The {FeO2}8 Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide
<p>TxtE is a cytochome P450 (CYP) homolog that mediates a nitric oxide (NO)-dependent direct nitration of l-tryptophan (l-Trp) to form 4-nitrotryptophan (4-NO<sub>2</sub>-l-Trp). This nitrated product is a precursor for thaxtomin A, a virulence factor produced by plant-pathogenic bacteria that causes the disease potato scab. A recent study provided the first characterization of intermediates along the TxtE nitration pathway.<sup>1</sup> The authors’ accumulated evidence supported a mechanism in which O<sub>2</sub> binds to Fe<sup>II</sup> TxtE to form an {FeO<sub>2</sub>}<sup>8</sup> intermediate, which subsequently reacted with NO to ultimately form Fe<sup>III</sup> TxtE and 4-NO<sub>2</sub>-l-Trp. Typical CYP mechanisms reduce and protonate the {FeO<sub>2</sub>}<sup>8</sup> intermediate to form a ferric-hydroperoxo species (Fe<sup>III</sup>–OOH) en route to formation of the active oxidant compound I. The previously reported lack of hydroxylated tryptophan resulting from TxtE turnover suggests that the TxtE cycle must stall at the {FeO<sub>2</sub>}<sup>8</sup> intermediate to avoid hydroxylation. Here we present LC-MS experiments showing suggesting that TxtE can hydroxylate l-Trp by the peroxide shunt but not via reduction of the {FeO<sub>2</sub>}<sup>8</sup> intermediate. Comparison of stopped-flow time courses in the presence and absence of excess reducing equivalents and common CYP electron transfer partners shown no spectral or kinetic evidence for reduction of the {FeO<sub>2</sub>}<sup>8</sup> intermediate. Furthermore, the electron coupling efficiency of TB14—a self-sufficient TxtE variant with C-terminal reductase domain—to form 4-NO<sub>2</sub>-l-Trp exhibits a 3% electron coupling efficiency when it is loaded with one reducing equivalent. This efficiency <i>increases</i> by 2-fold when TB14 is loaded with two or four reducing equivalents. This observation provides further evidence for our key conclusion that the TxtE {FeO<sub>2</sub>}<sup>8</sup> intermediate resists reduction. The resistance of the {FeO<sub>2</sub>}<sup>8</sup> intermediate to reduction is a key feature of TxtE, enabling reaction with NO and efficient nitration turnover.<b></b></p>