Purification and Properties of α-Ketoglutarate Synthase from a Photosynthetic Bacterium

Abstract α-Ketoglutarate synthase from the photosynthetic bacterium Chlorobium thiosulfatophilum, an enzyme that catalyzes the formation of α-ketoglutarate by a reductive carboxylation of succinyl-CoA, was purified about 120-fold by high speed centrifugation, fractionation with protamine sulfate, column chromatography on DEAE-Sephadex, and sedimentation in a sucrose gradient. α-Ketoglutarate synthase is readily inactivated by oxygen; strictly anaerobic conditions are essential for preserving enzyme activity during purification, handling, and storage. The reductive synthesis of α-ketoglutarate from succinyl-CoA and bicarbonate requires reduced ferredoxin. Of several ferredoxins tested, only those from the photosynthetic bacteria C. thiosulfatophilum, Chromatium, and Rhodospirillum rubrum were effective. The ferredoxins of Clostridium pasteurianum and spinach were found to be poor substitutes for those of photosynthetic bacteria. The reversibility of the α-ketoglutarate synthase reaction was demonstrated by the use of α-ketoglutarate, CoA, and one of several electron acceptors in substrate amounts: FAD, FMN, triphenyltetrazolium chloride, or ferredoxin. NAD and NADP were ineffective. Purified preparations of α-ketoglutarate synthase show, in the presence of either CoA or reduced ferredoxin, an active 14C exchange between [14C]bicarbonate and α-ketoglutarate. α-Ketoglutarate synthase was found to be distinct from pyruvate synthase with respect to molecular size and chromatographic properties. The molecular weights and sedimentation coefficients of α-ketoglutarate synthase and pyruvate synthase were found to be, respectively, 220,000 and 9.0 S and 300,000 and 10.7 S.