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 K(m) values for pyruvate, CoASH and P. furiosus ferredoxin at 80-degrees-C are 460, 100 and 70 muM, respectively. Carbon monoxide was a potent inhibitor of pyruvate oxidation (apparent K(i) = 7 muM). 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.