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.

Purification and characterization of two forms of hydrogenase isoenzyme 1 from Escherichia coli

“…Boxer and co-workers (4, 28) purified HYD1 and an active fragment of HYD2 and reported that these two isoenzymes are immunologically distinct. Other descriptions of hydrogenase purification from E. coli, to different levels of purity, can be found in the literature (1,5,13,14,31). Hydrogenases that had been purified to homogeneity had apparent molecular weights of 56,000 (1), 58,000 (14), and 200,000 (28).…”

Randomly induced Escherichia coli K-12 Tn5 insertion mutants defective in hydrogenase activity

“…Although several reports suggest a respiratory role for hydrogenase 2, our previous results (28) suggested that a labile hydrogenase species was involved in H, uptake. In this study, further evidence is provided in favor of the latter model.…”

“…The underlying genetic regulation mechanisms, however, are still poorly understood. Although more than 10 genetic loci have been described thus far, mapping near 58 min (11,15,22,24,30), 65 min (15,28), and 77 min (31), none of these searches was exhaustive, and it is still unknown whether these studies revealed all loci involved in H, metabolism. We therefore undertook a systematic screening of a large number of Tn5 insertion mutants; this search yielded one new locus, which was further characterized.…”

“…locus might make up the structural gene for the labile hydrogenase 2 (5), and hydL might regulate expression of the labile hydrogenase L and a second component, both required for Hup activity (28). In this paper, additional data are provided on hydC and a hypothetical function is assigned.…”

“…Thus far, at least three isoenzyme activities have been demonstrated: activities of the two electrophoretically stable hydrogenases 1 and 2 (3,4,26) and a third activity, which is inactivated upon gel electrophoresis and may consist of one or more isoenzymes (25,28). All of these isoenzymes probably contain nickel as a cofactor.…”