Original Paper

Archives of Microbiology

, Volume 193, Issue 12, pp 893-903

First online:

Efficient electron transfer from hydrogen to benzyl viologen by the [NiFe]-hydrogenases of Escherichia coli is dependent on the coexpression of the iron–sulfur cluster-containing small subunit

  • Constanze PinskeAffiliated withInstitute for Microbiology, Martin-Luther University Halle-Wittenberg
  • , Sara KrügerAffiliated withInstitute for Microbiology, Martin-Luther University Halle-Wittenberg
  • , Basem SobohAffiliated withInstitute for Microbiology, Martin-Luther University Halle-Wittenberg
  • , Christian IhlingAffiliated withInstitute of Pharmacy, Martin-Luther University Halle-Wittenberg
  • , Martin KuhnsAffiliated withInstitute for Microbiology, Martin-Luther University Halle-Wittenberg
  • , Mario BraussemannAffiliated withInstitute for Microbiology, Martin-Luther University Halle-Wittenberg
  • , Monique JaroschinskyAffiliated withInstitute for Microbiology, Martin-Luther University Halle-Wittenberg
  • , Christopher SauerAffiliated withInstitute for Microbiology, Martin-Luther University Halle-Wittenberg
  • , Frank SargentAffiliated withDivision of Molecular Microbiology, College of Life Sciences, University of Dundee
    • , Andrea SinzAffiliated withInstitute of Pharmacy, Martin-Luther University Halle-Wittenberg
    • , R. Gary SawersAffiliated withInstitute for Microbiology, Martin-Luther University Halle-Wittenberg Email author 

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Abstract

Escherichia coli can both oxidize hydrogen and reduce protons. These activities involve three distinct [NiFe]-hydrogenases, termed Hyd-1, Hyd-2, and Hyd-3, each minimally comprising heterodimers of a large subunit, containing the [NiFe] active site, and a small subunit, bearing iron–sulfur clusters. Dihydrogen-oxidizing activity can be determined using redox dyes like benzyl viologen (BV); however, it is unclear whether electron transfer to BV occurs directly at the active site, or via an iron–sulfur center in the small subunit. Plasmids encoding Strep-tagged derivatives of the large subunits of the three E. coli [NiFe]-hydrogenases restored activity of the respective hydrogenase to strain FTD147, which carries in-frame deletions in the hyaB, hybC, and hycE genes encoding the large subunits of Hyd-1, Hyd-2, and Hyd-3, respectively. Purified Strep-HyaB was associated with the Hyd-1 small subunit (HyaA), and purified Strep-HybC was associated with the Hyd-2 small subunit (HybO), and a second iron–sulfur protein, HybA. However, Strep-HybC isolated from a hybO mutant had no other associated subunits and lacked BV-dependent hydrogenase activity. Mutants deleted separately for hyaA, hybO, or hycG (Hyd-3 small subunit) lacked BV-linked hydrogenase activity, despite the Hyd-1 and Hyd-2 large subunits being processed. These findings demonstrate that hydrogenase-dependent reduction of BV requires the small subunit.

Keywords

[NiFe]-hydrogenase Iron–sulfur cluster Electron transfer Hydrogen evolution Hydrogen oxidation Viologen dyes