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

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 (S1) to more oxidized intermediates (S2 and S3), eventually cycling back to the most reduced S0. However, the interpretation of these models is controversial because geometric parameters within the Mn4CaO5 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, S1 → S2, 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 S2 state of the OEC. We show that the 1F/S2 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 S2 state and with the nature of the S1 → S2 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.借助X射线自由电子激光（X-ray free electron lasers, XFEL）实现的光系统II（photosystem II, PSII）飞秒串行晶体衍射（serial femtosecond crystallography, SFX）领域近期进展，首次获得了放氧复合物（oxygen-evolving complex, OEC）催化S态循环中不同中间体的几何模型。这些模型通过将OEC从暗稳定态（S1）闪击推进至更高氧化态中间体（S2与S3），最终循环回到还原程度最高的S0态而获得。但对这些模型的解读尚存争议，原因是OEC中Mn4CaO5簇的几何参数，与光谱学验证的不同S态中间体的锰氧化态所对应的配位化学预期参数并不完全吻合。本研究聚焦首个催化过渡过程S1→S2，该过程对应OEC的单电子氧化反应。结合包括新型有效氧化态方法在内的几何与电子结构判据，我们对本应表征OEC S2态的现有1次闪击（1-flash, 1F）SFX-XFEL晶体学模型展开分析。我们发现1F模型与S2态的对应关系并不明确，因为这些模型所蕴含的锰氧化态与总未配对电子数，既与纯S2态的参数不完全一致，也与S1→S2过渡过程的本质不符。此外，两次闪击（two-flashed, 2F）结构模型中的氧化态定义实际上难以阐明。本研究结果提示，不应仅通过对晶体学模型的字面解读来提取电子结构信息，同时呼吁对那些假定此类模型与OEC特定催化中间体完全对应的结构与机制解读展开重新评估。