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Literature summary extracted from

  • Van der Linden, E.; Burgdorf, T.; Bernhard, M.; Bleijlevens, B.; Friedrich, B.; Albracht, S.P.
    The soluble [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanides in its active site, one of which is responsible for the insensitivity towards oxygen (2004), J. Biol. Inorg. Chem., 9, 616-626.
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.12.1.2 FMN
-
 Cupriavidus necator

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.12.1.2 additional information release of one FMN reduces the NAD+ reduction by 90%, but is reversible by addition of excess FMN, insensitivity towards oxygen, with all 4 CN- groups bound to the enzyme, and towards CO Cupriavidus necator
1.12.1.2 O2 irreversible enzyme inhibition by oxygen occurs if the CN- bound to Ni2+ is irreversibly removed or if the enzyme is reduced by NADH Cupriavidus necator

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
1.12.1.2 cytoplasm
-
 Cupriavidus necator 5737
-
1.12.1.2 soluble
-
 Cupriavidus necator
-
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.12.1.2 CN- enzyme contains four cyanides in its active site, the Ni2+ bound one is responsible for the insensitivity towards oxygen, the active site is a (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO) centre, the CN- bound to the nickel ion can be irreversibly removed inducing enzyme inhibition by oxygen Cupriavidus necator
1.12.1.2 CO bound to the active site, the active site is a (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO) centre Cupriavidus necator
1.12.1.2 cyanide enzyme contains four cyanides in its active site, one of which is responsible for the insensitivity towards oxygen Cupriavidus necator
1.12.1.2 Fe2+ enzyme contains a [Ni-Fe] cluster Cupriavidus necator
1.12.1.2 Fe2+ the active site is a (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO) centre Cupriavidus necator
1.12.1.2 Ni2+ enzyme contains a [Ni-Fe] cluster Cupriavidus necator
1.12.1.2 Ni2+ the active site is a (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO) centre, H2 activation solely takes place on Ni2+ Cupriavidus necator

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.12.1.2 H2 + NAD+ Cupriavidus necator
-
 H+ + NADH
-
 ?
1.12.1.2 H2 + NAD+ Cupriavidus necator
-
 H+ + NADH
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.12.1.2 Cupriavidus necator
-
-
-
1.12.1.2 Cupriavidus necator
-
 facultative lithoautotrophic Knallgas bacterium
-

Oxidation Stability

EC Number Oxidation Stability Organism
1.12.1.2 insensitive to O2 Cupriavidus necator
1.12.1.2 insensitivity towards oxygen, irreversible enzyme inhibition by oxygen occurs if the CN- bound to Ni2+ is irreversibly removed or if the enzyme is reduced by NADH Cupriavidus necator

Purification (Commentary)

EC Number Purification (Comment) Organism
1.12.1.2
-
 Cupriavidus necator

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.12.1.2 additional information
-
-
 Cupriavidus necator
1.12.1.2 17 84 activity of different batches with NAD+ and H2 Cupriavidus necator
1.12.1.2 30 100 purified enzyme, forward reaction under aerobic conditions with NAD+ Cupriavidus necator
1.12.1.2 125 175 activity of different batches with NADH and ferricyanide Cupriavidus necator
1.12.1.2 1263
-
 activity of different batches with benzyl viologen and H2 Cupriavidus necator

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.12.1.2 ferrocyanide + NAD+
-
 Cupriavidus necator ferricyanide + NADH
-
 r
1.12.1.2 ferrocyanide + NAD+ diaphorase activity Cupriavidus necator ferricyanide + NADH
-
 r
1.12.1.2 H2 + NAD+
-
 Cupriavidus necator H+ + NADH
-
 ?
1.12.1.2 H2 + NAD+
-
 Cupriavidus necator H+ + NADH
-
 r
1.12.1.2 H2 + NAD+ H2 activation solely takes place on Ni2+ Cupriavidus necator H+ + NADH
-
 r
1.12.1.2 H2 + oxidized benzyl viologen
-
 Cupriavidus necator H+ + reduced benzyl viologen
-
 r
1.12.1.2 H2 + oxidized benzyl viologen hydrogenase activity Cupriavidus necator H+ + reduced benzyl viologen
-
 r
1.12.1.2 additional information addition of NADH prolonged the lag phase before H2 consumption Cupriavidus necator ?
-
 ?
1.12.1.2 additional information FMN release induces reduction with NADH, enzyme shows both hydrogenase and diaphorase activities, proton channeling Cupriavidus necator ?
-
 ?

Subunits

EC Number Subunits Comment Organism
1.12.1.2 More subunits HoxHY form a heterodimer which is responsible for the hydrogenase activity, subunits HoxFU form a heterodimer which is responsible for the NADH-dehydrogenase or diaphorase activity Cupriavidus necator
1.12.1.2 tetramer enzyme is composed of 4 Hox subunits, HoxF, HoxH, HoxU, and HoxY, with MWs of 67 kDa, 55 kDa, 26 kDa, and 23 kDa Cupriavidus necator

Synonyms

EC Number Synonyms Comment Organism
1.12.1.2 NAD+-reducing [NiFe]-hydrogenase
-
 Cupriavidus necator
1.12.1.2 SH
-
 Cupriavidus necator
1.12.1.2 SH i.e. soluble cytoplasmic hydrogenase Cupriavidus necator
1.12.1.2 soluble [NiFe]-hydrogenase
-
 Cupriavidus necator

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.12.1.2 30
-
 assay at Cupriavidus necator

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.12.1.2 8
-
 assay at Cupriavidus necator

Cofactor

EC Number Cofactor Comment Organism Structure
1.12.1.2 benzyl viologen oxidized and reduced Cupriavidus necator
1.12.1.2 FMN 2 molecules per enzyme molecule, one of 2 can be reversibly released upon reduction of the enzyme Cupriavidus necator
1.12.1.2 FMN required, 2 molecules per tetrameric enzyme complex, reduced enzyme reversibly releases half of the FMN bound, kinetics, FMN release induces reduction with NADH, overview Cupriavidus necator
1.12.1.2 NAD+
-
 Cupriavidus necator
1.12.1.2 NADH
-
 Cupriavidus necator