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

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Abstract
Reduced ferredoxin: CO2 oxidoreductase from Clostridium pasteurianum, 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∼–400 mV) in order to protect the CO2 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 CO2 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% Triton X‐100 into an enzymatically active species of molecular weight 118000. Treatment with sodium dodecl sulfate led to a dissociation of the enzyme into two enzymatically inactive polypeptide chains of molecular weights near 34000 and 86000. The purified enzyme will catalyze the following reactions at the same relative rates as the cell‐free extracts: (a) the reduction of CO2 to formate with reduced ferredoxin, (b) the oxidation of formate to CO2 with oxidized ferredoxin, (c) an isotopic exchange between 14CO2 and formate in the absence of ferredoxin, and (d) the reduction of methyl viologen with formate.It is concluded that CO2 reductase from C. pasteurianum is a molybdenum iron‐sulfur protein containing 1 mol of molybdenum, and 24 mol of non‐heme iron and acid‐labile sulfur per mol enzyme of molecular weight 118000.

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