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 FeII TxtE to form an {FeO2}8 intermediate, which subsequently reacted with NO to ultimately form FeIII TxtE and 4-NO2--Trp. The reaction of {FeO2}8 with NO was proposed to form a ferric-peroxynitrite (FeIII–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 (FeIII–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 FeIII–OOH, thereby avoiding hydroxylation of substrate. The implications of the results on TxtE efficiency and its pathway will be discussed.

The CYP homolog TxtE resists reduction of the {FeO2}8 intermediate, promoting efficient Trp nitration

ACS Spring Meeting 2021

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