This recombinant Pyrococcus furiosus NADP-specific NiFe-type cytoplasmic hydrogenase (SHI) is from the hyperthermophilic P. furiosus microorganism that grows optimally at 100°C. The enzyme is active over a broad temperature range, from ambient to 100°C, and is oxygen-sensitive, with a half-life in air of approximately 25 hours at room temperature.
The [NiFe] hydrogenases contain a minimum of two subunits known as the small (S) and large (L) subunits. The small subunit contains three iron-sulfur clusters while the large subunit contains the active site, a nickel-iron center which is connected to the solvent by a molecular tunnel. To date, periplasmic, cytoplasmic, and membrane-bound hydrogenases have been found. [NiFe] hydrogenases are known to be deactivated by molecular oxygen (O2). The [NiFe] hydrogenase of Pyrococcus furiosus is heterotetrameric wherein the additional two subunits allow the enzyme to use NAD(P)(H) as an electron carrier.
From the laboratory of Michael W. W. Adams, PhD, University of Georgia.
Part of The Investigator's Annexe program.
|Name:||Cytoplasmic [NiFe]-Hydrogenase (4 subunits; OE-SHI)|
|Accession ID:||alpha subunit (RefSeq: NP_578623.1; Locus Tag: PF0894) delta subunit (RefSeq: NP_578622.1; Locus Tag: PF0893)gamma subunit (RefSeq: NP_578621.1; Locus Tag: PF0892)beta subunit (RefSeq: NP_578620.1; Locus Tag: PF0891)|
|Molecular Weight:||Predicted: 155 kDa, Size Exclusion: 149 kDa +/- 5 kDa|
|Amino Acid Sequence:||Sequences available upon request|
|Fusion Tag(s):||His(x9): HHHHHHHHHG|
|Buffer:||50 mM Tris, 2 mM DT, 300 mM NaCl, pH 8.2|
|Concentration:||1mg/mL (>100 U/mL, see Additional Information below)|
|Comments:||NADP(H)-dependent reversible hydrogenase|
|Storage:||This item is oxygen sensitive. Stable when stored sealed in strictly anaerobic environment (<10 ppm O2) at room temperature for up to 6 months. For long-term storage, protein can be flash frozen in nitrogen and stored at -80C.|
This is a Histidine-tagged version of the published Strep-tag II version. This is an extremely thermostable, but oxygen sensitive enzyme. One unit (U) is 1 µmole of H2 evolved min-1 mg-1 using MV-linked assay.
The purified enzyme was analyzed by conventional SDS-PAGE except that the protein was incubated with the SDS-loading buffer for 10 min (lane 1) or for 60 min (lane 3) prior to electrophoresis. Native SHI (treated for 10 min) is shown in lane 2. The arrow indicates the high molecular weight catalytically active protein band seen in lanes 1 and 2. The center lane (M) contains the protein molecular weight ladder (Invitrogen) with corresponding masses as indicated in kDa.
Adapted from: Chandrayan, S. K., et al. J. Biol. Chem. 287, 3257-64.
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