Purification and characterization of pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus

Biochim Biophys Acta. 1993 Jan 15;1161(1):19-27. doi: 10.1016/0167-4838(93)90190-3.


Pyrococcus furiosus grows optimally at 100 degrees C by carbohydrate fermentation. It is thought to contain a novel tungsten-dependent, NAD(P)-independent glycolytic pathway in which one of the oxidation steps is catalyzed by a tungsten-containing aldehyde ferredoxin oxidoreductase. The enzyme that catalyzes the terminal oxidation step, pyruvate ferredoxin oxidoreductase (POR), has now been purified. POR has a molecular mass of 100 kDa and is comprised of three subunits (45, 31 and 24 kDa). It lacks tungsten but contains thiamine pyrophosphate (TPP) and two ferredoxin-type [4Fe-4S] clusters per molecule which, by EPR spectroscopy, can be differentiated by their relaxation properties. The enzyme requires CoASH but not TPP for pyruvate oxidation activity and will not use 2-oxoglutarate, phenyl pyruvate or indole pyruvate as substrates. POR is virtually inactive at 25 degrees C and shows a temperature optimum for pyruvate oxidation above 90 degrees C. The apparent Km values for pyruvate, CoASH and P. furiosus ferredoxin at 80 degrees C are 460, 100 and 70 microM, respectively. Carbon monoxide was a potent inhibitor of pyruvate oxidation (apparent Ki = 7 microM). The half-life of activity (t50%) in air at 25 degrees C was 15 min and the t50% value at 80 degrees C (under anaerobic conditions) was 23 min. Based on molecular comparisons with PORs from mesophilic organisms, it is proposed that P. furiosus POR may represent an ancestral form of a pyruvate-oxidizing enzyme.

Publication types

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

MeSH terms

  • Amino Acids / analysis
  • Archaea / enzymology*
  • Carbon Monoxide / pharmacology
  • Electron Spin Resonance Spectroscopy
  • Enzyme Stability
  • Hydrogen-Ion Concentration
  • Ketone Oxidoreductases / antagonists & inhibitors
  • Ketone Oxidoreductases / chemistry
  • Ketone Oxidoreductases / isolation & purification*
  • Molecular Weight
  • Pyruvate Synthase
  • Temperature


  • Amino Acids
  • Carbon Monoxide
  • Ketone Oxidoreductases
  • Pyruvate Synthase