Hyd-1 migrates as a single, fast-migrating activity band and introduction of a mutation in the hyaB gene, encoding the large subunit, abolished activity (Figure 1). Hyd-2, on the other hand, migrates as two more slowly-migrating activity bands and these are no longer detectable in hybC deletion mutant (Figure 1; [20]). Through the analysis of defined mutants lacking all 3 hydrogenases, it has been shown recently that the respiratory Fdh-N and Fdh-O enzymes also exhibit a H2:BV oxidoreductase activity, thus potentially defining a new class of hydrogenase [21]. The weak hydrogenase activity due to Fdh-N Cell Cycle inhibitor and Fdh-O is clearly visible

in a crude extract derived from strain HDK203, which lacks functional Hyd-2 and Hyd-3 enzymes (left lane of Figure 1). No other H2:BV oxidoreductase enzyme activity is discernible under the conditions used in the experiment shown in Figure 1. Figure 1 Identification of hydrogenases 1 and 2 in defined hydrogen metabolism mutants. Extracts from strains HDK203 (ΔhybBC hycA-H), which is Hyd-1+, HDK101 (Δhya hycA), which is Hyd-2+ and Hyd-3+ and HDK103 (Δhya hycA-H), which is Hyd-2+ were derived from cells after anaerobic growth in TGYEP, pH 6.5 and 25 μg of protein were applied to non-denaturating PAGE (7.5% w/v polyacrylamide). After

electrophoresis the gel was stained in an anaerobic glove box in the presence of ≤5% H2 with BV and TTC as described in the Methods section. On the right hand side of the figure the migration patterns of the selleck screening library formate dehydrogenases N and O (Fdh-N/O) and Interleukin-3 receptor the hydrogenases

(Hyd) 1 and 2 are given. The top of the gel is marked by an arrow. The conditions under which activity-staining is normally carried out involve long incubation times and a gas atmosphere of ≥ 95% nitrogen/≤ 5% hydrogen [20]. Because the Hyd-3 enzyme component of the FHL complex normally catalyzes proton reduction Selleckchem TPCA-1 rather than hydrogen oxidation in vivo and the spectrophotometric assay of this enzyme typically involves using saturating hydrogen concentrations, and consequently a very low redox potential in the assay, we decided to perform an in-gel activity stain under a 100% hydrogen gas atmosphere. Surprisingly, after exposure for only 10 minutes (see Methods) a prominent and highly active, high molecular weight complex showing H2:BV oxidoreductase activity appeared when the native gel was incubated in the presence of a 100% hydrogen atmosphere (Figure 2A, left panel). Although active Hyd-1 could also be detected, no activity bands corresponding to either Hyd-2 or the Fdh-N/O enzymes were observed under these conditions. The activity of this high-molecular weight complex was shown to be dependent on the presence of the hyc genes, as it was absent in extracts of strains CP971 (ΔhycA-I), FTD147 (ΔhyaB hybC hycE) and FTD150 (ΔhyaB hybC hycE hyfB-R) (Figure 2A).