In Situ Structural Observation of a Substrate- and Peroxide-Bound High-Spin Ferric-Hydroperoxo Intermediate in the P450 Enzyme CYP121

化学 光化学 过氧乙酸 立体化学 电子顺磁共振 基质(水族馆) 配体(生物化学) 结晶学 反应中间体 无机化学 过氧化氢 催化作用 有机化学 核磁共振 海洋学 地质学 生物化学 物理 受体
作者
Romie C. Nguyen,Ian Davis,M. Dasgupta,Yifan Wang,Philipp S. Simon,A. Butryn,Hiroki Makita,Isabel Bogacz,Kednerlin Dornevil,Pierre Aller,Asmit Bhowmick,Ruchira Chatterjee,In‐Sik Kim,Tiankun Zhou,Derek Mendez,Daniel W. Paley⧓,Franklin D. Fuller,Roberto Alonso‐Mori,A. Batyuk,Nicholas K. Sauter,Aaron S. Brewster,Allen M. Orville,Vittal K. Yachandra,Junko Yano,Jan Kern,Aimin Liu
出处
期刊:Journal of the American Chemical Society [American Chemical Society]
卷期号:145 (46): 25120-25133 被引量:11
标识
DOI:10.1021/jacs.3c04991
摘要

The P450 enzyme CYP121 from Mycobacterium tuberculosis catalyzes a carbon–carbon (C–C) bond coupling cyclization of the dityrosine substrate containing a diketopiperazine ring, cyclo(l-tyrosine-l-tyrosine) (cYY). An unusual high-spin (S = 5/2) ferric intermediate maximizes its population in less than 5 ms in the rapid freeze-quenching study of CYP121 during the shunt reaction with peracetic acid or hydrogen peroxide in acetic acid solution. We show that this intermediate can also be observed in the crystalline state by EPR spectroscopy. By developing an on-demand-rapid-mixing method for time-resolved serial femtosecond crystallography with X-ray free-electron laser (tr-SFX-XFEL) technology covering the millisecond time domain and without freezing, we structurally monitored the reaction in situ at room temperature. After a 200 ms peracetic acid reaction with the cocrystallized enzyme–substrate microcrystal slurry, a ferric-hydroperoxo intermediate is observed, and its structure is determined at 1.85 Å resolution. The structure shows a hydroperoxyl ligand between the heme and the native substrate, cYY. The oxygen atoms of the hydroperoxo are 2.5 and 3.2 Å from the iron ion. The end-on binding ligand adopts a near-side-on geometry and is weakly associated with the iron ion, causing the unusual high-spin state. This compound 0 intermediate, spectroscopically and structurally observed during the catalytic shunt pathway, reveals a unique binding mode that deviates from the end-on compound 0 intermediates in other heme enzymes. The hydroperoxyl ligand is only 2.9 Å from the bound cYY, suggesting an active oxidant role of the intermediate for direct substrate oxidation in the nonhydroxylation C–C bond coupling chemistry.
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