Catalytic Mechanism of Aromatic Nitration by Cytochrome P450 TxtE: Involvement of a Ferric-Peroxynitrite Intermediate

Abstract

The cytochromes P450 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 O 2 and NO to nitrate aromatic substrates such as L -tryptophan. A direct and selective aromatic 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 O 2 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 NO 2 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 reaction barriers for most steps in the catalytic cycle and, therefore, predict fast product formation from the 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.

The {FeO2}8 Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide

“…µM Fe II TxtE with 500 µM L-Trp in pH 8.0 buffer was mixed at a 1:1 (v/v) ratio with deoxygenated pH 8.0 buffer at 20 °C. The Fe II TxtE spectrum displayed absorbance features with maxima at 412 and 553 nm, consistent with previously reported spectra 1. Mixing the Fe II TxtE solution at a 1:1 (v/v) ratio with air-saturated pH 8.0 buffer at 20 °C resulted in complete disappearance of the Fe II TxtE absorption features within the mixing deadtime of 2 ms.…”

“…The kobs for the {FeO2} 8 reaction with NO at 20 °C was estimated at greater than 1700 s -1 , agreeing well with that previously reported. 1 Of note, the estimated kobs of this reaction is over 200,000-fold faster than the TxtE kautox of 0.008 s -1 . These results suggest that the reaction of NO with {FeO2} 8 is far too fast for autoxidation to account for the poor electron coupling observed under turnover conditions.…”

“…A recent mechanistic study of TxtE by Louka, et al provided the first direct characterization of TxtE pathway intermediates. 1 While the Fe III -OONO intermediate was not directly observed, the results support the feasibility of its intermediacy. It was shown that the Fe II TxtE reacted with O2…”

“…Our stopped-flow time course is consistent with data recently presented on the TxtE mechanism. 1 (Fig. 2).…”

The {FeO2}8 Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide

“…A recent study provided the first characterization of intermediates along the TxtE nitration pathway. 1 The authors' accumulated evidence supported a mechanism in which O2 binds to Fe II TxtE to form an {FeO2} 8 intermediate, which subsequently reacted with NO to ultimately form Fe III TxtE and 4-NO2-L-Trp. Typical CYP mechanisms reduce and protonate the {FeO2} 8 intermediate to form a ferric-hydroperoxo species (Fe III -OOH) en route to formation of the active oxidant compound I.…”