CHARACTERIZATION OF AN ANCESTRAL TYPE OF PYRUVATE FERREDOXIN OXIDOREDUCTASE FROM THE HYPERTHERMOPHILIC BACTERIUM, THERMOTOGA-MARITIMA

The hyperthermophilic bacterium, Thermotoga maritima, is a strict anaerobe that grows up to 90-degrees-C by carbohydrate fermentation. We report here on its pyruvate ferredoxin oxidoreductase (POR), the enzyme that catalyzes the oxidation of pyruvate to acetyl-CoA, the terminal oxidation step in the conversion of glucose to acetate. T. maritima POR was purified to electrophoretic homogeneity under strictly anaerobic conditions. It has a molecular weight of 113 000 and comprises four dissimilar subunits with M(r) values of approximately 43 000, 34 000, 23 000, and 13 000. It contains thiamine pyrophosphate (TPP) and at least two ferredoxin-type [4Fe-4S] clusters per molecule, as determined by iron analysis and EPR.spectroscopy. CoASH was absolutely required for pyruvate oxidation activity, while the addition of TPP was stimulatory. The apparent K(m) values at 80-degrees-C for pyruvate, CoASH, and TPP were 14.5, 0.34, and 0.043 mM; respectively, and the corresponding apparent V(m) values ranged from 154 to 170 mumol of pyruvate oxidized/min/mg (units/mg). The apparent K(m) and V(m), values for T. maritima ferredoxin, the proposed physiological electron carrier for POR, were 26 muM and 280 units/mg, respectively. POR did not use 2-oxoglutarate, phenyl pyruvate, or indolyl pyruvate as substrates. The enzyme was extremely thermostable: the temperature optimum for pyruvate oxidation was above 90-degrees-C, and the time for a 50% loss of activity (t50%) at 80-degrees-C (under anaerobic conditions) was 15 h. The enzyme was also very sensitive to inactivation by oxygen, with a t50% in air at 25-degrees-C of 70 min. Sodium nitrite was a weak inhibitor of POR activity (K(i) = 54 mM), while carbon monoxide (320 muM), sodium cyanide (20 mM), sodium fluoride (5 mM), and or sodium azide (2.5 mM) had no inhibitory effect. This is the first POR to be purified from a hyperthermophilic bacterium. Interestingly, its molecular properties are more similar to those of the POR from a hyperthermophilic archaeon than to those of PORs from mesophilic bacteria. The evolutionary significance of this is discussed.