Tetrazolium-based redox dyes are useful tools in zymographic detection of oxidoreductase enzyme activity in non-denaturing PAGE because upon irreversible reduction they generate coloured, insoluble formazan complexes, which are advantageous in cumulative staining procedures. Triphenyl tetrazolium has been used for a considerable time as a means of distinguishing the hydrogenase enzymes in E. coli
cell extracts [18
]. Measuring Hyd-3 activity in the presence of the H2
-oxidizing enzymes was problematic in the past and visualizing it had not been successfully accomplished until the current study was conducted. However, optimization of the in-gel assay conditions, together with the judicious use of defined mutants has allowed us for the first time to visualize Hyd-3 activity unequivocally after native-PAGE. The complexes exhibiting Hyd-3 activity migrate in native-PAGE at high molecular masses, similar to the trimer of trimers of the Fdh-N and Fdh-O with a mass of 500-550
]. This suggests that the stoichiometry of the individual components in the FHL complex might be greater than unity. Nothing is currently known about the stoichiometry of the FHL complex components or the architecture of the HycE/HycG large and small subunit within the complex, and this will form the subject of future studies.
The findings of the current study suggest that while the Fdh-H component of the FHL complex is required for maximal activity of the complex, in its absence activity of the Hyd-3 can still be detected and its migration position in the gel system is very similar in extracts of the wild-type and the fdhF
mutant. This suggests perhaps that the Fdh-H component is separated from the rest of the complex during electrophoresis. The lability of the Fdh-H activity has been noted previously [15
One possible reason why the Hyd-3 activity was previously overlooked after in-gel staining is the considerable overlap in the staining pattern of Fdh-N/O, Hyd-3 and Hyd-2. Alternatively, reliable detection of Hyd-3 activity appears to require hydrogen concentrations of minimally 5% in the gas phase and many of our previous studies used lower concentrations [20
]. 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.
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
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 Eh
]. For example a 100% hydrogen atmosphere will result in a Eh
mV in anaerobic cultures, while a 5% hydrogen concentration in the headspace equates to a redox potential of around -370
mV and 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 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
]) future studies will 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
]. PMS-mediated staining has been previously used for the oxygen-tolerant hydrogenases from R. eutropha
], 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 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 (Eh
value of -80
mV). The similar redox potential of NBT in our assay buffer with and without PMS (see Table ), indicates that Hyd-1 should reduce NBT directly, which is indeed what we have 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
]. The combined inclusion of BV (Eh
mV) and TTC (Eh
mV), along with 5% hydrogen in the headspace, of the assay was sufficient to maintain a low 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 ).
In stark contrast to Hyd-2 and Hyd-3, Hyd-1 shows a high activity at redox potentials above -100
]. 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
]. 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.