Amino acid variants of the HybB membrane subunit of <i>Escherichia coli</i> [NiFe]‐hydrogenase‐2 support a role in proton transfer

Lubek, Dorothea; Simon, Andreas H.; Pinske, Constanze

doi:10.1002/1873-3468.13514

Cited by 8 publications

(12 citation statements)

References 39 publications

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“…It can be further observed that the effect of CCCP addition is not immediate, but H 2 production rates shift rather gradually. For the direct dependence of an activity on pmf, we have previously observed immediate and complete effects of CCCP addition on the activity [2], suggesting an indirect effect of CCCP occurs here. Notably, the addition of other protonophores like 2,4-dinitrophenol (DNP) up to 1 mM had no effect on the H 2 production activity of the cells, similar to what was observed before at lower concentrations [8].…”

Section: Cccp Inhibits H 2 Production To Different Degrees Depending ...supporting

confidence: 66%

“…CCCP enhances Hyd-2 activity by removing the back-pressure of the pmf on the enzyme. When assayed directly, the effect is not very prominent because the enzyme works at its catalytic optimum, but in variants that exhibit difficulties in transferring electrons across the membrane, the effect is more pronounced [2]. Electrons from Hyd-2 are concomitantly channeled into the quinone pool for reduction of electron acceptors like fumarate, which can be internally produced during mixed-acid fermentation when glucose is provided to the cells [28].…”

Section: Hyd-1 Confers Resistance and Hyd-2 Sensitivity Of H 2 Produc...mentioning

confidence: 99%

“…The reaction of the formate hydrogenlyase (FHL) complex contributes indirectly to the generation of a proton gradient at the cytoplasmic membrane by consumption of protons (H + ) from the cytoplasm, diffusion of H 2 -gas across the membrane and subsequent oxidation by the two periplasmic H 2 -oxidizing hydrogenases (Hyd-1 and Hyd-2) [1] (Figure 1). Hyd-1 forms a redox loop, and Hyd-2 contributes directly to proton transfer from the cytoplasm to the quinone during oxidation of H 2 [1,2]. The hyaB and hybC genes encode the catalytic subunits of Hyd-1 and Hyd-2, respectively [3].…”

Section: Introductionmentioning

confidence: 99%

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Susceptibility of the Formate Hydrogenlyase Reaction to the Protonophore CCCP Depends on the Total Hydrogenase Composition

Marloth

Pinske

2020

Inorganics

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Fermentative hydrogen production by enterobacteria derives from the activity of the formate hydrogenlyase (FHL) complex, which couples formate oxidation to H2 production. The molybdenum-containing formate dehydrogenase and type-4 [NiFe]-hydrogenase together with three iron-sulfur proteins form the soluble domain, which is attached to the membrane by two integral membrane subunits. The FHL complex is phylogenetically related to respiratory complex I, and it is suspected that it has a role in energy conservation similar to the proton-pumping activity of complex I. We monitored the H2-producing activity of FHL in the presence of different concentrations of the protonophore CCCP. We found an inhibition with an apparent EC50 of 31 µM CCCP in the presence of glucose, a higher tolerance towards CCCP when only the oxidizing hydrogenase Hyd-1 was present, but a higher sensitivity when only Hyd-2 was present. The presence of 200 mM monovalent cations reduced the FHL activity by more than 20%. The Na+/H+ antiporter inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) combined with CCCP completely inhibited H2 production. These results indicate a coupling not only between Na+ transport activity and H2 production activity, but also between the FHL reaction, proton import and cation export.

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Section: Cccp Inhibits H 2 Production To Different Degrees Depending ...supporting

confidence: 66%

Section: Hyd-1 Confers Resistance and Hyd-2 Sensitivity Of H 2 Produc...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Susceptibility of the Formate Hydrogenlyase Reaction to the Protonophore CCCP Depends on the Total Hydrogenase Composition

Marloth

Pinske

2020

Inorganics

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“…The enzyme primarily sustains anaerobic hydrogenotrophic growth of E. coli using fumarate as an electron acceptor (77,214,219,227). It is thought that this hydrogenase can also generate PMF by coupling electron transfer to vectorial proton translocation via its transmembrane subunit (213,214,228). On some fermentable substrates, this complex can also act in reverse as a PMF-driven quinol-dependent proton reductase in a process thought to counterbalance an overreduced redox state of the quinone pool (206,214).…”

Section: Enterobacterialesmentioning

confidence: 99%

Molecular Hydrogen Metabolism: a Widespread Trait of Pathogenic Bacteria and Protists

Benoit

Maier

Sawers

et al. 2020

Microbiol Mol Biol Rev

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SUMMARY Pathogenic microorganisms use various mechanisms to conserve energy in host tissues and environmental reservoirs. One widespread but often overlooked means of energy conservation is through the consumption or production of molecular hydrogen (H2). Here, we comprehensively review the distribution, biochemistry, and physiology of H2 metabolism in pathogens. Over 200 pathogens and pathobionts carry genes for hydrogenases, the enzymes responsible for H2 oxidation and/or production. Furthermore, at least 46 of these species have been experimentally shown to consume or produce H2. Several major human pathogens use the large amounts of H2 produced by colonic microbiota as an energy source for aerobic or anaerobic respiration. This process has been shown to be critical for growth and virulence of the gastrointestinal bacteria Salmonella enterica serovar Typhimurium, Campylobacter jejuni, Campylobacter concisus, and Helicobacter pylori (including carcinogenic strains). H2 oxidation is generally a facultative trait controlled by central regulators in response to energy and oxidant availability. Other bacterial and protist pathogens produce H2 as a diffusible end product of fermentation processes. These include facultative anaerobes such as Escherichia coli, S. Typhimurium, and Giardia intestinalis, which persist by fermentation when limited for respiratory electron acceptors, as well as obligate anaerobes, such as Clostridium perfringens, Clostridioides difficile, and Trichomonas vaginalis, that produce large amounts of H2 during growth. Overall, there is a rich literature on hydrogenases in growth, survival, and virulence in some pathogens. However, we lack a detailed understanding of H2 metabolism in most pathogens, especially obligately anaerobic bacteria, as well as a holistic understanding of gastrointestinal H2 transactions overall. Based on these findings, we also evaluate H2 metabolism as a possible target for drug development or other therapies.

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“…The presence of an ion-conducting pathway in HybB is also supported by the observations that replacement of Arg89 HybB , Tyr99 HybB , Glu148 HybB and His184 HybB in E. coli HybB (Nside half-channel TMH 5-8) significantly decreased hydrogen oxidation (Lubek et al, 2019). Addition of a protonophore increases hydrogen oxidation in these mutated strains (Lubek et al, 2019), showing that ion translocation and catalytic activity are coupled.…”

Section: Ion Translocation Pathwaysmentioning

confidence: 67%

The Ion-Translocating NrfD-Like Subunit of Energy-Transducing Membrane Complexes

Calisto

Pereira

2021

Front. Chem.

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Several energy-transducing microbial enzymes have their peripheral subunits connected to the membrane through an integral membrane protein, that interacts with quinones but does not have redox cofactors, the so-called NrfD-like subunit. The periplasmic nitrite reductase (NrfABCD) was the first complex recognized to have a membrane subunit with these characteristics and consequently provided the family's name: NrfD. Sequence analyses indicate that NrfD homologs are present in many diverse enzymes, such as polysulfide reductase (PsrABC), respiratory alternative complex III (ACIII), dimethyl sulfoxide (DMSO) reductase (DmsABC), tetrathionate reductase (TtrABC), sulfur reductase complex (SreABC), sulfite dehydrogenase (SoeABC), quinone reductase complex (QrcABCD), nine-heme cytochrome complex (NhcABCD), group-2 [NiFe] hydrogenase (Hyd-2), dissimilatory sulfite-reductase complex (DsrMKJOP), arsenate reductase (ArrC) and multiheme cytochrome c sulfite reductase (MccACD). The molecular structure of ACIII subunit C (ActC) and Psr subunit C (PsrC), NrfD-like subunits, revealed the existence of ion-conducting pathways. We performed thorough primary structural analyses and built structural models of the NrfD-like subunits. We observed that all these subunits are constituted by two structural repeats composed of four-helix bundles, possibly harboring ion-conducting pathways and containing a quinone/quinol binding site. NrfD-like subunits may be the ion-pumping module of several enzymes. Our data impact on the discussion of functional implications of the NrfD-like subunit-containing complexes, namely in their ability to transduce energy.

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Amino acid variants of the HybB membrane subunit of Escherichia coli [NiFe]‐hydrogenase‐2 support a role in proton transfer

Cited by 8 publications

References 39 publications

Susceptibility of the Formate Hydrogenlyase Reaction to the Protonophore CCCP Depends on the Total Hydrogenase Composition

Susceptibility of the Formate Hydrogenlyase Reaction to the Protonophore CCCP Depends on the Total Hydrogenase Composition

Molecular Hydrogen Metabolism: a Widespread Trait of Pathogenic Bacteria and Protists

The Ion-Translocating NrfD-Like Subunit of Energy-Transducing Membrane Complexes

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