(469c) Unraveling the H-cluster Biosynthetic Pathway: Investigations of Radical SAM Chemistry Catalyzed by the Hydg Maturase

[FeFe] hydrogenases rapidly produce H₂, which has stimulated interest in their application for renewable energy technologies. In addition, their catalytic iron center termed the H-cluster has inspired the design of many synthetic catalysts, albeit few with ability to evolve H₂, and a better understanding of how the H-cluster is made could further the development of H₂-based technologies. HydE, HydF, and HydG are three enzymes that assemble the H-cluster and activate [FeFe] hydrogenases, and recent work has shed light on H-cluster biosynthesis such as the formation of the cofactor’s CO and CN ligands from free tyrosine. Nonetheless, challenges with experimentally probing the reaction pathway have left several aspects unresolved due in part to the O₂ sensitive nature of both the maturation proteins as well as the H-cluster. In this work, we combine multiple techniques to study the HydG maturase and its radical SAM chemistry. We use rapid freeze quench methods along with EPR spectroscopy in order to examine the radical SAM reaction and determine the kinetics of its products. In doing so, we identify a novel benzylomethyl-4-olate (BM4O) radical species that derives from free tyrosine radical, which presumably forms during the generation of CO and CN^– molecules. We also use stopped-flow infrared (SF-FTIR) spectroscopy to examine the HydG chemistry in situ. Specifically, we observe the concurrent formation of CO and CN^– species during the radical SAM reaction, which we attribute to a HydG-specific iron compound coordinated by at least two CO and one CN^–. We then utilize our cell-free hydrogenase maturation platform in conjunction with ENDOR spectroscopy to trace ⁵⁷Fe nuclei from HydG to the [FeFe] hydrogenase, providing conclusive evidence that the HydG maturase synthesizes an iron compound precursor of the H-cluster. From this work, and in contrast to the current paradigm, we present a new reaction sequence for the assembly of the H-cluster and the maturation of [FeFe] hydrogenases.