecules. Previously, binding of an additional oxygen was detected in the S 2 to S 3 transition. Here we demonstrate that early binding of the substrate oxygen to the five-coordinate Mn 1 center in the S 2 state is likely responsible for the S 2 high-spin EPR signal. Substrate binding in the Mn 1 −OH form explains the prevalence of the high-spin S 2 state at higher pH and its low-temperature conversion into the S 3 state. The given interpretation was confirmed by X-ray absorption spectroscopic measurements, DFT, and broken symmetry DFT calculations of structures and magnetic properties. Structural, electronic, and spectroscopic properties of the high-spin S 2 state model are provided and compared with the available S 3 state models. New interpretation of the high-spin S 2 state opens opportunity for analysis of factors controlling the oxygen substrate binding in PS II.

X-ray emission (XES) spectroscopy is an attractive technique for analysis of the electronic structure of molecules, materials, and metalloproteins. However, a better understanding of XES results is required. Using a combination of experiment and ground-state density functional theory analysis, we rationalize differences in the X-ray emission spectra of multinuclear Mn complexes. Model compounds, including dinuclear [Mn2O2L′4]­(ClO4)3 (L′= 2,2′-bipyridyl, [1]) and two examples from the Mn4O4L6 “cubane” family of model compounds (L = (p-R-C6H4)­PO2 −, R = OCH3 [2], CH3 [3] ), were compared with the Oxygen Evolving Complex of Photosystem II. Our analysis shows that changes in the structure of the Mn complexes, resulting in changes to the spin polarization, can introduce significant spectral shifts in compounds of the same formal redox state. The implications of changes in spin polarization for understanding photosynthetic water-splitting catalysis is discussed.