Mechanism of diatomic ligand biosynthesis by radical s-adenosylmethionine [FeFe]-hydrogenase maturase HydG
Duffus, Benjamin Richard.
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Iron-sulfur (Fe-S) clusters are ubiquitous in biology, and serve as catalysts in a vast array of chemical transformations that comprise central metabolic reactions and small molecule interconversions. Complex Fe-S clusters such as the [FeFe]-hydrogenase "Hcluster" cofactor are part of a distinct subgroup of metalloenzymes that have evolved from reduced Fe-S mineral phases, as the H-cluster catalyzes H-H bond formation through reduction of protons with electrons. Biosynthesis of this cofactor is unique in its involvement of two radical S-adenosylmethionine (SAM) enzymes HydG and HydE, and a scaffold GTPase HydF. Together, these proteins synthesize a unique Fe-S cluster that coordinates a bridging dithiolate ligand as well as two CN- and three CO ligands However, many mechanistic details relating to the biosynthesis are not well known. In this work, the radical SAM enzyme HydG has been shown to synthesize CO, CN-, and pcresol through a radical-initiated fragmentation of the substrate tyrosine. The catalytic mechanism is complex, because an accessory C-terminal Fe-S cluster is required for catalysis. The exact role of this cluster in the biosynthetic mechanism is unresolved, but is proposed to serve a modular role as a potential scaffold for diatomic ligand synthesis. To understand the catalytic mechanism, a combined biochemical and spectroscopic approach was applied. In this work, it is shown that the C-terminal Fe-S cluster is essential for the formation of both CO and CN- products. Spectral characterization of the enzyme has shown the formation of diatomic ligand products that are bound to the coordinated Fe-S clusters. Also, an H atom abstraction profile of HydG has been recently characterized to provide insight to the involvement of the 5'-deoxyadenosyl radical in catalysis. Further mechanistic insight into catalysis has also been investigated through site-directed mutagenesis and through using substrate analogues. The work presented as a whole, by establishing parallels to the radical SAM enzyme superfamily in character to biosynthesis, reveals unifying themes in complex metal cluster assembly related to radical-initiated modification of ordinary Fe-S clusters via product organometallic complex formation.