CHARACTERIZATION AND PURIFICATION OF PYRUVATE-FERREDOXIN OXIDOREDUCTASE FROM GIARDIA-DUODENALIS

The major 2-oxoacid oxidoreductase (2-OR), pyruvate:ferredoxin oxidore ductase (PFOR) from Giardia duodenalis has been purified to apparent h omogeneity. A second 2-OR with a preference for alpha-ketobutyrate as substrate was identified and was removed from PFOR containing fraction s during purification. Only PFOR and the second 2-OR were identified i n gels of crude Giardia extracts assayed for 2-OR activity. The native form of PFOR which is membrane associated, is a homodimer of 138 kDa subunits. Pyruvate is the preferred substrate: alpha-ketobutyrate and oxaloacetate, but not phenyl-pyruvate or a-ketoglutarate, are decarbox ylated. PFOR from Giardia is more stable than PFOR from most other org anisms and purified PFOR can be stored without deterioration at -70 de grees C. Purified PFOR donates electrons to Giardia ferredoxin (Fd I) with concomitant reduction of metronidazole. However, two other Giardi a ferredoxins did not accept electrons from PFOR. Consistent with the involvment of PFOR in metronidazole activation, the activity of pyruva te dependent 2-OR activity was decreased in all metronidazole-resistan t lines tested but not in furazolidone-resistant lines. The presence o f three different ferredoxins and two 2-ORs in Giardia suggests that a number of different electron transport pathways operate in this organ ism providing unusual metabolic flexibility for a eukaryote.