Mechanistic Determination of Fe(II)- and 2-oxo-gluterate-dependent Halogenases - Project Summary Halogenated organic compounds are essential to the pharmaceutical, agrochemical, and materials industries. Traditional halogenation methods, however, require hazardous reagents, such as dihalogen gases and chlorinated solvents, that pose environmental and safety concerns and often give poor selectivity, especially for unactivated C–H bonds. Enzyme-mediated halogenation provides a sustainable alternative, leveraging nature's ability to incorporate halides under mild conditions using benign salts (e.g. NaCl) as the halide source. Among halogenases, Fe/2OG halogenases stand out for their ability to functionalize unactivated sp3 C–H bonds, a reactivity rarely achieved synthetically. These enzymes share a common catalytic scaffold with Fe/2OG hydroxylases, yet selectively transfer halides instead of hydroxyl groups. The best-characterized members, SyrB2, WelO5, BesD, and AdaV/NTH, act on amino acids, natural products, and nucleotide substrates. Spectroscopic and computational studies suggest that halogenases may enforce an off-line ferryl geometry that suppresses rebound and favors halide transfer. Our recent structures of the nucleotide halogenase NTH bound to vanadyl, a ferryl mimic, unexpectedly reveals an in-line vanadyl-oxo position. Moreover, the substrate-metal- oxo distances and approach angles resemble those of Fe/2OG hydroxylases, which typically position substrates close to the metal center with relatively wide approach angles. However, stopped-flow UV-Vis measurements show that NTH undergoes slow HAT, inconsistent with Fe/2OG hydroxylases. To reconcile these observations, we propose that NTH initially forms an off-line ferryl but isomerizes into an in-line vanadyl prior to HAT, accounting for the sluggish HAT rate. This model prompted us to revisit our mechanistic interpretation for WelO5. Previously, we proposed that WelO5 begins in-line and then transitions off-line before HAT. Given that WelO5 exhibits faster HAT kinetic rates than NTH, we now suggest that WelO5 instead starts with an in-line ferryl, performs HAT, generates a ferric-hydroxide and only then isomerizes off-line to enable halide migration and transfer. This proposal aims to test these mechanistic hypotheses using stopped-flow UV-Vis, EPR, and Mössbauer spectroscopy to determine whether NTH and WelO5 share a common mechanism or represent distinct mechanistic strategies for halide transfer. Additionally, we plan to determine the cryo-EM structure of the SyrB1– SyrB2 complex using alternative substrates that allow for direct visualization of the ferryl intermediate. Ultimately, a deeper mechanistic understanding of Fe/2OG halogenases, including their ferryl kinetics, coordination geometries, and isomerization pathways, will illuminate the principles governing enzymatic halogenation and guide efforts to reprogram Fe/2OG hydroxylases into selective halogenases.
