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

Abstract

bEscherichia 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

Heterologous complementation studies in Escherichia coli with the Hyp accessory protein machinery from Chloroflexi provide insight into [NiFe]-hydrogenase large subunit recognition by the HypC protein family

“…2b, lane 1), as well as Hyd-3-dependent H 2 production (data not shown). Introduction of pSHH20 restored the slowly migrating isoforms of Hyd-2, which have been shown to comprise minimally the heterotrimer of HybOAC (Jack et al, 2004;Pinske et al, 2015). Strain DHP-B (DhypB), on the other hand, failed to show restoration of active Hyd-1 after transformation with pSHH20, despite Hyd-2 activity being restored (Fig.…”

“…These enzymes can either oxidize H 2 to generate a chemiosmotic proton gradient via a membrane-based electron transfer chain, as well as provide a source of reducing power, or dissipate accumulated intracellular reductant by reducing protons to produce H 2 ; some perform both functions under certain physiological conditions (Lubitz et al, 2014;Pinske et al, 2015;Vignais & Billoud, 2007 (Ogata et al, 2015). The biosynthesis of this cofactor is complicated, requiring the combined activities of six Hyp accessory proteins, whose functions include synthesis and insertion of an Fe(CN) 2 CO moiety into the apo-catalytic subunit followed by introduction of the nickel ion (Böck et al, 2006;Forzi & Sawers, 2007).…”

“…These enzymes can either oxidize H 2 to generate a chemiosmotic proton gradient via a membrane-based electron transfer chain, as well as provide a source of reducing power, or dissipate accumulated intracellular reductant by reducing protons to produce H 2 ; some perform both functions under certain physiological conditions (Lubitz et al, 2014;Pinske et al, 2015;Vignais & Billoud, 2007). The active site of all [NiFe]-hydrogenases studied to date has a bimetallic [NiFe]-cofactor that has one carbon monoxide and two cyanide moieties ligated to the iron ion (Lubitz et al, 2014;Böck et al, 2006).…”

Membrane Potential as a Regulation Mechanism of Periplasmic Nitrite Reductase Activity: A Mathematical Model

“…В результате сопряженной активности ферментов FHL-1 комплекса из формата в цитоплазме синтезируется молекулярный водород, который в отсутствие электронных акцепторов выводится из клетки и накапливается в среде. В респираторных условиях, в присутствии электронных акцепторов, этот водород может быть использован в качестве субстрата респираторными мембран-связанными гидрогеназами, которых у E. coli как минимум две, гидрогеназа Hyd-1 и Hyd-2 [21,17]. Это предположение не вступает в противоречие с каталитическими свойствами ферментов.…”

The Extended C-Terminal α-Helix of the HypC Chaperone Restricts Recognition of Large Subunit Precursors by the Hyp-Scaffold Machinery during [NiFe]-Hydrogenase Maturation in Escherichia coli

“…Gas chromatographic determination of hydrogen content in cultures was carried out in Hungate tubes filled with 5 mL of the respective medium and the 10-mL head space was flushed with nitrogen, as described [Pinske et al, 2015]. An aliquot of 200 µL gas phase from the head space was analyzed on a Shimadzu GC-2010 Plus gas chromatograph.…”