期刊
JOURNAL OF BACTERIOLOGY
卷 197, 期 2, 页码 296-306出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/JB.02335-14
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资金
- BBSRC [BB/I02008X/1, BB/L008521/1]
- Deutsche Forschungsgemeinschaft [SA 494/3-2]
- Biotechnology and Biological Sciences Research Council [BB/I02008X/1, BB/L008521/1] Funding Source: researchfish
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.
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