Zymographic differentiation of [NiFe]-Hydrogenases 1, 2 and 3 of Escherichia coli K-12
© Pinske et al.; licensee BioMed Central Ltd. 2012
Received: 6 May 2012
Accepted: 25 June 2012
Published: 6 July 2012
When grown under anaerobic conditions, Escherichia coli K-12 is able to synthesize three active [NiFe]-hydrogenases (Hyd1-3). Two of these hydrogenases are respiratory enzymes catalysing hydrogen oxidation, whereby Hyd-1 is oxygen-tolerant and Hyd-2 is considered a standard oxygen-sensitive hydrogenase. Hyd-3, together with formate dehydrogenase H (Fdh-H), forms the formate hydrogenlyase (FHL) complex, which is responsible for H2 evolution by intact cells. Hydrogen oxidation activity can be assayed for all three hydrogenases using benzyl viologen (BV; Eo′ = -360 mV) as an artificial electron acceptor; however ascribing activities to specific isoenzymes is not trivial. Previously, an in-gel assay could differentiate Hyd-1 and Hyd-2, while Hyd-3 had long been considered too unstable to be visualized on such native gels. This study identifies conditions allowing differentiation of all three enzymes using simple in-gel zymographic assays.
Using a modified in-gel assay hydrogen-dependent BV reduction catalyzed by Hyd-3 has been described for the first time. High hydrogen concentrations facilitated visualization of Hyd-3 activity. The activity was membrane-associated and although not essential for visualization of Hyd-3, the activity was maximal in the presence of a functional Fdh-H enzyme. Furthermore, through the use of nitroblue tetrazolium (NBT; Eo′ = -80 mV) it was demonstrated that Hyd-1 reduces this redox dye in a hydrogen-dependent manner, while neither Hyd-2 nor Hyd-3 could couple hydrogen oxidation to NBT reduction. Hydrogen-dependent reduction of NBT was also catalysed by an oxygen-sensitive variant of Hyd-1 that had a supernumerary cysteine residue at position 19 of the small subunit substituted for glycine. This finding suggests that tolerance toward oxygen is not the main determinant that governs electron donation to more redox-positive electron acceptors such as NBT.
The utilization of particular electron acceptors at different hydrogen concentrations and redox potentials correlates with the known physiological functions of the respective hydrogenase. The ability to rapidly distinguish between oxygen-tolerant and standard [NiFe]-hydrogenases provides a facile new screen for the discovery of novel enzymes. A reliable assay for Hyd-3 will reinvigorate studies on the characterisation of the hydrogen-evolving FHL complex.
KeywordsNiFe Hydrogenase Formate hydrogenlyase Formate dehydrogenase Non-denaturating polyacrylamide gel electrophoresis In-gel activity staining Redox-dyes
Under anaerobic conditions Escherichia coli synthesizes three membrane-associated [NiFe]-hydrogenases (Hyd), although its genome has the capacity to encode four of these enzymes [1, 2]. Hyd-1 and Hyd-2 are respiratory hydrogenases with their active sites facing the periplasm and the structural subunits of these are encoded within the hya and hyb operons [3, 4], respectively. The physiological role of both enzymes is to couple hydrogen oxidation to the reduction of the quinone pool in the inner membrane, and they can be readily isolated and characterised in an active form [5–8]. Hyd-1 is an oxygen-tolerant hydrogenase while Hyd-2 is a ‘standard’ oxygen-sensitive enzyme  and it has been proposed that Hyd-1 functions at more positive redox potentials, which are found at the aerobic-anaerobic interface [8–10].
Hyd-3 is encoded by the hyc operon [11, 12] and forms a key component of the formate hydrogenlyase (FHL) complex, which is predicted to be associated with the cytoplasmic side of the inner membrane and catalyses hydrogen and carbon dioxide production from formate. Expression of FHL is maximal under fermentative conditions in the absence of exogenous electron acceptors and is absolutely dependent on formate . Hyd-3 is considered a labile hydrogenase that has so far proven recalcitrant to isolation in an active form . The labile molybdenum- and selenium-dependent formate dehydrogenase-H (Fdh-H) is also associated with the FHL complex . Fdh-H represents one of the three formate dehydrogenase enzymes in E. coli (Fdh-H, Fdh-O, and Fdh-N) . Fdh-O and Fdh-N are membrane-bound and periplasmically-oriented respiratory enzymes that couple formate oxidation to quinone reduction and thus contribute directly to energy conservation.
Several methods have been described for visualizing the redox activity of hydrogenases. Most commonly, low-potential artificial redox-active viologen dyes such as methyl viologen (MV) and benzyl viologen (BV) have been used [17, 18]. All three E. coli hydrogenases can couple H2 oxidation to BV reduction in vitro and when extracts from fermentatively-grown cells are assayed Hyd-3 can contribute over 90% to the total activity [19, 20]. While Hyd-1- and Hyd-2-catalysed BV reduction can be readily visualised and the enzymes distinguished by use of an in-gel assay , Hyd-3 activity has so far proved recalcitrant to zymographic identification and this had been thought to be due to the instability of the large FHL complex (see ). Moreover, the large respiratory Fdh-N and Fdh-O enzyme complexes also contribute some background staining due to their inherent H2:BV oxidoreductase activities, thus making any assessment of a Hyd-3 associated activity potentially problematic . Alternative hydrogenase assays have been developed for other biological systems. For example, the oxygen-tolerant hydrogenases from Ralstonia eutropha H16 can be visualized with phenazine methosulfate (PMS)/nitroblue tetrazolium (NBT)  or PMS/triphenyl tetrazolium chloride (TTC)  combinations of redox dyes. Methylene blue has also been used extensively in hydrogenase research . However, the use of alternative redox-active electron acceptors has not really been extensively explored for the hydrogenases of E. coli.
The aim of this study, therefore, was to investigate the differential activities of the E. coli hydrogenases with a view to making it possible to distinguish all enzymes synthesized under anaerobic growth conditions. We describe here conditions that allow the unequivocal visualization of all three, membrane-associated, anaerobically inducible hydrogenase enzyme complexes.
Identification of Hyd-3 activity through an in-gel assay
Fdh-H is required to stabilize Hyd-3 but is not essential for activity
Strains and references
F-, araD139, Δ(argF-lac)U169, λ-, rpsL150, relA1 deoC1, flhD5301, Δ(fruK-yeiR)725(fruA25), rbsR22, Δ(fimB-fimE)632(::IS1)
MC4100 ΔhyaB hybC
MC4100 ΔhyaB hybC fdhE
MC4100 ΔhyaB hybC fdhE fdhF (KmR)
MC4100 ΔhyaB hybC fdoG fdnG (KmR)
MC4100 Δ(selC)400 (KmR)
MC4100 ΔfdhF recA56
MC4100 ΔhyaB ΔhybC ΔhycE
MC4100 ΔhyaB ΔhybC ΔhycE ΔhyfB-R
MC4100 coding for a chromosomal in-frame C-terminal His-tag on HyaA
MC4100 Δhya (KmR) ΔhycA
MC4100 Δhya (KmR) ΔhycA-H
MC4100 ΔhybBC (KmR) ΔhycA-H
FTH004 encoding C19G/C120G exchange in HyaA
FTH004 encoding a C120G exchange in HyaA
FTH004 encoding a C19G exchange in HyaA
The large Hyd-3 protein complex is active in a neutral pH gel-system and is membrane-associated
The FHL complex is associated with the cytoplasmic membrane and the active site of each enzyme component (Fdh-H and Hyd-3) faces the cytoplasm . To determine whether the Hyd-3 activity identified in this study was membrane-associated the crude extracts derived from anaerobically grown wild-type (MC4100), CP971 (ΔhycA-I) and CPD17 (ΔhyaB hybC fdhE) were separated into soluble and membrane fractions and an aliquot of each was separated in the high-pH gel-system and stained for Hyd-3 activity in an atmosphere of 100% hydrogen (Figure 3B). The results clearly demonstrate that Hyd-3 activity, along with that attributable to Hyd-1, was membrane-associated.
High hydrogen partial pressure facilitates detection of Hyd-3 activity after native-PAGE
Redox potentials of the assay buffers
Hydrogen in headspace
50 mM MOPS, pH 7
50 mM MOPS, pH 7, BV/TTCa
50 mM MOPS, pH 7, PMS/NBTb
50 mM MOPS, pH 7, NBT
+ 170 mV
+ 78 mV
+ 74 mV
+ 73 mV
- 120 mV
- 264 mV
- 38 mV
- 65 mV
- 349 mV
- 322 mV
- 92 mV
- 102 mV
Hyd-1 catalyzes the hydrogen-dependent reduction of nitroblue tetrazolium
Through the analysis of extracts derived from anaerobically grown E. coli strains specifically unable to synthesize Hyd-1 (FTD22), Hyd-2 (FTD67), Hyd-3 (CP971), Hyd-1/Hyd-2 (CP734) or all three [NiFe]-hydrogenases (FTD147 and DHP-F2), it was shown that only strains able to synthesize Hyd-1 were capable of reducing nitroblue tetrazolium (NBT) in a hydrogen-dependent manner (Figure 2C, left panel). Notably, intensely stained activity bands of Hyd-1 were observed after only 5 min incubation with 5% H2 in the gas phase. The redox potential of the assay buffer in the presence of 5% headspace hydrogen was determined to be – 38 mV (Table 2), decreasing to – 98 mV with 100% hydrogen in the headspace. Hyd-2 was unable to reduce NBT even after an incubation period of 3 h, as only Hyd-1 was visualized for the wild-type MC4100 (Figure 2A). Incubation for 16 h did not alter this pattern of staining (data not shown). Equally, Hyd-3 was also incapable of transferring electrons to NBT (Figure 2C). Similarly, deletion of the genes coding for the putative Hyd-4 enzyme  in strain FTD150 also did not result in a different pattern from strain FTD147, which suggests that Hyd-4 is not active under the conditions tested.
To analyse the specificity of the apparent Hyd-1-dependent NBT stain, the strain FM460 (ΔselC) was employed and a crude extract derived from this strain displayed a Hyd-1 activity band of similar intensity to that in MC4100 but the extract lacked the slower migrating activity band confirming that this was due to Fdh-N and Fdh-O (Figure 2C, right panel), as previously reported . A selC mutant is incapable of incorporating selenocysteine into proteins and so lacks all formate dehydrogenase activity . Moreover, strains CPD17 and CPD23, both carrying a deletion in fdhE, and strain CPD24, which carries deletions in the genes encoding the large subunit of Fdh-N and Fdh-O (Figure 2C, right panel) also lacked the Fdh-N and Fdh-O activity bands, as anticipated. Taken together, the fast-migrating, H2-dependent NBT-reducing activity band shown here is not linked to formate dehydrogenase activity and is Hyd-1.
Reduction of NBT by Hyd-1 variants with amino acid exchanges in the supernumerary cysteines near the proximal [4Fe-3 S] cluster
The core catalytic dimer of Hyd-1 reacts with NBT
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, 19]. 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 kDa . 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, 43].
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 . 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 fixation .
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 values . For example a 100% hydrogen atmosphere will result in a Eh = -420 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 .
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 . 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 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 . 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 .
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]. PMS-mediated staining has been previously used for the oxygen-tolerant hydrogenases from R. 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 . 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 . The structural and electronic properties of Hyd-1  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 2), 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 mV [8, 10]. The combined inclusion of BV (Eh = -360 mV) and TTC (Eh = -80 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 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 . This allows the bacterium to conduct its hydrogen metabolism over a very broad range of redox potentials.
Using increased partial pressure of dihydrogen in combination with the artificial electron acceptor combination benzyl viologen/triphenyl tetrazolium chloride, we defined conditions allowing the identification of an active Hyd-3 enzyme complex after non-denaturing gel electrophoresis. Moreover, by substituting BV/TTC with nitroblue tetrazolium as an electron acceptor we could demonstrate that only the oxygen-tolerant Hyd-1 enzyme could catalyse hydrogen-dependent dye reduction, suggesting that this facile assay could be used to identify oxygen-tolerant hydrogenases in other microorganisms. However, the ability of Hyd-1 to reduce NBT was not dependent on the oxygen-tolerance of the enzyme because an oxygen-sensitive Hyd-1 variant in which the supernumerary Cys-19 was substituted by Gly retained the ability to reduce the redox dye.
Strains and growth conditions
All strains used in this study are listed in Table 1. E. coli strains were routinely grown at 37°C on LB-agar plates or with shaking in LB-broth . Plates were solidified by adding 1.5% (w/v) agar to the media. Anaerobic growths were performed at 37°C as standing liquid cultures. Cultures for determination of enzyme activity were grown in TGYEP media  containing 1% (w/v) peptone, 0.5% (w/v) yeast extract, 0.1 M potassium buffer pH 6.5 and the cultures were supplemented with 0.8% (w/v) of glucose. When required, the antibiotics kanamycin and chloramphenicol were added to the culture media to the final concentration of 50 μg and 12 μg per ml, respectively. The strains CPD17, CPD23 and CPD24 were constructed using P1kc phage transduction to move the respective defined deletion mutation from the appropriate strains obtained from the Keio collection [48, 50]. When required the plasmid pCP20 was used to remove the antibiotic resistance cassette as described .
Polyacrylamide gel electrophoresis
Non-denaturing PAGE was performed using a discontinuous system with 7.5% (w/v) polyacrylamide separating gels in 250 mM Tris/HCl buffer, pH 8.5 including 0.1% (w/v) Triton X-100 . As running buffer 0.1 M Tris/0.1 M glycine buffer was used. After reaching mid-exponential phase of growth cells were harvested from cultures by centrifugation at 10,000 x g for 15 min at 4 °C and after washing once in the same volume of 50 mM MOPS buffer pH 7.0, cells were resuspended in a tenth of their volume of 50 mM MOPS buffer pH 7.0, broken by sonification and cell debris and unbroken cells removed as described . Samples of crude extract were resuspended at a protein concentration of 10 mg ml-1 in 50 mM MOPS buffer pH 7.0 and incubated with a final concentration of 5% (w/v) Triton X-100 prior to application of the solubilized sample (usually 25 μg of protein) to the gels. Alternatively, for neutral pH analyses the barbitone gel system was used. This system uses final concentrations of 34 mM Tris-phosphate buffered stacking gel, pH 5.5 and 62.5 mM Tris-HCl resolving gel pH 7.5. The running buffer consists of 82.5 mM Tris and 26.8 mM diethylbarbituric acid, pH 7.0. Hydrogenase activity-staining was done as described in  with 0.5 mM benzyl viologen (BV) and 1 mM 2,3,5,-triphenyltetrazolium chloride (TTC) and continuous flushing with highly pure hydrogen gas until the activity bands appeared except that the buffer used was 50 mM MOPS pH 7.0. Alternatively, staining was done in hydrogen-flushed buffer using 0.3 mM phenazine methosulfate (PMS) as mediator and 0.2 mM nitroblue tetrazolium (NBT) as electron acceptor . When formate was added as substrate to the buffer, a final concentration of 50 mM was used. When used in native-PAGE molecular mass standard proteins from a gel filtration markers kit 29-700 kDa (Sigma) were mixed in equal amounts and 6 μg of each were loaded on the gel.
Immunological and enzymic methods
Western blotting was performed as described in  by transferring proteins to nitrocellulose membranes and challenging them with monoclonal penta-His antibody from mouse (Qiagen) or polyclonal anti-Hyd-1 antibody (1:20000). Secondary goat-anti-mouse or anti-rabbit antibody, respectively conjugated with HRP enzyme (Bio-Rad, USA) was used for visualisation with the Immobilon Western chemiluminescent HRP substrate (Millipore, USA). Purification of active Hyd-1 from a 5 L culture of strain FTH004 (His-HyaA) grown in TGYEP, pH 6.5 supplemented with 5 μM Ni2+ was carried out as described . Determination of protein concentration was done by the method of Bradford (Bio-Rad, USA) .
Measurement of redox potential
Aliquots of 50 mM MOPS buffer pH 7.0 containing the concentrations of the respective redox dyes indicated above were either incubated overnight in an anaerobic chamber with an atmosphere containing 5% hydrogen for 6 h or was bubbled with hydrogen gas (100% atmosphere) for 30 min and the redox potential determined using a EMC 30-K010-D redox micro-electrode (Sensortechnik Meinsburg GmbH, Germany) attached to a Lab850 pH/redox meter (Schott Instruments, Germany). The electrode was standardized using a redox buffer provided by the company. Measurements were performed two times.
We are grateful to Alison Parkin for providing the oxygen-sensitive hydrogenase 1 strains and to Stefanie Hartwig for help with the redox potential measurements. Martin Sauter is thanked for providing strain HDK101. This work was supported by the BBSRC grant BB/I02008X/1 to FS and DFG grant SA 494/3-1 to RGS.
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