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Updated: Sep 9, 2025

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
Published on: June 20, 2019
The Radical S-Adenosyl-l-methionine Enzyme HydE Forms an Fe(I)Fe(I) Dimer En Route to the [FeFe] Hydrogenase
Guodong Rao1, Lizhi Tao1, Xin Yu2
1Department of Chemistry, University of California, Davis, California 95616, United States.
Abstract:
[FeFe] hydrogenases are highly efficient metalloenzymes that catalyze hydrogen conversion via a sophisticated active site cofactor known as the H-cluster. Biosynthesis of its [2Fe]H subcluster, which contains CO, CN-, and azadithiolate ligands, requires the action of several dedicated enzymes, including the radical S-adenosyl-l-methionine (rSAM) enzyme HydE. HydE has been proposed to convert a mononuclear [Fe(II)(cysteinate)(CO)2(CN)]- precursor into a dimeric [FeI2(SH)2(CO)4(CN)2]2- complex, yet direct characterization of this product species has remained elusive. Here, we report spectroscopic identification of the dimeric product of the HydE reaction in its one-electron oxidized, mixed-valent Fe1.5+Fe1.5+ (S = 1/2) state. Continuous wave and pulse 13C ENDOR spectroscopy confirm the presence of two nonequivalent CN- ligands and delocalized spin density across the diiron core. Oxidation of a synthetic [Fe2(SH)2(CO)4(CN)2]2- complex reproduces similar electron paramagnetic resonance features only when it is preincubated with HydE, supporting in situ formation and stabilization of the dimer by the enzyme. These findings implicate the FeIFeI dimer as the terminal product of HydE and strongly suggest its central role in H-cluster assembly, expanding our understanding of rSAM enzyme catalysis in metallocofactor biosynthesis.
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