Abstract
BackgroundEscherichia coli synthesizes three membrane-bound molybdenum- and selenocysteine-containing formate dehydrogenases, as well as up to four membrane-bound [NiFe]-hydrogenases. Two of the formate dehydrogenases (Fdh-N and Fdh-O) and two of the hydrogenases (Hyd-1 and Hyd-2) have their respective catalytic subunits located in the periplasm and these enzymes have been shown previously to oxidize formate and hydrogen, respectively, and thus function in energy metabolism. Mutants unable to synthesize the [NiFe]-hydrogenases retain a H2: benzyl viologen oxidoreductase activity. The aim of this study was to identify the enzyme or enzymes responsible for this activity.ResultsHere we report the identification of a new H2: benzyl viologen oxidoreductase enzyme activity in E. coli that is independent of the [NiFe]-hydrogenases. This enzyme activity was originally identified after non-denaturing polyacrylamide gel electrophoresis and visualization of hydrogen-oxidizing activity by specific staining. Analysis of a crude extract derived from a variety of E. coli mutants unable to synthesize any [NiFe]-hydrogenase-associated enzyme activity revealed that the mutants retained this specific hydrogen-oxidizing activity. Enrichment of this enzyme activity from solubilised membrane fractions of the hydrogenase-negative mutant FTD147 by ion-exchange, hydrophobic interaction and size-exclusion chromatographies followed by mass spectrometric analysis identified the enzymes Fdh-N and Fdh-O. Analysis of defined mutants devoid of selenocysteine biosynthetic capacity or carrying deletions in the genes encoding the catalytic subunits of Fdh-N and Fdh-O demonstrated that both enzymes catalyze hydrogen activation. Fdh-N and Fdh-O can also transfer the electrons derived from oxidation of hydrogen to other redox dyes.ConclusionsThe related respiratory molybdo-selenoproteins Fdh-N and Fdh-O of Escherichia coli have hydrogen-oxidizing activity. These findings demonstrate that the energy-conserving selenium- and molybdenum-dependent formate dehydrogenases Fdh-N and Fdh-O exhibit a degree of promiscuity with respect to the electron donor they use and identify a new class of dihydrogen-oxidizing enzyme.
Highlights
Escherichia coli synthesizes three membrane-bound molybdenum- and selenocysteine-containing formate dehydrogenases, as well as up to four membrane-bound [NiFe]-hydrogenases
Metabolism of hydrogen and formate is often highly interlinked in many bacteria that can oxidize both compounds. This is exemplified in the fermentative metabolism of the enterobacterium Escherichia coli where up to one third of the carbon from glucose is converted to formate; formate is disproportionated to H2 and CO2 [1,2,3]
Hydrogenase-independent hydrogen: BV oxidoreductase activity in E. coli membranes Membrane fractions derived from anaerobically cultured wild-type E. coli K-12 strains such as P4X [12,15] and MC4100 [16] exhibit a slowly migrating hydrogen: benzyl viologen (BV) oxidoreductase activity that cannot be assigned to either Hyd-1 or Hyd-2
Summary
Escherichia coli synthesizes three membrane-bound molybdenum- and selenocysteine-containing formate dehydrogenases, as well as up to four membrane-bound [NiFe]-hydrogenases. Fdh-N (FdnGHI) and Fdh-O (FdoGHI) are highly related enzymes at both the amino acid sequence and functional levels [1,4] They are multi-subunit oxidoreductases each comprising a large catalytic subunit (FdnG or FdoG), an electron-transfer subunit (FdnH or FdoH) and a membrane-anchoring subunit (FdnI or FdoI); the latter has a quinone-binding site that allows transfer of electrons derived from formate oxidation into the respiratory chain [4,5,6]. Both enzymes have their respective active site located on the periplasmic face of the cytoplasmic membrane and they couple formate oxidation to energy conservation. Fdh-O is synthesized constitutively and is present at low levels aerobically, during fermentative growth and nitrate respiration [6,9]
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