Does Serial Femtosecond Crystallography Depict State-Specific Catalytic Intermediates of the Oxygen-Evolving Complex?

[Image: see text] Recent advances in serial femtosecond crystallography (SFX) of photosystem II (PSII), enabled by X-ray free electron lasers (XFEL), provided the first geometric models of distinct intermediates in the catalytic S-state cycle of the oxygen-evolving complex (OEC). These models are obtained by flash-advancing the OEC from the dark-stable state (S(1)) to more oxidized intermediates (S(2) and S(3)), eventually cycling back to the most reduced S(0). However, the interpretation of these models is controversial because geometric parameters within the Mn(4)CaO(5) cluster of the OEC do not exactly match those expected from coordination chemistry for the spectroscopically verified manganese oxidation states of the distinct S-state intermediates. Here we focus on the first catalytic transition, S(1) → S(2), which represents a one-electron oxidation of the OEC. Combining geometric and electronic structure criteria, including a novel effective oxidation state approach, we analyze existing 1-flash (1F) SFX-XFEL crystallographic models that should depict the S(2) state of the OEC. We show that the 1F/S(2) equivalence is not obvious, because the Mn oxidation states and total unpaired electron counts encoded in these models are not fully consistent with those of a pure S(2) state and with the nature of the S(1) → S(2) transition. Furthermore, the oxidation state definition in two-flashed (2F) structural models is practically impossible to elucidate. Our results advise caution in the extraction of electronic structure information solely from the literal interpretation of crystallographic models and call for re-evaluation of structural and mechanistic interpretations that presume exact correspondence of such models to specific catalytic intermediates of the OEC.