Electrochemical characterization of Sav Hppd Az1 Luke Kays, Dylan Boucher, Julian Buchholz, Shelley Minteer University of Utah, USA
Directed evolution has been well documented as an efficient and powerful tool to engineer enzymes for specific functions, such as enzyme stability, or reprogramming an enzyme’s selectivity for a different substrate beyond its natural scope. Based on Darwinian evolution, directed evolution generates a series of genetic mutants via mutagenesis and screens them for a desired trait. The improved variant then acts as a starting point for the next cycle, and this continues until the desired trait is exhibited satisfactorily. Despite the synthetic achievements due to directed evolution it has not been paired with electrosynthetic methods. Such a pairing could provide access to novel and sustainable transformations with unprecedented and tunable selectivity. Sav HppD Az1 is a non heme iron enzyme designed via direct evolution to catalyze a C(sp 3 )-H azidation reaction. This process occurs through an iron-catalyzed radical transfer by an initial homogeneous N-F bond splitting. This N-F bond is required for the reaction to drive the necessary redox cycling of the iron active site. Direct electrochemical oxidation of the enzyme would eliminate the need for the N-F bond and greatly expand the scope of possible substrates. We investigated the possibility of adapting the reactivity of Sav HppD Az1 for an electrochemical system using less complex substrate structures. The enzyme was immobilized on the electrode surface using pyrene-functionalized hydrogels, which eliminates the need for a mediator. The redox features of the enzyme were examined via cyclic voltammetry and square-wave voltammetry in anaerobic conditions, with a distinct oxidation signal observed for the iron active site in the presence of azide, suggesting coordination into the active site. This methodology offers a way to investigate the reactivity of non-native enzymes and represents an electrosynthetic application of directed evolution.
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