Exploring Reaction Pathway Promiscuity of Diiron Oxygenase AetD

Heme oxidase-like diiron oxidases (HDOs) constitute an expanding superfamily with intricate and diverse catalytic functions. Among them, AetD has been identified as a nitrile-forming enzyme, although its catalytic mechanism remains poorly understood. In this study, AetDs unexpected reaction pathway promiscuity is demonstrated, showcasing its ability to convert (bromine-substituted) l -tryptophan into not only indole-3-carbonitrile but also a range of 3′-C functionalized indole derivatives, including amine, alcohol, aldehyde, and carboxylic acid. Through the analysis of product distributions, interconversions, isotope-labeling experiments, and QM/MM simulations, it is revealed that the highly reactive diiron (III, IV) X species, featuring an Fe2(IV) = O moiety, is capable of initiating hydrogen atom transfer from distinct sites on the substrate. These findings highlight how the flexible diiron structure of AetD enables diverse functional group transformations, establishing this enzyme as a powerful catalytic tool for complex and challenging biotransformations.