Photosynthesis fuels life on Earth by storing solar energy in chemical form. Today’s oxygen-rich atmosphere has resulted from the splitting of water at the protein-bound manganese cluster of photosystem II during photosynthesis. Formation of molecular oxygen starts from a state with four accumulated electron holes, the S4 state—which was postulated half a century ago1 and remains largely uncharacterized. Here we resolve this key stage of photosynthetic O2 formation and its crucial mechanistic role. We tracked 230,000 excitation cycles of dark-adapted photosystems with microsecond infrared spectroscopy. Combining these results with computational chemistry reveals that a crucial proton vacancy is initally created through gated sidechain deprotonation. Subsequently, a reactive oxygen radical is formed in a single-electron, multi-proton transfer event. This is the slowest step in photosynthetic O2 formation, with a moderate energetic barrier and marked entropic slowdown. We identify the S4 state as the oxygen-radical state; its formation is followed by fast O–O bonding and O2 release. In conjunction with previous breakthroughs in experimental and computational investigations, a compelling atomistic picture of photosynthetic O2 formation emerges. Our results provide insights into a biological process that is likely to have occurred unchanged for the past three billion years, which we expect to support the knowledge-based design of artificial water-splitting systems.

Note: This paper is part of the JCP Special Topic on 65 Years of Electron Transfer.

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Picosecond optical parametric oscillators (OPOs) with broad wavelength tunability are frequently used as light sources in hyperspectral coherent Raman scattering (CRS) microscopy. We investigate how changes in the pulse length during OPO wavelength tuning of the pump beam affect hyperspectral CRS imaging. We find that significant distortions of the resulting CRS spectra occur if the OPO is operated without monitoring pulse length variations. By utilizing a custom-written MATLAB based control program to counteract changes in pulse length, normalized and reproducible data sets can be acquired. We demonstrate this by comparing hyperspectral data obtained from pure substances, as well as relevant biological specimens.

Photosynthesis fuels life on Earth by storing solar energy in chemical form, inspiring technological schemes for sustainable fuel production. Today’s oxygen-rich atmosphere results from photosynthetic O2-production during water-splitting at the protein-bound manganese cluster of photosystem II. Formation of the O2 molecule starts from a state with four accumulated electron holes, the S4-state, postulated half a century ago1 and remaining enigmatic ever since. Here we resolve this missing key element in photosynthetic O2-formation and its crucial mechanistic role. We tracked 230,000 excitation cycles of dark-adapted photosystems with microsecond infrared spectroscopy. Combing these results with computational chemistry reveals that in S4 not only are four electron holes accumulated by metal ion and protein sidechain oxidation, but also a crucial proton vacancy is created through gated sidechain deprotonation. Subsequently, a reactive oxygen radical is formed in an astonishing single-electron multi-proton transfer event. This is the slowest step in photosynthetic O2-formation – despite its low energetic barrier – due to entropic slowdown. In conjunction with previous breakthroughs in experimental and computational investigations, a compelling atomistic picture of photosynthetic O2-formation emerges. Our results provide insight into a biological process that has probably operated in the same unique way for three billion years and are expected to support the knowledge-based design of artificial water-splitting systems.