title: 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.
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.
authors: Institute for Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120, Halle, Saale, Germany.
authors: Constanze
authors: C
authors: Pinske
authors: Sara
authors: S
authors: Krüger
authors: Basem
authors: B
authors: Soboh
authors: Christian
authors: C
authors: Ihling
authors: Martin
authors: M
authors: Kuhns
authors: Mario
authors: M
authors: Braussemann
authors: Monique
authors: M
authors: Jaroschinsky
authors: Christopher
authors: C
authors: Sauer
authors: Frank
authors: F
authors: Sargent
authors: Andrea
authors: A
authors: Sinz
authors: R Gary
authors: RG
authors: Sawers
mesh_terms: D001594:Benzyl Viologen / Q000378:metabolism*; D020134:Catalytic Domain; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids
chemical_list: D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D001594:Benzyl Viologen; D006859:Hydrogen; D010088:Oxidoreductases; C043296:nickel-iron hydrogenase; C029746:uptake hydrogenase; D006864:Hydrogenase