How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase

Lisa Bowman, Lindsey Flanagan, Paul K. Fyfe, Alison Parkin, William N. Hunter, Frank Sargent*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydrogenase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe-3S] cluster in the small subunit. In addition, His 229 was observed to lie close to a buried glutamic acid (Glu 73 ), which is conserved in oxygen-tolerant hydrogenases. His 229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observedwith oxygen-tolerant [NiFe]-hydrogenases. It is possible thatHis229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73 could play a supporting role in fine-tuning the chemistry of His229 to enable this function.

Original languageEnglish
Pages (from-to)449-458
Number of pages10
JournalBiochemical journal
Issue number3
Publication statusPublished - 15 Mar 2014


  • Hydrogen metabolism
  • Iron-sulphur cluster [NiFe]-hydrogenase
  • Oxygen-tolerance
  • Protein film electrochemistry (PFE)
  • Salmonella enterica

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