Purification and characterization of pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus.
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.
Blamey
J M
JM
Department of Biochemistry, University of Georgia, Athens 30602.
Adams
M W
MW
D000596:Amino Acids / Q000032:analysis; D001105:Archaea / Q000201:enzymology*; D002248:Carbon Monoxide / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007658:Ketone Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D050743:Pyruvate Synthase; D013696:Temperature
D000596:Amino Acids; D002248:Carbon Monoxide; D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase