from Peptostreptococcus productus (strain Marburg). Ulrike Reubelt 1, Gert Wohlfarth 1, Roland Sehmid 2, and Gabriele Diekert 1 i Institut f/Jr Mikrobiologie, ...
Arch Microbiol (1991) 156:422-426
Archives of
Hicrobiology
030289339100155C
9 Springer-Verlag 1991
Purification and characterization of ferredoxin from Peptostreptococcusproductus (strain Marburg) Ulrike Reubelt 1, Gert Wohlfarth 1, Roland Sehmid 2, and Gabriele Diekert 1 i Institut f/Jr Mikrobiologie, Universit/it Stuttgart, Azenbergstrasse 18, W-7000 Stuttgart 1, Federal Republic of Germany 2 Institut fiir Mikrobiologie, Universit/it Osnabrfick, Barbarastrasse 11, W-4500 Osnabrfick, Federal Republic of Germany Received June 4, 1991/Accepted Juli 19, 1991
Abstract. Ferredoxin was purified to apparent homogeneity from cell extracts of the homoacetogen Peptostreptococcus productus (strain Marburg). The yield was 70 gg ferredoxin per g wet cells o f P. productus. The UV-vis spectrum exhibited characteristics of a typical clostridial ferredoxin spectrum with a molar extinction coefficient ~385 o f ~ 30000 M - 1 c m - 1 and an A385/Azs o ratio of 0.76. The molecular weight Mr was near 5700 as calculated from the amino acid composition. The protein contained per mol 9.9 mol iron, 8.2 mol acid-labile sulfide, and near 7 mol cysteine indicating the presence of two 4 Fe/4 S clusters. The redox potential was determined to be - 4 1 0 inV. The purified ferredoxin was reduced with carbon monoxide by the carbon monoxide dehydrogenase from crude extracts and by the partially enriched enzyme o f P. productus. Key words: Peptostreptococcus productus (strain Marburg) - Homoacetogenic bacteria - Ferredoxin - Carbon monoxide dehydrogenase
Peptostreptococcus productus (strain Marburg) is a strictly anaerobic, homoacetogenic bacterium (for reviews on homoacetogens see Fuchs 1986; Ljungdahl 1986; Diekert 1990, 1991 ; Wood 1991), which is able to grow on H2 plus CO2 or on carbon monoxide as the sole energy source (Geerligs et al. 1987). The bacterium catalyzes the synthesis of acetate from carbon monoxide via the acetyl CoA pathway in its energy metabolism (Ma et al. 1987, 1991). The methyl group o f acetate is formed from carbon dioxide, which is derived from carbon monoxide oxidation. CO2 is then reduced to formate, which is subsequently activated to formyl tetrahydrofolate, followed by conversion to methenyl tetrahydrofolate. The latter compound is reduced to methyl tetrahydrofolate via methylene tetrahydrofolate as the intermediate
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(Wohlfarth et al. 1990, 1991). The methyl group is incorporated into acetyl CoA by a corrinoid protein. The carboxyl group of acetate is derived from carbon monoxide, which is incorporated directly into acetyl CoA (Ma et al. 1987). Since most homoacetogens are able to grow o n H 2 plus CO2 as the sole energy source, the reduction o f CO2 to acetate must be coupled with a net formation of ATP. Evidence was presented, that ATP is generated by electron transport phosphorylation (Diekert et al. 1986; Geerligs et al. 1989; Heise et al. 1989). It was suggested, that redox reactions participating in the electron flow from H2 or CO, respectively, to intermediates in methyl group formation are involved in energy conservation o f these bacteria (Diekert et al. 1986). Since ferredoxin plays a major role as an electron carrier in acetate synthesis from CO2 (Ragsdale et al. 1983; Ragsdale and Ljungdahl 1984), characterization of this protein was a prerequisite for further investigations on redox reactions in carbon monoxide utilizing homoacetogens. In this respect, the question, whether in the CO utilizing P. productus ferredoxin is the physiological electron acceptor for carbon monoxide oxidation, is of special interest. In the present communication the purification and characterization of ferredoxin from P. productus is described.
Materials and methods
Source of materials All chemicals and biochemicals used were of the highest available purity and were purchased from Boehringer (Mannheim, FRG), Merck (Darmstadt, FRG), Serva (Heidelberg, FRG), or Sigma (Mfinchen, FRG). Column materials were obtained from Pharmacia (Uppsala, Sweden). Centricon tubes were purchased from Amicon (Danvers, USA). Cellosyl was a gift from Dr. Br/iu, Hoechst AG (Frankfurt/Main, FRG). Polymin P (polyethyleneimine, molecular weight 20000 Dalton) was obtained from BASF AG (Ludwigshafen, FRG). Bio-Rad silver stain was from Bio-Rad Laboratories (Mfinchen, FRG).
423
Purification of ferredoxin Peptostreptococcusproductus (ATCC 43917) was grown on glucose medium, which was supplemented with 0.14 mM (NH4)zFe(SO4)2 (Geerligs et al. 1987). Cells were harvested by centrifugation in the late logarithmic growth phase and stored frozen at -20~ The first three steps of the purification procedure were performed according to Sch6nheit et al. (1978). However, the whole procedure was carried out under anaerobic conditions (anaerobic chamber or rubber stoppered bottles).
Step 1 Preparation of crude extract. Frozen cells were suspended in 1.5 volumes of H20 containing 10 mg/ml cellosyl and 1 mM phenylmethylsulfonyl fluoride. The suspension was incubated at 37~ for 30 rain. The crude extract was cooled on ice and adjusted to pH 6.5 with 2 M Tris-base. Then H20 was added until the conductivity of the extract was equal to that of a 0.1 M NaC1 solution. Step 2: Acetone precipitation. Acetone ( - 2 0 ~ was added to the crude extract upon rapid stirring to a final concentration of 55% (v/v). The mixture was centrifuged at 20 000 x g and 4~ for 20 min to remove precipitated protein. Step 3: Polyethyleneimineprecipitation. Ferredoxin was precipitated from the acetone supernatant by the addition of polymin P to a final concentration of 0.5% (w/v). After stirring on ice for 10 rain, the mixture was centrifuged at 20 000 x g and 4 ~C for 20 min. Step 4: Chromatography on Phenyl Sepharose. The brown pellet containing ferredoxin was dissolved in 20 mM Tris-HC1 pH 7.5 containing 2.5 M ammonium sulfate. After sterile filtration the solution was passed through a Phenyl Sepharose Fast Flow column (1.0x15cm) preequilibrated with 2.5M ammoniumsulfate in 20 mM Tris-HC1 pH 7.5. The column was washed with a decreasing stepwise gradient of ammonium sulfate in buffer. Ferredoxin was eluted from the column with 1.5 M ammonium sulfate in buffer. Step 5: Chromatography on Mono Q. Fractions containing ferredoxin were pooled and 9 volumes 20 mM Tris-HCl pH 7.5 were added. The solution was apphed to a Mono Q column (1.0 x 10 cm) preequilibrated with 20 mM Tris-HCl pH 7,5. Ferredoxin was eluted with a linear gradient from 0 - 1 M KC1 in 20 mM Tns-HC1 pH 7.5 at a KC1 concentration of approximately 0.72 M. Fractions containing ferredoxin were pooled, concentrated with Centricon 3, and stored frozen at - 2 0 ~ under N 2 a s the gas phase.
methyl viologen, 10% (v/v) ethylene glycol, 0.5 mM phenyl methyl sulfonyl fluoride, 5 mM dithiothreitol, and 5 gM dithionite]. After application of crude extract (30 ml; near 15 mg protein/ml) the column was washed with buffer (see above), and subsequently with a stepwise gradient of buffer containing increasing concentrations of NaC1 (100 raM/step; 100 ml/step). The enzyme was eluted at a NaCI concentration of 300 raM. The specific activity of the partially purified enzyme was 55 U/rag (with 10 mM methyl viologen), the yield was near 50%.
Preparation of crude extracts from Clostridium pasteurianum Clostridium pasteurianum (DSM 525; obtained from the Deutsche Sammlung yon Mikroorganismen, Braunschweig, FRG) was grown on glucose medium as described by Scherer and Thauer (1978). The cells were harvested by centrifugation in the late logarithmic growth phase and stored frozen at - 2 0 ~C. 1 g frozen cells were suspended in 2 ml 100 mM potassium phosphate buffer pH 7.5 containing 1 mg lysozyme, 0.5 nag deoxyribonuclease II, and 5 mM MgC12 with N 2 as the gas phase. The suspension was incubated at 37~ for I h. The crude extract was cooled on ice and centrifuged for 20 rain at 5000 x g in the anaerobic chamber. The supernatant containing hydrogenase was stored at - 2 0 ~ in a rubber stoppered serum bottle with Nz as the gas phase.
Determination of the redox potential The standard redox potential was calculated from the equilibrium constant of the hydrogenase reaction. The reduction of ferredoxin with hydrogenase from C. pasteurianum was conducted anaerobically under hydrogen (100 kN/m 2) at 25~ in rubber stoppered glass cuvettes filled with 1 ml 100 mM potassium phosphate buffer pH 7.2 containing 0.043 mM ferredoxin. The reaction was started by the addition of 5 gl crude extract from C. pasteurianum using a glass syringe. The reduction of ferredoxin was followed photometrically at 405 rim. After the equilibrium was reached as indicated by the termination of the reaction, the pH of the reaction mixture was measured for the calculation of the proton concentration.
Determination of carbon monoxide dehydrogenase activity Analytical procedures SDS polyacrylamide gel electrophoresis was performed as described by Schfigger and von Jagow (1987). Iron was determined as described earlier (Fish 1988). Acid labile sulfide was estimated as described by Rabinowitz (1978). Amino acid analysis was kindly performed by Dr. R. Schmid (University of Osnabriick, FRG). The apparent molecular mass of ferredoxin was determined by gel filtration on a Superdex 75 column (1.6 x 60 cm). The sample and elution buffer was 20 mM Tris-HC1 containing 0.15 M KC1. The following molecular weight standards were used: bovine serum albumin, Mr 66000; ovalbumin, Mr 43000; chymotrypsinogen A, Mr 25000; ribonuclease, Mr 14700; trypsin inhibitor, 5890.
Carbon monoxide dehydrogenase was assayed spectrophotometrically following either the reduction of methyl viologen at 578 nm, or the reduction of ferredoxin at 405 nm with carbon monoxide as the electron donor. The assay was conducted anaerobically at 37~ in glass cuvettes containing either 10 mM methyl viologen or 0.03 mM ferredoxin in I ml 100 mM potassium phosphate buffer pH 7.2. The gas phase was carbon monoxide. The reaction was started by the addition of crude extract from P. productus.
Results
Purification of ferredoxin Partial enrichment of CO dehydrogenase of Peptostreptococcus productus The extremely oxygen-sensitive carbon monoxide dehydrogenase was enriched by chromatography of crude extracts on DEAE Sepharose. The column (1.6 x 30 cm) was preequilibrated with buffer [50 mM Tris-HC1 pH 8.0 containing 10 mM MgClz, 0.1 mM
F e r r e d o x i n was p u r i f i e d f r o m Peptostreptococcus productus (strain M a r b u r g ) . T h e o r g a n i s m was g r o w n in the presence o f high c o n c e n t r a t i o n s o f i r o n in the m e d i u m to increase the yield o f f e r r e d o x i n ( R a g s d a l e a n d L j u n g d a h l 1984). T h e p u r i f i c a t i o n p r o c e d u r e is s u m m a r i z e d in
424 Table 1. Purification of ferredoxin from 25 g wet cells of Peptostreptococcus productus (strain Marburg). For experimental conditions see 'Materials and methods'
Aass
10
Purification step
A280
Crude extract Acetone precipitation Polymin P precipitation Phenyl sepharose Mono Q
4763.0
168
0.04
1.0
100
n.d.
n.d.
n.d.
n.d.
n.d.
385.0
41
0.11
2.8
24
26.0 6.7
13 5
0.50 0.75
12.5 18.8
8 3
Table 2. Amino acid analysis of ferredoxin purified from Peptostreptoeoccus productus. The samples contained 6 lag protein. The data were obtained from triple experiments
Ala Arg Asx Cys Glx Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val
\ \\\
g 05
~x ,
