Challenges of observing O-O bond formation in the Mn₄Ca cluster of photosystem II

Photosystem II (PS II) sustains the biosphere by enabling oxygen evolution in the S₃-to-S₀ transition (duration ~1-2 ms) of its catalytic cycle. Using time-resolved Mn K_β X-ray emission spectroscopy, we observed spectroscopic changes (which were unimpeded in D₂O buffer) consistent with a modified electronic structure as early as ~50 μs in the transition. These changes are consistent with the reduction of the Mn centers at the ~50-200 μs time window in H₂O and at ~50-500 μs in D₂O, ahead of the reduction of the redox-active Tyr_Z ^•+. These results indicate the multi-step nature of O-O bond formation and O₂ release by the oxygen-evolving complex (OEC), where an O-O bond is most likely formed prior to the final electron-transfer step. The D₂O-induced extension of up to ~500 μs in the lifetime of the early transient state of a possible peroxo nature opens it up to further spectroscopic and structural analysis.