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, T(op t) = 100-degrees-C) and the bacterium Thermotoga maritima (Tm, T(opt) = 80-degrees-C). Each catalyzes the oxidative decarboxylation of pyruv ate to acetyl-CoA and CO2 near the optimal growth temperature of the o rganism and are virtually inactive at 25-degrees-C. Both PORs contain a thiamine pyrophosphate (TPP) cofactor and at least two [4Fe-4S] ferr edoxin-type clusters. We have now shown, using EPR spectroscopy and me tal analyses, that Pf POR also contains an unusual copper center that is not present in Tm POR. In addition, distinct catalytic intermediate s were generated in both enzymes by the addition, separately and in co mbination, of the substrates pyruvate and CoASH, and these were examin ed by EPR spectroscopy. The addition of pyruvate to oxidized Pf POR pr oduced an isotropic signal centered at g = 2.01, which was measurably broader in the presence of pyruvate-2-C-13. This signal, which was ass igned to a (hydroxyethyl)thiamine pyrophosphate radical intermediate, was not observed in Tm POR under the same experimental conditions. Inc ubation of the oxidized enzymes with CoASH resulted in the partial red uction of the copper site in Pf POR and the partial reduction of a nov el 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-sulf ur centers that are reduced by sodium dithionite. On the basis of thes e observations, two distinct catalytic mechanisms are proposed for pyr uvate oxidation by these two hyperthermophilic enzymes.