The Ferric-Superoxo Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide

Abstract

TxtE is a cytochrome P450 (CYP) homologue that mediates the nitric oxide (NO)-dependent direct nitration of l-tryptophan (Trp) to form 4-nitro-l-tryptophan (4-NO₂-Trp). A recent report showed evidence that TxtE activity requires NO to react with a ferric-superoxo intermediate. Given this minimal mechanism, it is not clear how TxtE avoids Trp hydroxylation, a mechanism that also traverses the ferric-superoxo intermediate. To provide insight into canonical CYP intermediates that TxtE can access, electron coupling efficiencies to form 4-NO₂-Trp under single- or limited-turnover conditions were measured and compared to steady-state efficiencies. As previously reported, Trp nitration by TxtE is supported by the engineered self-sufficient variant, TB14, as well as by reduced putidaredoxin. Ferrous (Fe^II) TxtE exhibits excellent electron coupling (70%), which is 50-fold higher than that observed under turnover conditions. In addition, two- or four-electron reduced TB14 exhibits electron coupling (∼6%) that is 2-fold higher than that of one-electron reduced TB14 (3%). The combined results suggest (1) autoxidation is the sole TxtE uncoupling pathway and (2) the TxtE ferric-superoxo intermediate cannot be reduced by these electron transfer partners. The latter conclusion is further supported by ultraviolet-visible absorption spectral time courses showing neither spectral nor kinetic evidence for reduction of the ferric-superoxo intermediate. We conclude that resistance of the ferric-superoxo intermediate to reduction is a key feature of TxtE that increases the lifetime of the intermediate and enables its reaction with NO and efficient nitration activity.

Figure 1.

Reaction mechanism for canonical substrate (Sub) hydroxylation by CYPs (left cycle) and the minimal mechanism for nitration by the CYP homologue TxtE (right cycle).

Figure 2.

Representative LC-MS extracted ion chromatograms (EICs) of TxtE and TB14 reactions monitoring (A) Trp-OH (m/z 219) or (B) 4-NO₂-Trp (m/z 248) formation. All reaction mixtures contained 500 μM Trp in 100 or 200 mM Tris buffer (pH 8.0) with additional components: 3 mM H₂O₂ and denatured TxtE (Boiled TxtE/H₂O₂); 5 μM hemin and 3 mM H₂O₂ (Hemin/H₂O₂); 5 μM TB14 and 5 mM H₂O₂ (TB14/H₂O₂); 5 μM TB14, 500 μM NADPH, and 5 μM catalase (TB14 O₂-turnover + cat); 5 μM TB14 and 500 μM NADPH (TB14 O₂-turnover); 0.5 μM TB14, 2 mM NADPH, and 1.33 mM DEA NONOate (TB14 Nitration turnover); 100 μM Fe^II TxtE and 0.9 mM PROLI-NONOate (Fe^IITxtE); and 100 μM Fe^III TxtE, 200 μM PdX_red, and 0.75 mM PROLI-NONOate (Fe^IIITxtE + 0.2 eq. PdX_red). All samples were incubated at room temperature for 120 min (TB14 samples) or 30 min (all others) prior to analysis.

Figure 3.

Stopped-flow spectral time courses of anaerobic Fe^II TxtE mixed with O₂ in the (A) presence or (B) absence of Trp at pH 8.0. Dashed traces obtained by mixing an Fe^II TxtE solution against deoxygenated buffer. Solid black and red traces are the first and last collected spectra in the time courses, respectively. Gray traces were collected at intermediate times. The inset shows representative single-wavelength traces collected with low-intensity light time courses and fit with single-exponent functions. Final conditions: (A) 5 μM Fe^II TxtE, 250 μM Trp, and 130 μM O₂ and (B) 5 μM Fe^II TxtE and 130 μM O₂. All solutions were in 100 mM Tris (pH 8.0), and all reactions performed at 20 °C in a 1 cm path length cuvette.

Figure 4.

Stopped-flow sequential-mixing spectral time course of the TxtE ferric-superoxo intermediate vs NO over 10 ms at pH 8.0 and 20 °C. The dashed trace is the spectrum of Fe^II TxtE acquired from mixing Fe^II TxtE with deoxygenated buffer in both mixing steps. Conditions after stopped-flow mixing: 10 μM Fe^II TxtE, 250 μM Trp,70 μM O₂, and 600 μM NO in 100 mM Tris (pH 8.0) at 20 °C in a 1 cm path length cuvette.

Figure 5.

Stopped-flow spectral time courses of (A and B) anaerobic Fe^II TxtE and PdX_red or (C and D) TB14_4e‑red mixed with O₂ in the presence of excess Trp at pH 8.0 and 21 °C. Each time course is divided into (A and C) a fast phase and (B and D) a slow phase. The dashed trace was obtained by mixing the anaerobic solution against deoxygenated buffer. Solid black, red, or blue traces were recorded at times indicated in the figure legends. Gray traces were recorded at intermediate times. The inset shows single-exponent fits to representative A₅₆₁ or A₃₉₈ traces. Conditions after mixing: (A and B) 12.5 μM Fe^II TxtE, 50 μM PdX_red, 250 μM Trp, and 130 μM O₂ and (C and D) 20 μM TB14_4e‑red, 250 μM Trp, and 130 μM O₂. All solutions were in 100 mM Tris (pH 8.0), and all reactions performed at 20 °C in a 1 cm path length cuvette.

Figure 6.

Summary of results from this study. The resistance of the TxtE ferric-superoxo intermediate to reduction avoids formation of Trp-OH and leaves autoxidation as the only uncoupling pathway available to TxtE. Brackets surround proposed intermediates not yet characterized for TxtE.

Figure 7.

Crystal structures of the CYP101A1 ferric-superoxo intermediate (Protein Data Bank entry 1DZ8) and Trp-bound Fe^III TxtE (Protein Data Bank entry 4TPO). Carbon atoms are colored green for CYP101A1 and gray for TxtE, oxygen atoms red, nitrogen atoms dark blue, and sulfur atoms yellow, and the iron atom is colored orange. H-Bonds are represented by dashed yellow lines.