The catalytic mechanism of aromatic nitration by cytochrome P450 TxtE: Involvement of a ferric-peroxynitrite intermediate

Savvas Louka, Sarah M. Barry, Derren J. Heyes, M. Qadri E. Mubarak, Hafiz Saqib Ali, Lona Alkhalaf, Andrew W. Munro, Nigel S. Scrutton, Gregory L. Challis, Sam P. De Visser

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The cytochrome P450s are heme-dependent enzymes that catalyze many vital reaction processes in the human body related to biodegradation and biosynthesis. They typically act as mono-oxygenases; however, the recently discovered P450 subfamily TxtE utilizes O2 and NO to nitrate aromatic substrates such as L-tryptophan. A direct and selective ar-omatic nitration reaction may be useful in biotechnology for the synthesis of drugs or small molecules. Details of the catalytic mechanism are unknown and it has been suggested that the reaction should proceed through either an iron(III)-superoxo or an iron(II)-nitrosyl intermediate. To resolve this controversy, we used stopped-flow kinetics to provide evidence for a catalytic cycle where dioxygen binds prior to NO to generate an active iron(III)-peroxynitrite species that is able to nitrate L-Trp efficiently. We show that the rate of binding of O2 is faster than that of NO and also leads to L-Trp nitration, while little evidence of product formation is observed from the iron(II)-nitrosyl complex. To support the experimental studies, we performed density functional theory studies on large active site cluster models. The studies suggest a mechanism involving an iron(III)-peroxynitrite that splits homolytically to form an iron(IV)-oxo heme (Compound II) and a free NO2 radical via a small free energy of activation. The latter activates the substrate on the aromatic ring, while Compound II picks up the ipso-hydrogen to form the product. The calculations give small re-action barriers for most steps in the catalytic cycle and, therefore, predict fast product formation after the formation of an iron(III)-peroxynitrite complex. These findings provide the first detailed insight into the mechanism of nitration by a member of the TxtE subfamily and highlight how the enzyme facilitates this novel reaction chemistry.
Original languageEnglish
JournalJournal of the American Chemical Society
Early online date19 Aug 2020
Publication statusE-pub ahead of print - 19 Aug 2020

Research Beacons, Institutes and Platforms

  • Manchester Institute of Biotechnology


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