Insights Into the Redox Sensitivity of Chloroflexi Hup-Hydrogenase Derived From Studies in Escherichia coli: Merits and Pitfalls of Heterologous [NiFe]-Hydrogenase Synthesis.

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.

A H2 -oxidizing, 1,2,3-trichlorobenzene-reducing multienzyme complex isolated from the obligately organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1.

Dehalococcoides mccartyi is a small, slow-growing bacterium of the phylum Chloroflexi that conserves energy using aliphatic and aromatic organohalides as electron acceptors, and hydrogen as sole electron donor. A recent study identified a protein complex in the membrane of strain CBDB1 comprising a Hup hydrogenase, a complex iron-sulphur molybdoprotein and a reductive dehalogenase (RdhA) that catalyses reduction of 1,2,3,4-tetrachlorobenzene. Using a combination of size-exclusion chromatography, in-gel hydrogenase activity-staining, immunological analysis and mass spectrometry, we identified here a large molecular mass protein complex solubilized from the cytoplasmic membrane of D. mccartyi strain CBDB1 that catalysed H2 -dependent reduction of 1,2,3-trichlorobenzene (1,2,3-TCB) to 1,3-DCB. In-gel zymographic staining revealed H2 :benzyl viologen oxidoreductase activity associated with the complex and immunological analysis identified co-elution of CdbdA195, the predicted catalytic subunit of the iron-sulphur molybdoenzyme, the chlorobenzene-specific RdhA, CbrA, and traces of HupL, the catalytic subunit of the Hup hydrogenase. Quantitative reverse transcriptase PCR analyses indicated that the expression of the hupL and cbdbA195 genes was induced by 1,2,3-TCB but not by hydrogen. Together, these data identify and describe a protein-based electron-transfer complex catalysing H2 oxidation coupled to chlorobenzene reduction.