Electron inventory of the iron-sulfur scaffold complex HypCD essential in [NiFe]-hydrogenase cofactor assembly

Biochem J. 2021 Sep 17;478(17):3281-3295. doi: 10.1042/BCJ20210224.


The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN- and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system producing the HypCD complex from E. coli with complete metal content. This enabled in-depth spectroscopic characterizations. The results obtained by EPR and Mössbauer spectroscopy demonstrate that the [Fe](CN)2CO cofactor and the [4Fe-4S] cluster of the HypCD complex are redox active. The data indicate a potential-dependent interconversion of the [Fe]2+/3+ and [4Fe-4S]2+/+ couple, respectively. Moreover, ATR FTIR spectroscopy reveals potential-dependent disulfide formation, which hints at an electron confurcation step between the metal centers. MicroScale thermophoresis indicates preferable binding between the HypCD complex and its in vivo interaction partner HypE under reducing conditions. Together, these results provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions required for the assembly of the CN- and CO ligands on the scaffold complex HypCD.

Keywords: Fe-S proteins; biosynthesis; carbon monoxide; cyanide; maturation; redox activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbon Monoxide / metabolism
  • Catalytic Domain
  • Disulfides / metabolism
  • Electron Spin Resonance Spectroscopy / methods
  • Electrons
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / metabolism*
  • Hydrogenase / metabolism*
  • Ions / metabolism
  • Iron / metabolism*
  • Iron-Sulfur Proteins / metabolism*
  • Ligands
  • Oxidation-Reduction
  • Protein Binding
  • Proteins / metabolism*
  • Spectroscopy, Fourier Transform Infrared / methods
  • Spectroscopy, Mossbauer / methods
  • Sulfur / metabolism*


  • Disulfides
  • Escherichia coli Proteins
  • HypC protein, E coli
  • HypD protein, Bacteria
  • HypE protein, E coli
  • Ions
  • Iron-Sulfur Proteins
  • Ligands
  • Proteins
  • Sulfur
  • Carbon Monoxide
  • Iron
  • nickel-iron hydrogenase
  • Hydrogenase