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Pyruvate-Ferredoxin Oxidoreductase

III. PURIFICATION AND PROPERTIES OF THE ENZYME

From the ¹ From Basic Biochemistry, Veterans Administration Hospital and the University of Texas Medical School, Dallas, Texas 75216, and the Department of Biochemistry, University of California, Berkeley, California 94720

Pyruvate-ferredoxin oxidoreductase has been purified to 90% homogeneity from Clostridium acidi-urici. Multiple forms of the enzyme were detected after acrylamide gel electrophoresis by the use of an enzyme-specific staining method. The enzyme catalyzes a pyruvate-CO₂ exchange reaction, oxidation of pyruvate with the reduction of various electron acceptors, and acetoin formation from pyruvate and acetaldehyde. The enzyme contains non-heme iron, sulfide, thiamine, and a chromophore that absorbs at 400 nm. The three enzymic activities determined are not dependent on the addition of thiamine pyrophosphate when tested with the purified enzyme, but they are dependent on the addition of the cofactor when an aged crude preparation is used. The enzyme requires a high concentration of sulfhydryl compound for maximum activity and is also activated by these compounds. Coenzyme A (2 µm) is required for the pyruvate-CO₂ exchange reaction, but higher concentrations of CoA inhibit this reaction. Kinetic studies demonstrate that CoA decreases the apparent K_m for bicarbonate about 5-fold without affecting the K_m for pyruvate. The enzyme reduces ferredoxin, rubredoxin, FAD, and artificial electron acceptors, but not pyridine nucleotides. The stoichiometry of the reaction was demonstrated with ferredoxin or benzylviologen as an electron acceptor. Clostridial ferredoxin was found to transfer two electrons in the pyruvate-ferredoxin oxidoreductase reaction. The molar extinction coefficient of reduced ferredoxin was determined to be 19.6 x 10³ m^-1 cm^-1 at 390 nm.

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