A Cytochrome P450 TxtE Model System with Mechanistic and Theoretical Evidence for a Heme Peroxynitrite Active Species

The cytochrome P450 homolog, TxtE, efficiently catalyzes the direct and regioselective aromatic nitration of the indolyl moiety of L-tryptophan to 4-nitro-L-tryptophan, using nitric oxide (NO) and dioxygen (O₂) as co-substrates. Pathways for such direct and selective nitration of heteroaromatic motifs present platforms for engineering new nitration biocatalysts for pharmacologically beneficial targets, among a medley of other pivotal industrial applications. Precise mechanistic details concerning this pathway are only weakly understood, albeit a heme iron(III)-peroxynitrite active species has been postulated. To shed light on this unique reaction landscape, we investigated the indole nitration pathway of a series of biomimetic ferric heme superoxide mimics, [(Por)Fe^III(O₂–•)], in the presence of NO. Therein, our model systems gave rise to three distinct nitroindole products, including 4-nitroindole, the product
analogous to that obtained with TxtE. Moreover, ¹⁵N and ¹⁸O isotope labeling studies, along with meticulously designed control experiments lend credence to a heme peroxynitrite active nitrating agent, drawing close similarities to the tryptophan nitration mechanism of TxtE. All organic and inorganic reaction components have been fully characterized using spectroscopic methods. Theoretical investigation into several mechanistic possibilities deem a unique indolyl radical based reaction pathway as the most energetically favorable, products of which, are in excellent agreement with experimental findings.

Scab is a disease situation that causes lesions in root vegetables which drastically reduces their marketability, where potato scab is by far the most economically impactful. Thaxtomin A, which inhibits cellulose biosynthesis^[28] is predominantly produced by Streptomyces scabies, and is essentially effected by the condensation of nitrotryptophan and phenylalanine, followed by a hydroxylation step. (Figure 1A).^[29-31] The very first step in this particular pathway involves the direct and regioselective nitration of the indolyl moiety of L-tryptophan (L-Trp) to 4-nitro-L-tryptophan (4-NO₂-Trp; Figure 1A) using nitric oxide (NO)^[32] and dioxygen (O₂),^[17] which is catalyzed by the aforementioned TxtE P450 enzyme. Indeed, the nitro group of thaxtomin A has been deemed indispensable for its observed virulence against the host. However, the precise mechanistic details surrounding how these primarily monooxygenase enzymes facilitate the high-fidelity insertion of -NO₂ groups into various biorelevant organic moieties remain enigmatic to-date.^[33] This lack of comprehension in turn severely limits the prospects of corresponding biosynthetic pathways being targeted by prolific bactericides with significant
agricultural and health benefits.