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

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 X-100 into an enzymatically active species of molecular weight 118 000. Treatment with sodium dodecyl 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 C02 to formate with reduced ferredoxin, (b) the oxidation of formate to C02 with oxidized ferredoxin, (c) an isotopic exchange between I4CO2 and formate in the absence of ferredoxin, and (d) the reduction of methyl viologen with formate.It is concluded that C02 reductase from C. pusteurianum is a molybdenum iron-sulfur protein containing 1 mol of molybdenum, and 24mol of non-heme iron and acid-labile sulfur per mol enzyme of molecular weight 1 18 000.Clostridium pasteurianum is a strictly anaerobic bacterium that can grow on mineral salts media with glucose as the sole carbon and energy source. The organism is known to have an unusual pathway of heterotrophic C02 fixation: C02 is reduced to formate which is assimilated into one-carbon units [1,2]. The reduction of C 0 2 to formate is catalyzed by a ferredoxin-dependent formate dehydrogenase, which has been named C02 reductase (reduced ferredoxin : C02 oxidoreductase) in order to indicate that the physiological function of the enzyme is to catalyze the formation of formate from C02 rather than the reverse reaction [3].The C02 reductase activity was found to be very labile, and to be rapidly inactivated by only trace amounts of molecular oxygen. The enzyme has been reported to catalyze the following reactions : the reduction of C02 to formate with reduced ferredoxin or reduced methyl viologen, the oxidation of formate to COZ with oxidized ferredoxin or oxidized methyl viologen,...

Dissection of the Caffeate Respiratory Chain in the Acetogen Acetobacterium woodii : Identification of an Rnf-Type NADH Dehydrogenase as a Potential Coupling Site

“…A. woodii can couple the oxidation of various donors, such as hexoses, methyl group-containing compounds, or H 2 -CO 2 , with caffeate reduction. Oxidation of hexoses generates NADH as well as reduced ferredoxin, and methyl group oxidation yields reduced pyridine nucleotides and probably ferredoxin (47,55). Therefore, oxidation of these substrates can easily be coupled to caffeate reduction directly via NADH.…”

A rapid procedure for the purification of ferredoxin from clostridia using polyethyleneimine

“…Clostridium pasteurianurn was grown on glucose medium [8], Clostridium kluyveri on crotonate medium [9], Clostridium acidi-urici on urate medium [ I 0] and Clostridium perfringens on glutamate medium [11]. After harvesting, the cells were frozen in liquid nitrogen and stored at -70°C.…”

Formate production through biocatalysis

“…FDHs from such sources are therefore naturally geared toward the generation of formate from CO 2 . Accordingly, there have been several reports of acetogen FDHs showing a higher than normal preference for the reduction of CO 2 coupled to the oxidation of various cofactors, [11][12][13] but an FDH where this surpasses the ability to catalyze formate oxidation has not previously been reported.…”

“…16 The production and isolation of the enzyme was performed in parallel with that of the well-characterized FDH from Candida boidinii (FDH_CanBo) for comparison and was performed aerobically. As opposed to other highly oxygen-labile acetogen FDHs, [11][12][13] FDH H _CloCa showed increased stability, retaining activity over a few days. To assay FDH H _CloCa and FDH_CanBo in both reaction directions, the equilibrium was approached from either side by using high excesses of formate or CO 2 (in the form of bicarbonate) with a much lower concentration of the corresponding nicotinamide cofactor (NAD + /NADH).…”