title: How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase.
abstract: 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 hydro-genase 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, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 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 observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function.
authors.affiliation: *Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Lisa
authors.initials: L
authors.lastname: Bowman
authors.affiliation: †Department of Chemistry, University of York, Heslington, York YO10 5DD, England, U.K.
authors.forename: Lindsey
authors.initials: L
authors.lastname: Flanagan
authors.affiliation: ‡Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Paul K
authors.initials: PK
authors.lastname: Fyfe
authors.affiliation: †Department of Chemistry, University of York, Heslington, York YO10 5DD, England, U.K.
authors.forename: Alison
authors.initials: A
authors.lastname: Parkin
authors.affiliation: ‡Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
authors.forename: William N
authors.initials: WN
authors.lastname: Hunter
authors.affiliation: *Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Frank
authors.initials: F
authors.lastname: Sargent
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D018360:Crystallography, X-Ray; D005818:Genetic Engineering; D018698:Glutamic Acid / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D019779:Salmonella enterica / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D021122:Protein Subunits; D018698:Glutamic Acid; D006639:Histidine; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen