Tuesday, April 1, 2025
4/1/2025
Spectroscopic Characterization of Enzymatic Intermediates with FeS Clusters - Project Summary Iron-sulfur clusters are found in all kingdoms of life, used by nature for electron transfer, for performing highly specific organometallic chemistry in the biosynthesis of natural products, and for catalysis of challenging redox transformations of small molecules. Elucidating the mechanisms of these reactions is critical for understanding the fundamental biochemistry of these enzymes, which could inspire future drug and (bio)catalyst design. Spectroscopic techniques to probe enzymatic mechanisms and reactive intermediates under physiological conditions and in nondestructive manners are desired but can be difficult to achieve. Advanced electron paramagnetic resonance (EPR) and parahydrogen induced polarization (PHIP) nuclear magnetic resonance (NMR) techniques, along with appropriate isotopic labeling, will be utilized to study the mechanisms and reactive intermediates of several enzymes containing unique iron-sulfur clusters. Herein, three systems suitable for these techniques are discussed. The last step of biotin synthesis is catalyzed by BioB, where a radical S-adenosyl-L-methionine enzyme sacrificially uses a [2Fe-2S] auxiliary cluster for S-atom transfer. A new, “Type II” class of BioB has been recently discovered, utilizing an uncommon [4Fe-5S] auxiliary cluster that may not be destroyed during turnover, though the mechanism is unestablished. Advanced EPR studies with appropriate isotopic labeling will help characterize the paramagnetic intermediates, providing hypotheses for their structures in the absence of crystallographic data. NMR hyperpolarization techniques (PHIP) will be employed to study the H-cluster of [FeFe]-hydrogenase, where the Britt group’s abilities to isotope-edit the 13C, 15N, and 57Fe nuclei of the diiron subcluster is hypothesized to allow for polarization transfer from parahydrogen, demonstrating the utility of PHIP in studying enzymatic mechanisms. Signal enhancement will overcome NMR’s inherent low sensitivity and allow for site-specific characterization of the structure and dynamics of the H-cluster under physiological conditions. The FeMo cofocator (FeMoco) of nitrogenase uses “green” sources of H2 in the form of H+ and e− to catalyze the difficult reduction of nitrogen into ammonia, which is used as fertilizer. Despite herculean efforts to understand this mechanism, much of it remains unestablished; the structure of the substrate-bound species is widely debated, and even less is known about the mechanism after substrate binding. FeMoco also generates one equivalent of H2 per turnover, and the H2 release is thought to relate to N2 binding in a reversible fashion. Thus, it is hypothesized PHIP is a technique well suited for probing this mechanistic step. Polarization transfer to other magnetic nuclei including 15N is expected to add direct evidence regarding the binding mode of substrate in FeMoco. Alternative strategies involving the design of polarization transfer catalysts that can produce hyperpolarized nitrogenase substrates are also proposed. Expected outcomes of this research will add to the rich field of iron-sulfur biochemistry and provide new spectroscopic tools for studying enzymatic mechanisms under physiological conditions.
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