Fully Refined Semisynthesis of the [FeFe] Hydrogenase H-Cluster

Guodong Rao, Xin Yu, Yu Zhang, Thomas B. Rauchfuss, R. David Britt

Research output: Contribution to journalArticlepeer-review


[FeFe] hydrogenases contain a 6-Fe cofactor that serves as the active site for efficient redox interconversion between H2 and protons. The biosynthesis of the so-called H-cluster involves unusual enzymatic reactions that synthesize organometallic Fe complexes containing azadithiolate, CO, and CN- ligands. We have previously demonstrated that specific synthetic [Fe(CO)x(CN)y] complexes can be used to functionally replace proposed Fe intermediates in the maturation reaction. Here, we report the results from performing such cluster semisynthesis in the context of a recent fully defined cluster maturation procedure, which eliminates unknown components previously employed from Escherichia coli cell lysate and demonstrate this provides a concise route to H-cluster synthesis. We show that formaldehyde can be used as a simple reagent as the carbon source of the bridging adt ligand of H-cluster in lieu of serine/serine hydroxymethyltransferase. In addition to the actual H-cluster, we observe the formation of several H-cluster-like species, the identities of which are probed by cryogenic photolysis combined with EPR/ENDOR spectroscopy.

Original languageEnglish (US)
Pages (from-to)2868-2877
Number of pages10
Issue number19
StatePublished - Oct 3 2023

ASJC Scopus subject areas

  • Biochemistry


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