Using high concentrations of hydrogen in the staining procedure has the advantage that Hyd-3 activity is detectable after a few minutes’ exposure, while Hyd-2 is not detectable under these conditions, possibly due to the low abundance of the enzyme in extracts of E. coli coupled with the brief exposure to hydrogen. Hyd-3, like Hyd-1, is a more abundant

enzyme and this possibly explains the rapid visualization of both these enzymes after only 10 min exposure to high hydrogen concentrations. AZD1390 datasheet The fact that the FHL complex is active in H2 oxidation contrasts the physiological direction of the reaction in the E. coli cell. This, therefore, might be an explanation for the comparatively high H2 concentrations required to drive the reaction in the direction of hydrogen oxidation. The similar redox potentials of formate and hydrogen do, however, indicate that this reaction should be freely reversible, possibly pointing to a role of a progenitor of the FHL complex in CO2 fixation [44]. Another possible explanation for the effect of hydrogen concentration on Hyd-3 activity is that high hydrogen concentrations drive the redox potential of a solution to more negative E h values [10]. For example

a 100% hydrogen atmosphere will result in a E h = -420 mV in anaerobic cultures, while a 5% hydrogen concentration in the headspace equates to a redox potential of around -370 mV and BLZ945 nmr a dissolved hydrogen concentration in cultures of maximally 40 μM at 25°C [36]. Our recent studies have shown that the [Fe-S]-cluster-containing small selleckchem subunit of the hydrogenase must be associated with the large subunit in order for hydrogen-dependent BV reduction to occur [20]. It is possible that BV receives electrons from a [Fe-S] cluster. If this is the case, then hydrogen-dependent BV reduction by a component of Hyd-3 also possibly occurs via a [Fe-S] cluster; however, due to the considerable number of [Fe-S] cluster-containing subunits in the complex (HycB, HycF, HycG and the Fdh-H enzyme itself [20, 45]) future studies will aminophylline be required to elucidate whether BV can interact with one or several

sites in the complex. The use of the electron acceptor NBT enabled a clear distinction between Hyd-1 and Hyd-2 activities. Previous experiments have shown that PMS/NBT staining is sometimes non-specific due to interaction with protein-bound sulfhydryl groups and even BSA was shown to be capable of staining gels incubated with PMS/NBT [46]. We could clearly show in this study, however, that, of the hydrogenases in E. coli, only Hyd-1 was capable of the specific, hydrogen-dependent reduction of PMS/NBT. Notably, both respiratory Fdhs also showed a strong NBT-reducing activity, which correlates well with previous findings for these enzymes [21]. Hyd-1 is similar to the oxygen-tolerant hydrogenases of R. eutropha and it is equipped with two supernumerary cysteinyl residues, which coordinate the proximal [4Fe-3S]-cluster [9, 47].