Physiology and bioenergetics of [NiFe]-hydrogenase 2-catalyzed H2-consuming and H2-producing reactions in Escherichia coli

Constanze Pinske, Monique Jaroschinsky, Sabine Linek, Ciarán L. Kelly, Frank Sargent (Lead / Corresponding author), R. Gary Sawers (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

59 Citations (Scopus)


Escherichia coli uptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H2 to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochrome b membrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H2 evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H2 evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H2-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H2 oxidation or H2 evolution in whole cells, reversible H2-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H2 evolution mode. Our findings suggest that Hyd-2 switches between H2-consuming and H2-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H2 evolution from glycerol requires reverse electron transport.

Original languageEnglish
Pages (from-to)296-306
Number of pages11
JournalJournal of Bacteriology
Issue number2
Publication statusPublished - Jan 2015


  • Carbonyl Cyanide m-Chlorophenyl Hydrazone
  • Escherichia coli
  • Escherichia coli Proteins
  • Hydrogen
  • Hydrogenase


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