Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H₂-Consuming and H₂-Producing Reactions in Escherichia coli

• Published in: Journal of bacteriology 2015, Vol.197 (2), p.296-306
• Main Authors: Pinske, Constanze, Jaroschinsky, Monique, Linek, Sabine, Kelly, Ciarán L, Sargent, Frank, Sawers, R. Gary
• Format: Article
• Language: English
• Subjects: bacteriology; Carbonyl Cyanide m-Chlorophenyl Hydrazone - metabolism; casamino acids; cyanides; cytochrome b; dyes; electron transfer; electrons; Escherichia coli; Escherichia coli - metabolism; Escherichia coli Proteins - metabolism; evolution; fermentation; ferredoxin hydrogenase; glycerol; hydrogen; Hydrogen - metabolism; Hydrogenase - metabolism; menaquinones; oxidation; protons
• ISSN: 0021-9193; 1098-5530
• DOI: 10.1128/jb.02335-14

Escherichia coli uptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H ₂ 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. H ₂ 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 H ₂ evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H ₂-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H ₂ oxidation or H ₂ evolution in whole cells, reversible H ₂-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 H ₂ evolution mode. Our findings suggest that Hyd-2 switches between H ₂-consuming and H ₂-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H ₂ evolution from glycerol requires reverse electron transport.