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[FeFe]-Hydrogenase Cofactor Assembly

  1. Eric M. Shepard,
  2. Amanda S. Byer,
  3. Eric S. Boyd,
  4. Kevin D. Swanson,
  5. John W. Peters,
  6. Joan B. Broderick

Published Online: 5 DEC 2013

DOI: 10.1002/9781119951438.eibc2153

Encyclopedia of Inorganic and Bioinorganic Chemistry

Encyclopedia of Inorganic and Bioinorganic Chemistry

How to Cite

Shepard, E. M., Byer, A. S., Boyd, E. S., Swanson, K. D., Peters, J. W. and Broderick, J. B. 2013. [FeFe]-Hydrogenase Cofactor Assembly. Encyclopedia of Inorganic and Bioinorganic Chemistry. 1–15.

Author Information

  1. Montana State University, Bozeman, MT, USA

Publication History

  1. Published Online: 5 DEC 2013


This article highlights recent advances in our understanding of the biosynthetic pathway for active site H-cluster assembly in [FeFe]-hydrogenases. The H-cluster is composed of a [4Fe–4S] cubane bridged via one cysteine thiolate to a 2Fe subcluster; the 2Fe subcluster is further coordinated by carbon monoxide, cyanide, and bridging dithiolate ligands. Biosynthesis of the H-cluster occurs through stepwise modification of simple Fe–S cluster precursors; these chemical modifications are carried out by three gene products denoted HydE, HydF, and HydG. Maturation of the [FeFe]-hydrogenase requires the presence of a preformed [4Fe–4S] cluster, indicating that HydE, HydF, and HydG are directed toward the biosynthesis of the 2Fe subcluster. HydE and HydG are both radical S-adenosylmethionine (SAM) enzymes and utilize a CX3CX2C motif to bind site-differentiated [4Fe–4S]2+/+ clusters that promote the reductive cleavage of SAM into methionine and a 5′-deoxyadenosyl radical responsible for hydrogen atom abstraction from substrate. In an extraordinary biochemical reaction, HydG uses this chemistry to catalyze the radical degradation of tyrosine into p-cresol and a glycine-like intermediate; an accessory, C-terminal [4Fe–4S] cluster then degrades the latter intermediate into cyanide and carbon monoxide. HydE is presumed to be responsible for bridging dithiolate ligand biosynthesis from an unknown substrate. HydF contains an N-terminal GTPase domain and a C-terminal Fe–S-cluster-binding domain and binds both [2Fe–2S] and [4Fe–4S] clusters; evidence suggests that the former cluster is modified by HydE and HydG to form a 2Fe H-cluster precursor on HydF. The role of GTP hydrolysis in H-cluster maturation remains largely unexplained; however, it may be related to gating the protein–protein interactions between HydF and HydE and HydG. The reactions catalyzed by HydE and HydG can be thought of as specific ligand modification events designed to fine-tune H-cluster reactivity; not only do the chemical transformations themselves have direct links to reactions important on early Earth, but radical SAM enzymes with their canonical CX3CX2C motif are likely very ancient. The modern diversity that is observed in chemical reactions catalyzed by the radical SAM superfamily can be explained evolutionarily through the recruitment of distinct protein domains that impart specific substrate activation capabilities. Recruitment of these domains is discussed in the context of HydE and HydG and important links are made to the nitrogenase system where intriguing parallels between [FeFe]-hydA H-cluster biosynthesis and nitrogenase FeMo-cofactor assembly clearly exist.


  • hydrogenase;
  • H-cluster;
  • radical SAM;
  • S-adenosylmethionine;
  • GTPase;
  • cluster assembly;
  • cluster biosynthesis