Though reactive flavin‐N5/C4α‐oxide intermediates can be spectroscopically profiled for some flavin‐assisted enzymatic reactions, their exact chemical configurations are hardly visualized. Structural systems biology and stable isotopic labelling techniques were exploited to correct this stereotypical view. Three transition‐like complexes, the α‐ketoacid…N5‐FMN_ox complex (I ), the FMN_ox‐N5‐aloxyl‐C′α⁻‐C4α⁺ zwitterion (II ), and the FMN‐N5‐ethenol‐N5‐C4α‐epoxide (III ), were determined from mandelate oxidase (Hmo) or its mutant Y128F (monooxygenase) crystals soaked with monofluoropyruvate (a product mimic), establishing that N5 of FMN_ox an alternative reaction center can polarize to an ylide‐like mesomer in the active site. In contrast, four distinct flavin‐C4α‐oxide adducts (IV –VII ) from Y128F crystals soaked with selected substrates materialize C4α of FMN an intrinsic reaction center, witnessing oxidation, Baeyer–Villiger/peroxide‐assisted decarboxylation, and epoxidation reactions. In conjunction with stopped‐flow kinetics, the multifaceted flavin‐dependent reaction continuum is physically dissected at molecular level for the first time.

中文翻译：

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黄素氧化物中间体的结构和化学捕集揭示了底物导向的反应多样性。

尽管可以对某些黄素辅助的酶促反应进行光谱分析，确定反应性黄素-N5 /C4α-氧化物中间体，但几乎看不到其确切的化学构型。利用结构系统生物学和稳定的同位素标记技术来纠正这种定型观点。三种类似过渡的配合物，即α-酮酸…N5-FMN _ox配合物（I），FMN _ox ‐N5-aloxyl-C'α ^-- C4α ⁺两性离子（II）和FMN-N5-乙烯醇N5- C4α-环氧化物（III）是从扁桃酸盐氧化酶（Hmo）或其突变体Y128F（单加氧酶）晶体中浸入一氟丙酮酸盐（模拟产物）确定的，确定FMN _ox的N5另一个反应中心可以在活性位点极化成类似叶立德的同向异构体。相反，从Y128F晶体中浸入了选定的底物的四个截然不同的黄素C4α-氧化物加合物（IV – VII）使FMN的C4α成为本征反应中心，见证了氧化，Baeyer-Villiger /过氧化物辅助的脱羧和环氧化反应。结合流阻动力学，首次在分子水平上对多黄素依赖性反应连续体进行了物理剖析。