C-terminal extension of the H2-activating subunit, HoxH, directs maturation of the NAD-reducing hydrogenase in Alcaligenes eutrophus

Eur J Biochem. 1997 Apr 15;245(2):441-8. doi: 10.1111/j.1432-1033.1997.t01-3-00441.x.


Formation of enzymatically active [NiFe] hydrogenases is dependent on a number of posttranslational steps, including metal attachment to a precursor of the catalytic subunit, truncation of a small C-terminal peptide from the precursor, and oligomerisation of the subunits. Two amino acid replacements were introduced by site-directed mutagenesis at the C-terminal proteolytic cleavage site of HoxH, the Ni-containing subunit of the cytoplasmic NAD-reducing hydrogenase of Alcaligenes eutrophus H16. Replacement of Ala465, the first residue of the 24-amino-acid cleaved polypeptide, by Pro yielded a form of HoxH that was blocked in C-terminal proteolysis. This HoxH subunit, although capable of binding Ni, was blocked in formation of a stable tetrameric holoenzyme. In the second mutant, the C-terminal extension of HoxH was eliminated by substituting the Ala codon for a translational stop codon. Although this mutant subunit was able to form the oligomeric holoenzyme, it was devoid of Ni. Both mutant proteins contained only traces of H2-activating functions. H2-dependent reduction of NAD and benzylviologen, and D2/H+-exchange activity were almost completely abolished, while the NADH oxidoreductase activity, mediated by the diaphorase moiety of the hydrogenase, was retained. These results allow the following conclusions: the C-terminal extension of HoxH is neccessary to direct specific Ni insertion into the hydrogenase; subunit assembly to the holoenzyme is not dependent on Ni insertion; and a precursor with the C-terminal peptide is not competent for assembly.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcaligenes / enzymology*
  • Amino Acid Sequence
  • Amino Acids / analysis
  • Binding Sites
  • DNA, Bacterial / chemistry
  • Electrophoresis, Polyacrylamide Gel
  • Hydrogen / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nickel / metabolism
  • Operon
  • Oxidoreductases / biosynthesis
  • Oxidoreductases / chemistry*
  • Oxidoreductases / genetics
  • Protein Conformation
  • Protein Processing, Post-Translational
  • Restriction Mapping
  • Solubility


  • Amino Acids
  • DNA, Bacterial
  • Nickel
  • Hydrogen
  • Oxidoreductases
  • hydrogen dehydrogenase