E. coli has also the coding capacity to synthesize four membrane-associated, multi-subunit Hyd enzymes, which are termed Hyd-1 through

Hyd-4 [2, 10]. Hyd-1, Hyd-2 and Hyd-3 have been characterized in detail. Like Fdh-N and Fdh-O, Hyd-1 and Hyd-2 have their active sites located facing the periplasm [11]. Both enzymes oxidize hydrogen and contribute to energy conservation. Due to the fact that hydrogenases catalyze the reversible oxidation of dihydrogen in vitro, the activities of all three characterized [NiFe]-hydrogenases of E. coli can be determined simultaneously in a single reaction using hydrogen as electron donor and the artificial electron acceptor benzyl viologen (BV) [12, 13]. Moreover, the hydrogen-oxidizing activities

of Hyd-1 and Hyd-2 can also be visualized after electrophoretic separation www.selleckchem.com/products/Bortezomib.html under non-denaturing conditions in the presence of detergent [12]. Because of its apparent labile nature the activity of Hyd-3 cannot be visualized after gel electrophoresis. It was noted many years ago [14] that in non-denaturing polyacrylamide gels a slowly-migrating protein complex with a hydrogen: BV oxidoreductase enzyme activity, apparently unrelated to either Hyd-1 or Hyd-2, could be visualized after electrophoretic separation of membrane fractions derived from E. coli grown under anaerobic conditions. In this study, this hydrogenase-independent enzyme activity could be identified as being catalyzed by the highly related Fdh-N and Fdh-O enzymes. Results Hydrogenase-independent hydrogen: BV oxidoreductase https://www.selleckchem.com/products/dibutyryl-camp-bucladesine.html Bacterial neuraminidase 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 NVP-BGJ398 concentration assigned to either Hyd-1 or Hyd-2. Previous findings based on non-denaturing PAGE [16] estimated a size of approximately 500 kDa for this complex. To demonstrate the hydrogenase-independent nature of this enzyme activity, extracts derived from a hypF mutant, which lacks the central hydrogenase maturase HypF and consequently is unable to synthesize active [NiFe]-hydrogenases [17], retained this single slowly migrating species exhibiting hydrogen:BV oxidoreductase activity, while the activity bands corresponding to Hyd-1 and Hyd-2 were no longer visible (Figure 1). This result demonstrates that the activity of this slowly migrating band is completely unrelated to the [NiFe]-hydrogenases Hyd-1, Hyd-2, Hyd-3 or Hyd-4. Note that no active, stained bands were observed when this experiment was performed with a nitrogen gas atmosphere (data not shown). Figure 1 A hypF mutant retains hydrogenase-independent H 2 : BV oxidoreductase activity.