Purification and Properties of Reduced Ferredoxin: CO2 Oxidoreductase from Clostridium pasteurianum, a Molybdenum Iron‐Sulfur‐Protein

  • SCHERER P
  • THAUER R
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Abstract

Reduced ferredoxin : C02 oxidoreductase from Clostridium pusteurianum, a ferredoxin-dependent soluble formate dehydrogenase, was purified 3000-fold with an overall yield ranging between 10 and 20 ' %;. The enzyme was at least 85 ' %; pure as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and by the molybdenum content. The enrichment was performed in the presence of 10 mM azide, a competitive inhibitor of the enzyme, and under strictly anaerobic conditions (E E -400 mV) in order to protect the C02 reductase from rapid inactivation. The purification procedure included a heat step, a precipitation by polyethyleneimine and by ammonium sulfate, a sedimentation by ultracentrifugation, and a chromatography on DEAE-cellulose in 39 ammonium sulfate. The highly purified C02 reductase contained 7.2 nmol of molybdenum, 170 nmol of non-heme iron and 150 nmol of acid-labile sulfur per mg of protein. The spectrum of the enzyme was typical of an iron-sulfur protein. Selenium, tungsten, flavins or heme could not be detected in significant amounts. The enzyme consisted of a number of aggregated forms with molecular weights near 700000, 460000, and 240000 which could be partially dissociated in the presence of 1 o/, Triton

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