PURIFICATION AND CHARACTERIZATION OF PYRUVATE-FERREDOXIN OXIDOREDUCTASE FROM HYDROGENOBACTER-THERMOPHILUS TK-6

Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic hom ogeneity from an aerobic, thermophilic, obligately chemolithoautotroph ic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, b y precipitation with ammonium sulfate and fractionation by DEAE-Sephar ose CL-GB, polyacrylate-quaternary amine, hydroxyapatite, and Superdex -200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular m asses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an alpha beta gamma delta-structure. The activity was dete cted with pyruvate, coenzyme A, and one of the following electron acce pters in substrate amounts: ferredoxin isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP , and ferredoxins from Chlorella spp. and Clostridium pasteurianum wer e ineffective as the electron acceptor. The temperature optimum for py ruvate oxidation was approximately 80 degrees C. The pH optimum was 7. 6-7.8. The apparent K-m values for pyruvate and coenzyme A at 70 degre es C were 3.45 mM and 54 mu M, respectively. The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activ ity (t(50%)) at 70 degrees C was approximately 8 h.