Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12

Ballantine, Stuart P.; Boxer, David H.

doi:10.1128/jb.163.2.454-459.1985

Cited by 205 publications

(124 citation statements)

References 23 publications

(33 reference statements)

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“…Protein concentration was determined [22] with bovine serum albumin as standard. Hydrogenase enzyme activity was determined according to [23] except that the buffer used was 50 mM MOPS, pH 7.5. Activities were measured using 1 cm path-length anaerobic cuvettes using an Uvicon 900 dual-wavelength spectrophotometer.…”

Section: Other Methodsmentioning

confidence: 99%

Development of a cell‐free system reveals an oxygen‐labile step in the maturation of [NiFe]‐hydrogenase 2 of Escherichia coli

Soboh¹,

Krüger²,

Kuhns³

et al. 2010

FEBS Letters

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a b s t r a c tBy combining extracts from strains lacking genes encoding either the maturation enzymes or the large subunits of hydrogenases 1, 2 and 3 we could reconstitute in vitro under strictly anaerobic conditions 10-15% of the hydrogenase activity present in wild type Escherichia coli extracts. Purified, unprocessed Strep-tagged variants of the hydrogenase 2 large subunit, HybC, isolated from either a DhybD (encoding the hydrogenase 2-specific protease) mutant or a strain deficient in HypF could also be matured to active, processed enzyme using this system. These studies reveal that minimally one step early on the hydrogenase maturation pathway is oxygen-labile.

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Section: Other Methodsmentioning

confidence: 99%

Development of a cell‐free system reveals an oxygen‐labile step in the maturation of [NiFe]‐hydrogenase 2 of Escherichia coli

Soboh¹,

Krüger²,

Kuhns³

et al. 2010

FEBS Letters

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“…All assays were performed at 2 5 T . Hydrogenase activity was routinely measured as H2 : benzyl viologen oxidoreductase exactly as described previously [6]. One unit of activity is the amount catalysing the reduction of 1 p o l benzyl viologen min-'.…”

Section: Methodsmentioning

confidence: 99%

“…This induced the expression of isoenzyme 1 without loss of isoenzyme 2 content. The cells were harvested after 5 h of growth and membrane vesicles prepared as previously described [6]. The 63Ni-labelled membrane vesicles were added to the membrane fraction prepared from 300 g wet weight of cells and the preparation carried out as described above.…”

Section: Preparation Of Hydrogenase Isoenzyme Imentioning

confidence: 99%

Purification and properties of membrane-bound hydrogenase isoenzyme 1 from anaerobically grown Escherichia coli K12

1986

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Hydrogenase isoenzyme 1 from the membrane fraction of anaerobically grown Escherichia coli has been purified to near homogeneity. The preparation involved dispersion of the membrane fraction with deoxycholate followed by ammonium sulphate precipitation, ion-exchange, hydroxyapatite and gel filtration chromatography steps. The enzyme was assayed by quantification of the H2 : benzyl viologen oxidoreductase activity immunoprecipitated by a non-inhibitory antiserum specific for the enzyme. The enzyme constituted about 8 % of the hydrogenase activity found in the detergent-dispersed membranes, the remainder being attributable to hydrogenase isoenzyme 2. Isoenzyme 1 was purified 130-fold and the specific activity of the final preparation was 10.6 pmol benzyl viologen reduced min-(mg protein)-' (H2 : benzyl viologen oxidoreductase). The final preparation contained polypeptides of apparent M , 64000, 31 000 and 29000. Antibodies were raised both to the final preparation and to immunoprecipitation arcs containing hydrogenase isoenzyme 1, excised from crossed immunoelectrophoresis plates. The former cross-reacted with all three polypeptides in the enzyme preparation but the latter recognised only the M,-64000 polypeptide. Immunological analysis revealed that the polypeptides of apparent M , 31000 and 29000 are fragments of a single polypeptide of M, 35000 which is present in the detergent-dispersed membranes. The fragmentation of the M,-35 000 polypeptide during the preparation correlates with a change in the electrophoretic mobility of the enzyme. A similar electrophoretic mobility change was observed, accompanied by cleavage of the Mr-35000 polypeptide to one of 32000 when the enzyme was analysed after exposure of detergent-dispersed membranes to trypsin. The enzyme in the detergent-dispersed membranes consists minimally of two subunits of M , 64000 and two subunits of M , 35000. It contained 12.2 mol Fe and 9.1 mol acid-labile S2-/200000 g enzyme. The enzyme, purified from bacteria grown in the presence of 63Ni, was found to contain 0.64 (+ 0.20) mol Ni/200 000 g enzyme. A constant ratio of 63Ni immunoprecipitated to hydrogenase isoenzyme 1 activity immunoprecipitated by antiserum specific for the enzyme was observed during the preparation, consistent with Ni being part of the enzyme. The enzyme has a low K , for H2 (2.0 pM) in the H2 : benzyl viologen oxidoreductase assay. It catalyses H2 evolution employing reduced methyl viologen as electron donor. It is inhibited reversibly by CO and irreversibly by N-bromosuccinimide.Hydrogenase (EC 1.12. -. -) in Escherichia coli is involved in two distinct modes of H2 metabolism [l]. The formate hydrogenlyase system catalyses the oxidation of endogenously produced formate to C 0 2 and H2 during anaerobic growth on glucose in the absence of a terminal respiratory electron acceptor. An hydrogenase forms part of the formate hydrogenlyase system [2]. E. coli is also able to derive energy to support anaerobic growth from the oxidation of H2 coupled to the reduction of fumarate, a non...

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“…To detect HYD1 and HYD2 activities specifically, crude extracts were separated on 7.5% non-denaturing polyacrylamide gels and HYD1 and HYD2 were visualized with a benzyl viologen-linked hydrogen uptake assay [14]. The activity bands were fixed by the addition of 1 mM 2,3,5-triphenyltetrazolium chloride.…”

Section: Activity Stainingmentioning

confidence: 99%

“…In contrast, HYD1 and HYD2 are integral membrane proteins [6,7]. HYD2 is particular in that an active HYD2 can be solubilized following treatment of the isolated membrane fraction with trypsin [14]. However, it remained unclear to which surface of the cytoplasmic membrane HYD2 is attached.…”

Section: Introductionmentioning

confidence: 99%

Requirement for nickel of the transmembrane translocation of NiFe‐hydrogenase 2 in Escherichia coli

Rodrigue¹,

Boxer

Mandrand‐Berthelot³

et al. 1996

FEBS Letters

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The cellular location of membrane-bound NiFehydrogenase 2 (HYD2) from Escherichia coil was studied by immunobiot analysis and by activity staining. Treatment of spheroplasts with trypsin was able to release active HYD2 into the soluble fraction, indicating that HYD2 is attached to the periplasmic side of the cytoplasmic membrane and that HYD2 undergoes a trans-membrane translocation during its biosynthesis. By using a n/k mutant deficient in the high affinity specific nickel transport system, we show that the intracellniar availability of nickel is essential for the processing of the large subunit and for the transmembrane translocation of HYD2. We also demonstrate that the processing of the precursor, which is related with nickel incorporation, can occur in the membrane-depleted soluble fraction and that it is associated with the increase in resistance to proteolysis of the processed form of the large subunit. The mechanism of the transmembrane translocation of HYD2 is discussed.

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Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12

Cited by 205 publications

References 23 publications

Development of a cell‐free system reveals an oxygen‐labile step in the maturation of [NiFe]‐hydrogenase 2 of Escherichia coli

Development of a cell‐free system reveals an oxygen‐labile step in the maturation of [NiFe]‐hydrogenase 2 of Escherichia coli

Purification and properties of membrane-bound hydrogenase isoenzyme 1 from anaerobically grown Escherichia coli K12

Requirement for nickel of the transmembrane translocation of NiFe‐hydrogenase 2 in Escherichia coli

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