title: Physiology and bioenergetics of [NiFe]-hydrogenase 2-catalyzed H2-consuming and H2-producing reactions in Escherichia coli.
abstract: 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.
authors.affiliation: Division of Molecular Microbiology, University of Dundee, College of Life Sciences, Dundee, Scotland, United Kingdom.
authors.forename: Constanze
authors.initials: C
authors.lastname: Pinske
authors.affiliation: Institute for Biology/Microbiology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
authors.forename: Monique
authors.initials: M
authors.lastname: Jaroschinsky
authors.affiliation: Institute for Biology/Microbiology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Linek
authors.affiliation: Division of Molecular Microbiology, University of Dundee, College of Life Sciences, Dundee, Scotland, United Kingdom.
authors.forename: Ciarán L
authors.initials: CL
authors.lastname: Kelly
authors.affiliation: Division of Molecular Microbiology, University of Dundee, College of Life Sciences, Dundee, Scotland, United Kingdom f.sargent@dundee.ac.uk gary.sawers@mikrobiologie.uni-halle.de.
authors.forename: Frank
authors.initials: F
authors.lastname: Sargent
authors.affiliation: Institute for Biology/Microbiology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany f.sargent@dundee.ac.uk gary.sawers@mikrobiologie.uni-halle.de.
authors.forename: R Gary
authors.initials: RG
authors.lastname: Sawers
mesh_terms: D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase