Mechanism for Six-Electron Aryl-N-Oxygenation by the Non-Heme Diiron Enzyme CmlI

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• 作者：Anna J. Komor ; Brent S. Rivard ; Ruixi Fan ; Yisong Guo ; Lawrence Que Jr. ; John D. Lipscomb
• 刊名：Journal of the American Chemical Society
• 出版年：2016
• 出版时间：June 15, 2016
• 年：2016
• 卷：138
• 期：23
• 页码：7411-7421
• 全文大小：569K
• 年卷期：0
• ISSN：1520-5126

文摘

The ultimate step in chloramphenicol (CAM) biosynthesis is a six-electron oxidation of an aryl-amine precursor (NH₂-CAM) to the aryl-nitro group of CAM catalyzed by the non-heme diiron cluster-containing oxygenase CmlI. Upon exposure of the diferrous cluster to O₂, CmlI forms a long-lived peroxo intermediate, P, which reacts with NH₂-CAM to form CAM. Since P is capable of at most a two-electron oxidation, the overall reaction must occur in several steps. It is unknown whether P is the oxidant in each step or whether another oxidizing species participates in the reaction. Mass spectrometry product analysis of reactions under ¹⁸O₂ show that both oxygen atoms in the nitro function of CAM derive from O₂. However, when the single-turnover reaction between ¹⁸O₂-P and NH₂-CAM is carried out in an ¹⁶O₂ atmosphere, CAM nitro groups contain both ¹⁸O and ¹⁶O, suggesting that P can be reformed during the reaction sequence. Such reformation would require reduction by a pathway intermediate, shown here to be NH(OH)-CAM. Accordingly, the aerobic reaction of NH(OH)-CAM with diferric CmlI yields P and then CAM without an external reductant. A catalytic cycle is proposed in which NH₂-CAM reacts with P to form NH(OH)-CAM and diferric CmlI. Then the NH(OH)-CAM rereduces the enzyme diiron cluster, allowing P to reform upon O₂ binding, while itself being oxidized to NO-CAM. Finally, the reformed P oxidizes NO-CAM to CAM with incorporation of a second O₂-derived oxygen atom. The complete six-electron oxidation requires only two exogenous electrons and could occur in one active site.