Purifications and Characterizations of a Ferredoxin and Its Related 2-oxoacid:ferredoxin Oxidoreductase From the Hyperthermophilic Archaeon, Sulfolobus Solfataricus P1

J Biochem Mol Biol. 2006 Jan 31;39(1):46-54. doi: 10.5483/bmbrep.2006.39.1.046.

Abstract

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of 11,180+/-50 Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S], type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa alpha and 34 kDa beta subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at 55 degrees C, pH 7.0. Maximum activity was observed at 70 degrees C and the optimum pH for enzymatic activity was 7.0 -8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with Km and kcat values of 163 microM and 452 min(-1), respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.

MeSH terms

  • Amino Acid Sequence
  • Archaeal Proteins* / chemistry
  • Archaeal Proteins* / genetics
  • Archaeal Proteins* / isolation & purification
  • Archaeal Proteins* / metabolism
  • Ferredoxins*
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Molecular Weight
  • Pyruvate Synthase* / chemistry
  • Pyruvate Synthase* / genetics
  • Pyruvate Synthase* / isolation & purification
  • Pyruvate Synthase* / metabolism
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Sulfolobus solfataricus* / chemistry
  • Sulfolobus solfataricus* / enzymology
  • Temperature

Substances

  • Archaeal Proteins
  • Ferredoxins
  • Pyruvate Synthase