High yield purification of a tagged cytoplasmic [NiFe]-hydrogenase and a catalytically-active nickel-free intermediate form

Protein Expr Purif. 2015 Mar;107:90-4. doi: 10.1016/j.pep.2014.10.018. Epub 2014 Nov 21.


The cytoplasmic [NiFe]-hydrogenase I (SHI) of the hyperthermophile Pyrococcus furiosus evolves hydrogen gas (H2) from NADPH. It has been previously used for biohydrogen production from sugars using a mixture of enzymes in an in vitro cell-free synthetic pathway. The theoretical yield (12 H2/glucose) is three times greater than microbial fermentation (4 H2/glucose), making the in vitro approach very promising for large scale biohydrogen production. Further development of this process at an industrial scale is limited by the availability of the H2-producing SHI. To overcome the obstacles of the complex biosynthetic and maturation pathway for the [NiFe] site of SHI, the four gene operon encoding the enzyme was overexpressed in P. furiosus and included a polyhistidine affinity tag. The one-step purification resulted in a 50-fold increase in yield compared to the four-step purification procedure for the native enzyme. A trimeric form was also identified that lacked the [NiFe]-catalytic subunit but catalyzed NADPH oxidation with a specific activity similar to that of the tetrameric form. The presence of an active trimeric intermediate confirms the proposed maturation pathway where, in the terminal step, the NiFe-containing catalytic subunit assembles with NADPH-oxidizing trimeric form to give the active holoenzyme.

Keywords: Affinity purification; Enzyme maturation; Hydrogen; Hydrogenase; Pyrococcus.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / genetics
  • Archaeal Proteins / isolation & purification*
  • Archaeal Proteins / metabolism
  • Biocatalysis
  • Catalytic Domain
  • Cytoplasm / chemistry
  • Cytoplasm / enzymology*
  • Cytoplasm / genetics
  • Cytoplasm / metabolism
  • Hydrogenase / chemistry*
  • Hydrogenase / genetics
  • Hydrogenase / isolation & purification*
  • Hydrogenase / metabolism
  • Kinetics
  • NADP / metabolism
  • Nickel / metabolism
  • Protein Multimerization
  • Pyrococcus furiosus / chemistry
  • Pyrococcus furiosus / enzymology*
  • Pyrococcus furiosus / genetics


  • Archaeal Proteins
  • NADP
  • Nickel
  • nickel-iron hydrogenase
  • Hydrogenase