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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

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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.

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Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (Grant SA 494/3-1 to RGS and grant SI 867/13-1 to AS) and the region of Sachsen-Anhalt.

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Authors and Affiliations

  1. Institute for Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120, Halle (Saale), Germany

    Constanze Pinske, Sara Krüger, Basem Soboh, Martin Kuhns, Mario Braussemann, Monique Jaroschinsky, Christopher Sauer & R. Gary Sawers

  2. Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 1, 06120, Halle (Saale), Germany

    Christian Ihling & Andrea Sinz

  3. Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, UK

    Frank Sargent

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  1. Constanze Pinske
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  2. Sara Krüger
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  3. Basem Soboh
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  4. Christian Ihling
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  5. Martin Kuhns
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  10. Andrea Sinz
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  11. R. Gary Sawers
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Correspondence to R. Gary Sawers.

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Communicated by William Metcalf.

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Pinske, C., Krüger, S., Soboh, B. et al. 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. Arch Microbiol 193, 893–903 (2011). https://doi.org/10.1007/s00203-011-0726-5

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  • DOI: https://doi.org/10.1007/s00203-011-0726-5

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