In Vitro Activation of [FeFe] Hydrogenase: New Insights into Hydrogenase Maturation

Shawn E. McGlynn, Shane S. Ruebush, Anatoli Naumov, Lauren E. Nagy, Alexandra Dubini, Paul W. King, Joan B. Broderick, Matthew C. Posewitz, John W. Peters

Research output: Contribution to journalArticlepeer-review

105 Scopus Citations


The in vitro activation of the [FeFe] hydrogenase is accomplished by combining Escherichia coli cell extracts containing the heterologously expressed inactive HydA with extracts in which hydrogenase-specific maturation proteins HydE, HydF, and HydG are expressed in concert. Interestingly, the process of HydA activation occurs rapidly and in the absence of potential substrates, which suggests that the hydrogenase accessory proteins synthesize an H-cluster precursor that can be quickly transferred to the hydrogenase enzyme to affect activation. HydA activity is observed to be dependent on the protein fraction containing all three accessory proteins expressed in concert and cannot be accomplished with addition of heat-treated extract or extract filtrate, suggesting that the activation of the hydrogenase structural protein is mediated by interaction with the accessory assembly protein(s). These results represent the first important step in understanding the process of H-cluster assembly and provide significant insights into hydrogenase maturation.

Original languageAmerican English
Pages (from-to)443-447
Number of pages5
JournalJournal of Biological Inorganic Chemistry
Issue number4
StatePublished - 2007

NREL Publication Number

  • NREL/JA-270-41769


  • H-cluster
  • Hydrogenase
  • Iron-sulfur cluster assemblies
  • Metalloenzymes
  • Radical S-adenosyl methionine enzymes


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