title: The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen: benzyl viologen oxidoreductase activity.
abstract: BACKGROUND
  Escherichia 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.
  
  
  RESULTS
  Here 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Institute for Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str, 3, 06120 Halle (Saale), Germany.
authors.forename: Basem
authors.initials: B
authors.lastname: Soboh
authors.forename: Constanze
authors.initials: C
authors.lastname: Pinske
authors.forename: Martin
authors.initials: M
authors.lastname: Kuhns
authors.forename: Mandy
authors.initials: M
authors.lastname: Waclawek
authors.forename: Christian
authors.initials: C
authors.lastname: Ihling
authors.forename: Karen
authors.initials: K
authors.lastname: Trchounian
authors.forename: Armen
authors.initials: A
authors.lastname: Trchounian
authors.forename: Andrea
authors.initials: A
authors.lastname: Sinz
authors.forename: Gary
authors.initials: G
authors.lastname: Sawers
mesh_terms: D001594:Benzyl Viologen / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005560:Formate Dehydrogenases / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism*; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D051140:Selenoproteins / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D051140:Selenoproteins; D001594:Benzyl Viologen; D006859:Hydrogen; D010088:Oxidoreductases; D005560:Formate Dehydrogenases