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Abstract
The highly oxygen-sensitive hydrogen uptake (Hup) hydrogenase from Dehalococcoides mccartyi forms part of a protein-based respiratory chain coupling hydrogen oxidation with organohalide reduction on the outside of the cell. The HupXSL proteins were previously shown to be synthesized and enzymatically active in Escherichia coli. Here we examined the growth conditions that deliver active Hup enzyme that couples H2 oxidation to benzyl viologen (BV) reduction, and identified host factors important for this process. In a genetic background lacking the three main hydrogenases of E. coli we could show that additional deletion of genes necessary for selenocysteine biosynthesis resulted in inactive Hup enzyme, suggesting requirement of a formate dehydrogenase for Hup activity. Hup activity proved to be dependent on the presence of formate dehydrogenase (Fdh-H), which is typically associated with the H2-evolving formate hydrogenlyase (FHL) complex in the cytoplasm. Further analyses revealed that heterologous Hup activity could be recovered if the genes encoding the ferredoxin-like electron-transfer protein HupX, as well as the related HycB small subunit of Fdh-H were also deleted. These findings indicated that the catalytic HupL and electron-transferring HupS subunits were sufficient for enzyme activity with BV. The presence of the HupX or HycB proteins in the absence of Fdh-H therefore appears to cause inactivation of the HupSL enzyme. This is possibly because HupX or HycB aided transfer of electrons to the quinone pool or other oxidoreductase complexes, thus maintaining the HupSL heterodimer in a continuously oxidized state causing its inactivation. This proposal was supported by the observation that growth under either aerobic or anaerobic respiratory conditions did not yield an active HupSL. These studies thus provide a system to understand the redox sensitivity of this heterologously synthesized hydrogenase.
Highlights
The bacterial genus Dehalococcoides belongs to the phylum Chloroflexi and the type species D. mccartyi is completely dependent on hydrogen for growth (Löffler et al, 2013; Schubert et al, 2018)
The membrane-associated, ferredoxin-like protein HupX is encoded within the operon of the Hup hydrogenase, but seems to associate more tightly with the core OmeAB-reductive dehalogenases (Rdh) complex (Hartwig et al, 2017; Seidel et al, 2018), suggesting that it is the main mediator of electron transfer and acts as a “connector” protein between HupSL and the rest of the complex
The activity resulting from introduction of the plasmid lacking the hupX gene was apparently weaker than that resulting from introduction of phupXSL, both enzyme activities showed very similar migration characteristics, indicating that HupX is neither necessary for the ability of the enzyme to reduce benzyl viologen (BV) nor seems to co-migrate with HupSL in this particular activity band
Summary
The bacterial genus Dehalococcoides belongs to the phylum Chloroflexi and the type species D. mccartyi is completely dependent on hydrogen for growth (Löffler et al, 2013; Schubert et al, 2018). The Hup enzyme is found in a respiratory supercomplex comprising a two-subunit complex iron-sulfur molybdoprotein, OmeAB (organohalide molybdoenzyme) and one of a number of reductive dehalogenases (Rdh), which catalyze the reduction of particular organohalides that function as electron acceptors for the bacterium (Fincker and Spormann, 2017; Schubert et al, 2018). The membrane-associated, ferredoxin-like protein HupX is encoded within the operon of the Hup hydrogenase, but seems to associate more tightly with the core OmeAB-Rdh complex (Hartwig et al, 2017; Seidel et al, 2018), suggesting that it is the main mediator of electron transfer and acts as a “connector” protein between HupSL and the rest of the complex. HupX is homologous to HybA, a component of the Hyd-2 H2-oxidizing hydrogenase of Escherichia coli (Sargent et al, 1998; Beaton et al, 2018) and recent studies have provided strong evidence indicating that HybA is responsible for coupling electron transfer to the quinone pool, as Hyd-2 has no membrane subunit with a recognized heme cofactor, necessary for electron transfer into the membrane (Dubini et al, 2002; Pinske et al, 2015; Beaton et al, 2018)
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