title: Partial characterization of an electrophoretically labile hydrogenase activity of Escherichia coli K-12.
abstract: A mutant of Escherichia coli K-12 is described that is specifically impaired in only one hydrogenase isoenzyme. By means of Tn5-mediated insertional mutagenesis, a class of mutants was isolated (class I) that had retained 20% of the overall hydrogenase activity. As determined by neutral polyacrylamide gel electrophoresis, the mutant contained normal amounts of the hydrogenase isoenzymes 1 and 2. Therefore, the hydrogenase activity affected seemed to be electrophoretically labile and was called hydrogenase L. The presence of such an activity was recently suggested in various papers and was called isoenzyme 3. Hydrogenase L might be identical or part of the latter isoenzyme. By DEAE ion-exchange chromatography it could be separated from hydrogenases 1 and 2. Hydrogenase activity in the parent strain HB101, determined manometrically with cell-free preparations and methylviologen as the electron acceptor, immediately showed maximal activity. However, class I mutants showed a lag phase which was dependent on the protein concentration utilized in the assay. This suggested that the fast initial activity of HB101 was due to hydrogenase L. The enzyme or enzyme complex showed an Mr around 300,000 and a pH optimum between 7 and 8. Strong indications about its physiological role were provided by the finding that in class I mutants H2 production by the formate-hydrogen lyase pathway was unimpaired, whereas fumarate-dependent H2 uptake was essentially zero. Complementation with F-prime factor F'116 but not with F'143 and coconjugation and cotransduction experiments localized the mutation (hydL) close to metC at approximately 64.8 min.
authors.affiliation: Department of Microbiology, Vrije Universiteit, Amsterdam, The Netherlands.
authors.forename: K
authors.initials: K
authors.lastname: Stoker
authors.forename: L F
authors.initials: LF
authors.lastname: Oltmann
authors.forename: A H
authors.initials: AH
authors.lastname: Stouthamer
mesh_terms: D002852:Chromatography, Ion Exchange; D002874:Chromosome Mapping; D003227:Conjugation, Genetic; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008040:Genetic Linkage; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation; D011489:Protein Denaturation
chemical_list: D007527:Isoenzymes; D006864:Hydrogenase