Cytochromes P450 (CYPs) catalyze a remarkable breadth of chemistries. One CYP of interest to our groups is TxtE a homolog that mediates a nitric oxide (NO)-dependent direct nitration of -tryptophan (-Trp) to form 4-nitrotryptophan (4-NO₂--Trp). This product is a precursor for thaxtomin A, a virulence factor produced by plant-pathogenic bacteria that causes the potato disease scab. The NO required for TxtE activity is derived from -arginine via the activity of a bacterial nitric oxide synthase, TxtD. Another NO-dependent CYP nitration to form 3-nitrotyrosine was discovered in the rufomycin biosynthesis pathway from Streptomyces atratus. A recent collaborative study between the Scrutton, Challis, and de Visser groups provided the first characterization of intermediates along the TxtE nitration pathway. The 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--Trp. The reaction of {FeO₂}⁸ with NO was proposed to form a ferric-peroxynitrite (Fe^III–OONO) intermediate. Computational data from this study proposed the intermediate decays to form nitrogen dioxide (NO₂) and a ferryl intermediate, which together, mediate -Trp nitration.

Our labs sought to understand how TxtE promotes NO reaction with the {FeO₂}⁸ intermediate while avoiding reduction of the intermediate to form a ferric-hydroperoxo (Fe^III–OOH)—the pathway for canonical CYPs. We compared LC-MS and stopped-flow experiments of TxtE in the absence vs. presence of excess reducing equivalents and typical CYP electron transfer partners. Our cumulative results strongly suggest that the TxtE {FeO₂}⁸ intermediate cannot be reduced to the Fe^III–OOH, thereby avoiding hydroxylation of substrate. The implications of the results on TxtE efficiency and its pathway will be discussed.

Yousong Ding

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