The electron-proton bottleneck of photosynthetic oxygen evolution

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.