JBIC Journal of Biological Inorganic Chemistry

, Volume 24, Issue 6, pp 783–792 | Cite as

H-cluster assembly intermediates built on HydF by the radical SAM enzymes HydE and HydG

  • Amanda S. Byer
  • Eric M. Shepard
  • Michael W. Ratzloff
  • Jeremiah N. Betz
  • Paul W. King
  • William E. Broderick
  • Joan B. BroderickEmail author
Original Paper
Part of the following topical collections:
  1. Joan Broderick: Papers in Celebration of Her 2019 ACS Alfred Bader Award in Bioinorganic or Bioorganic Chemistry


[FeFe]-hydrogenase catalyzes the reversible reduction of protons to H2 at a complex metallocofactor site, the H-cluster. Biosynthesis of this active-site H-cluster requires three maturation enzymes: the radical S-adenosylmethionine enzymes HydE and HydG synthesize the nonprotein ligands, while the GTPase HydF provides a scaffold for assembly of the 2Fe subcluster of the H-cluster ([2Fe]H) prior to its transfer to hydrogenase. To delineate the assembly and delivery steps for the 2Fe precursor cluster coordinated to HydF ([2Fe]F), we have heterologously expressed HydF in the presence of HydE alone (HydFE) or HydG alone (HydFG), and characterized the resulting purified HydFE and HydFG using UV–visible, EPR, and FTIR spectroscopies and biochemical assays. The iron–sulfur clusters on HydF are modified by co-expression with HydE or HydG, as evidenced by the changes in the visible, EPR, and FTIR spectral features. Further, biochemical assays show that HydFE is capable of activating HydAΔEFG to a limited extent (~ 1% of WT) even though the normal source of CO and CN ligands of [2Fe]H (HydG) was absent. Activation assays performed with HydFG, in contrast, exhibit no ability to mature HydAΔEFG. It appears that in the case of HydFE, trace diatomics from the cellular environment are incorporated into a [2Fe]F-like precursor on HydF in the absence of HydG. We conclude that the product of HydE, presumably the dithiomethylamine ligand of [2Fe]H, is absolutely essential to the activation process, while the diatomic products of HydG can be provided from alternate sources.

Graphic abstract


[FeFe] hydrogenase H-cluster Maturation Biosynthesis Radical SAM HydF 



The authors gratefully acknowledge the U.S. Department of Energy, Office of Basic Energy Sciences (DE-SC0005404 to J.B.B. and E.M.S.), for supporting all experimental work other than the FTIR spectroscopy. FTIR spectroscopy was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Chemical Sciences, Geosciences and Biosciences Division, and under U.S. Department of Energy Contract No. DE-AC36-08-GO28308 with the National Renewable Energy Lab.

Supplementary material

775_2019_1709_MOESM1_ESM.pdf (5.2 mb)
Supplementary material 1 (PDF 5277 kb)


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Copyright information

© Society for Biological Inorganic Chemistry (SBIC) 2019

Authors and Affiliations

  • Amanda S. Byer
    • 1
  • Eric M. Shepard
    • 1
  • Michael W. Ratzloff
    • 2
  • Jeremiah N. Betz
    • 1
  • Paul W. King
    • 2
  • William E. Broderick
    • 1
  • Joan B. Broderick
    • 1
    Email author
  1. 1.Department of Chemistry and BiochemistryMontana State UniversityBozemanUSA
  2. 2.Biosciences Center, National Renewable Energy LaboratoryGoldenUSA

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