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 Pinske, Sara Krüger, Basem Soboh, Christian Ihling, Martin Kuhns, Mario Braussemann, Monique Jaroschinsky, Christopher Sauer, Frank Sargent, Andrea Sinz, R. Gary Sawers

    Research output: Contribution to journalArticlepeer-review

    35 Citations (Scopus)


    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.

    Original languageEnglish
    Pages (from-to)893-903
    Number of pages11
    JournalArchives of Microbiology
    Issue number12
    Publication statusPublished - Dec 2011


    • [NiFe]-hydrogenase
    • Iron-sulfur cluster
    • Electron transfer
    • Hydrogen evolution
    • Hydrogen oxidation
    • Viologen dyes
    • OPERON
    • K-12
    • STEP

    Cite this