Pyruvate ferredoxin oxidoreductases of the hyperthermophilic archaeon, Pyrococcus furiosus, and the hyperthermophilic bacterium, Thermotoga maritima, have different catalytic mechanisms.
Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from two hyperthermophiles, the archaeon Pyrococcus furiosus (Pf, Topt = 100 degrees C) and the bacterium Thermotoga maritima (Tm, Topt = 80 degrees C). Each catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 near the optimal growth temperature of the organism and are virtually inactive at 25 degrees C. Both PORs contain a thiamine pyrophosphate (TPP) cofactor and at least two [4Fe-4S] ferredoxin-type clusters. We have now shown, using EPR spectroscopy and metal analyses, that PfPOR also contains an unusual copper center that is not present in Tm POR. In addition, distinct catalytic intermediates were generated in both enzymes by the addition, separately and in combination, of the substrates pyruvate and CoASH, and these were examined by EPR spectroscopy. The addition of pyruvate to oxidized Pf POR produced an isotropic signal centered at g = 2.01, which was measurably broader in the presence of pyruvate-2(13)C. This signal, which was assigned to a (hydroxyethyl)thiamine pyrophosphate radical intermediate, was not observed in Tm POR under the same experimental conditions. Incubation of the oxidized enzymes with CoASH resulted in the partial reduction of the copper site in Pf POR and the partial reduction of a novel iron-sulfur center in Tm POR, which was not seen in the dithionite-reduced enzyme. The addition of both pyruvate and CoASH to the PORs in their oxidized states resulted in the reduction of the same iron-sulfur centers that are reduced by sodium dithionite.(ABSTRACT TRUNCATED AT 250 WORDS)
Smith
E T
ET
Department of Biochemistry, University of Georgia, Athens 30602.
Blamey
J M
JM
Adams
M W
MW
D001105:Archaea / Q000201:enzymology*; D002384:Catalysis; D003065:Coenzyme A / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D050743:Pyruvate Synthase; D011773:Pyruvates / Q000737:chemistry; D019289:Pyruvic Acid
D011773:Pyruvates; D019289:Pyruvic Acid; D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase; D003065:Coenzyme A