title: The soluble NAD+-Reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH.
abstract: The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD(+) at the expense of H(2). We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).
authors.affiliation: Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, D-10115 Berlin, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Burgdorf
authors.forename: Eddy
authors.initials: E
authors.lastname: van der Linden
authors.forename: Michael
authors.initials: M
authors.lastname: Bernhard
authors.forename: Qing Yuan
authors.initials: QY
authors.lastname: Yin
authors.forename: Jaap W
authors.initials: JW
authors.lastname: Back
authors.forename: Aloysius F
authors.initials: AF
authors.lastname: Hartog
authors.forename: Anton O
authors.initials: AO
authors.lastname: Muijsers
authors.forename: Chris G
authors.initials: CG
authors.lastname: de Koster
authors.forename: Simon P J
authors.initials: SP
authors.lastname: Albracht
authors.forename: Bärbel
authors.initials: B
authors.lastname: Friedrich
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D020118:Cupriavidus necator / Q000201:enzymology*; D042967:Electron Transport Complex I / Q000737:chemistry; D004789:Enzyme Activation; D004795:Enzyme Stability; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D005810:Multigene Family; D009249:NADP / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
chemical_list: D021122:Protein Subunits; D009249:NADP; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D042967:Electron Transport Complex I