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

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Published copy (DOI)


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


Publication details

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


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.

    Research areas

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

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