The binuclear cluster of [FeFe] hydrogenase is formed with sulfur donated by cysteine of an [Fe(Cys)(CO)2(CN)] organometallic precursor

Guodong Rao, Scott A. Pattenaude, Katherine Alwan, Ninian J. Blackburn, R. David Britt, Thomas B. Rauchfuss

Research output: Contribution to journalArticlepeer-review


The enzyme [FeFe]-hydrogenase (HydA1) contains a unique 6-iron cofactor, the H-cluster, that has unusual ligands to an Fe–Fe binuclear subcluster: CN, CO, and an azadithiolate (adt) ligand that provides 2 S bridges between the 2 Fe atoms. In cells, the H-cluster is assembled by a collection of 3 maturases: HydE and HydF, whose roles aren’t fully understood, and HydG, which has been shown to construct a [Fe(Cys)(CO)2(CN)] organometallic precursor to the binuclear cluster. Here, we report the in vitro assembly of the H-cluster in the absence of HydG, which is functionally replaced by adding a synthetic [Fe(Cys)(CO)2(CN)] carrier in the maturation reaction. The synthetic carrier and the HydG-generated analog exhibit similar infrared spectra. The carrier allows HydG-free maturation to HydA1, whose activity matches that of the native enzyme. Maturation with 13CN-containing carrier affords 13CN-labeled enzyme as verified by electron paramagnetic resonance (EPR)/electron nuclear double-resonance spectra. This synthetic surrogate approach complements existing biochemical strategies and greatly facilitates the understanding of pathways involved in the assembly of the H-cluster. As an immediate demonstration, we clarify that Cys is not the source of the carbon and nitrogen atoms in the adt ligand using pulse EPR to target the magnetic couplings introduced via a 13C3 15N-Cys–labeled synthetic carrier. Parallel mass-spectrometry experiments show that the Cys backbone is converted to pyruvate, consistent with a cysteine role in donating S in forming the adt bridge. This mechanistic scenario is confirmed via maturation with a seleno-Cys carrier to form HydA1–Se, where the incorporation of Se was characterized by extended X-ray absorption fine structure spectroscopy.

Original languageEnglish (US)
Pages (from-to)20850-20855
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number42
StatePublished - Oct 15 2019


  • Cysteine
  • H-cluster biosynthesis
  • Iron carbonyl cyanides

ASJC Scopus subject areas

  • General


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