Abstract：
Copper nitrite reductases (CuNiRs) exhibit a strong pH dependence of their catalytic activity. Structural movies can be obtained by serially recording multiple structures (frames) from the same spot of a crystal using the MSOX serial crystallography approach. This method has been combined with on-line single crystal optical spectroscopy to capture the pH-dependent structural changes that accompany during turnover of CuNiRs from two Rhizobia species. The structural movies, initiated by the redox activation of a type-1 copper site (T1Cu) via X-ray generated photoelectrons, have been obtained for the substrate-free and substrate-bound states at low (high enzymatic activity) and high (low enzymatic activity) pH. At low pH, formation of the product nitric oxide (NO) is complete at the catalytic type-2 copper site (T2Cu) after a dose of 3 MGy (frame 5) with full bleaching of the T1Cu ligand-to-metal charge transfer (LMCT) 455 nm band (S(σ)Cys → T1Cu2+) which in itself indicates the electronic route of proton-coupled electron transfer (PCET) from T1Cu to T2Cu. In contrast at high pH, the changes in optical spectra are relatively small and the formation of NO is only observed in later frames (frame 15 in Br2DNiR, 10 MGy), consistent with the loss of PCET required for catalysis. This is accompanied by decarboxylation of the catalytic AspCAT residue, with CO2 trapped in the catalytic pocket.
摘要：
亚硝酸铜还原酶（CuNiRs）的催化活性具有很强的pH依赖性。可以通过使用MSOX串行晶体学方法从晶体的同一点串行记录多个结构(帧)来获得结构电影。该方法已与在线单晶光谱法结合使用，以捕获两种根瘤菌中CuNiRs周转过程中伴随的pH依赖性结构变化。结构电影，通过X射线产生的光电子对1型铜位点（T1Cu）进行氧化还原活化，已在低（高酶活性）和高（低酶活性）pH下获得无底物和底物结合状态。在低pH值，在剂量为3MGy（框架5）并完全漂白T1Cu配体到金属的电荷转移（LMCT）455nm之后，在催化2型铜位点（T2Cu）上完成了产物一氧化氮（NO）的形成（S（σ）Cys→T1Cu2），这本身就表明了质子耦合电子转移（PCET）从T1Cu到T2Cu的电子路线。相反，在高pH下，光谱的变化相对较小，NO的形成仅在以后的帧中观察到(Br2DNiR中的第15帧，10MGy)，与催化所需的PCET损失一致。这伴随着催化AspCAT残留物的脱羧，二氧化碳被困在催化袋中。
