eutropha[22, 23], which led to the suggestion that particular structural features of oxygen-tolerant hydrogenases accounted for the differences in dye-reducing activity of the oxygen-tolerant and sensitive enzymes. The supernumerary Cys-19 of the small subunit, when exchanged for a glycine was shown to convert Hyd-1 from an oxygen-tolerant to an oxygen-sensitive enzyme [9]. This amino acid exchange did not affect NBT reduction in our assay system, thus indicating that the

oxygen-tolerance is not the sole reason for the ability of Hyd-1 to reduce NBT. This finding is also in agreement with the recent observation Selleckchem Fedratinib that the exchange of the supernumerary cysteines does not affect the catalytic bias of Hyd-1 to function in hydrogen-oxidation [9]. The structural and electronic properties of Hyd-1 [40] probably

govern its ability to transfer electrons from hydrogen to comparatively high-potential redox dyes such as NBT (E h value of -80 mV). The similar redox potential of NBT in our assay buffer with and without PMS (see Table 2), indicates that Hyd-1 should reduce NBT directly, which is indeed what we have see more observed (data not shown). Neither Hyd-3 nor Hyd-2 can reduce NBT and this is presumably because they function optimally at very low redox potentials, although potential steric effects restricting interaction of the enzymes with the dye cannot be totally excluded at this stage. Hyd-2 is a classical hydrogen-oxidizing enzyme that functions optimally at redox potentials lower than -100 to -150 mV [8, 10]. The selleck chemicals combined inclusion of BV (E

h = -360 mV) and TTC (E h = -80 mV), along with 5% hydrogen in the headspace, of the assay was sufficient to maintain a low Resminostat redox potential to detect Hyd-2 readily. This also explains why long incubation times are required for visualization of Hyd-1 activity with the BV/TTC assay. Increasing the hydrogen concentration in the assay to 100% drives the redox potential below -320 mV and explains why the Hyd-3 activity was readily detectable at hydrogen concentrations above 25% (see Figure 4). In stark contrast to Hyd-2 and Hyd-3, Hyd-1 shows a high activity at redox potentials above -100 mV [8, 10]. In the assay system used in this study, the presence of NBT in the buffer system resulted in a redox potential of -65 mV in the presence 5% hydrogen and -92 mV when the hydrogen concentration was 100%, both of which are optimal for Hyd-1 activity and well above that where the Hyd-2 is enzymically active [8, 10]. Placed in a cellular context, this agrees perfectly with the roles of Hyd-2 in coupling hydrogen oxidation to fumarate reduction, of Hyd-1 in scavenging hydrogen during microaerobiosis and of Hyd-3 in functioning at very low redox potentials in proton reduction [1]. This allows the bacterium to conduct its hydrogen metabolism over a very broad range of redox potentials.