- Email: [email protected]
[38] Preparation and propertiesof clostridial ferredoxins
[38]
CLOSTRIDIALFERREDOXINS
431
upon spectrophotometric measurement of the oxidation of Na2S204 (315 nm peak) while it serves as the reductant for nitrogenase or hydrogenase. The method is particularly useful for kinetic studies of these enzyme systems.
[38] P r e p a r a t i o n
and Properties
of Clostridial
Ferredoxins By JESSE RABINOWlTZ Ferredoxin was first isolated from Clostridiumpasteurianumby Mortenson, Valentine, and Carnahan. 1 The protein functions as an electron transfer factor in the enzymatic formation of acetyl phosphate and hydrogen from pyruvate by extracts of that organism treated with DEAE-cellulose and in a variety of other enzymatic reactions subsequently described. 2 It was characterized as an iron-sulfur protein when the presence of acid-labile sulfur was recognized. 3 Ferredoxin has been found in all clostridial species that have been examined. Procedures for the purification of clostridial ferredoxin have been based on the acidic nature of the protein and its resultant high affinity for DEAEcellulose, 3 its small size relative to other proteins and resultant solubility in acetone, 4 and the relative ease of crystallization of the protein with ammonium sulfate. Clostridial ferredoxin has a molecular weight of about 6000 and is composed of a single polypeptide chain containing 55 or fewer amino acid residues, 8 of which are cysteine, s The protein also contains 8 iron atoms and 8 atoms of acid-labile sulfur. 6 The biological activity of this protein is associated with its unusually low redox potential. Although the amino acid sequence of the protein isolated from several clostridial species is known (Table IV), the molecular structure with respect to the iron and acid-labile sulfur atoms is not known at this time. na The iron and sulfur atoms can be removed by 1L. E. Mortenson, R. C. Valentine, and J. E. Carnahan, Biochem. Biophys. Res. Commun. 7, 448 (1962). 2R. C. Valentine, Bacteriol. Rev. 28, 497 (1964). 3B. B. Buchanan, W. Lovenberg, and J. C. Rabinowitz, Proc. Nat. Acad. Sci. U.S. 49, 345 ( 1963). 4L. E. Mortenson, Biochim. Biophys. Acta 81, 71 (1964). 5W. Lovenberg, B. B. Buchanan, and J. c. Rabinowitz, J. Biol. Chem. 238, 3899 (1963). 6J.-S. Hong, Ph.D. Thesis, Univ. of California, Berkeley (1969). eaNote added in proof: This is no longer true. The structure of the iron-sulfur complex of the ferredoxin isolated from Micrococcus aerogenes, which is a clostridial-type ferredoxin, has been determined: L. C. Sieker, E. Adman, and L. H. Jensen, Nature 235, 40 (1972).
432
NITROGEN FIXATION
[38]
t r e a t m e n t o f f e r r e d o x i n with mercurial sulfhydryl reagents, 7 by treatm e n t with acid, s or by heat. ° T h e a p o f e r r e d o x i n f o r m e d by these treatments can be isolated, and f e r r e d o x i n can be reconstituted f r o m this material p r e p a r e d in this m a n n e r by using 5°Fe-labeled salts or [aSS]sodium sulfide in the reconstitution reactions. Growth of Clostridium pasteurianum Potato Tube Cultures. Lyophilized cultures o f C. pasteurianum (American T y p e Culture Collection No. 6013) or spore stocks o f this organism in soil are most easily revived by growth on "potato m e d i u m . " This is p r e p a r e d in the following manner: Solid CaCO3 is a d d e d to cover the bottom o f a test tube (18 × 150 mm). Fresh potatoes, peeled, washed, and finely diced, are a d d e d to the test tube to give a layer about 3 cm high, and 10-12 ml o f a 2% sucrose solution in tap water is a d d e d to the tubes. T h e tubes are plugged with n o n a b s o r b e n t cotton and autoclaved for 20 minutes at 120 °. After cooling, the tubes are sealed by adding a plug o f absorbent cotton and 2 d r o p s o f 2 N K2COz followed by 2 drops o f 4 M pyrogallol. T h e tube is immediately closed with a r u b b e r stopper and may be stored for 2 weeks. T o inoculate, the absorbent cotton is discarded; it is replaced with a fresh seal after inoculation. T h e inoculated tubes are incubated at 30 ° . It may take 30-40 hours for a p o t a t o - m e d i u m culture to start growing when inoculated f r o m soil or lyophilized spore stocks, and about 15 hours when an inoculum grown on the p o t a t o - m e d i u m culture is used. Since there is a vigorous evolution o f h y d r o g e n d u r i n g growth, tubes should not be s t o p p e r e d too tightly. T h e fully grown p o t a t o - m e d i u m culture may be stored in the r e f r i g e r a t o r u p to a m o n t h and used for subsequent inoculations into liquid media. Growth on Synthetic Medium. In o r d e r to grow a 40-liter culture o f the organism, f o u r 500-ml cultures are used as an inoculum. This m e d i u m 1° is p r e p a r e d in the following m a n n e r : Solid CaCO3 (1.5 g) and 250 ml o f tap water are a d d e d to each o f four 750-ml E r l e n m e y e r flasks. T h e following c o m p o n e n t s are dissolved in 900 ml o f tap water:
Sucrose, 40 g MgCI2.6H20, 7.4%, 4 ml NaCI, 20%, 1 ml Na~MoO4.2H20, 10%, 0.2 ml 7Richard Malkin and J. C. Rabinowitz, Biochem. Biophys. Res. Commun. 23, 822 (1966), 8J.-S. Hong and J. C. Rabinowitz, Biochem. Biophys. Res. Commun. 29, 246 (1967). 9T. Devanathan, J. M. Akagi, R. T. Hersh, and R. H. Himes,J. Biol. Chem.244, 2846 (1969). 10j. E. Carnahan and J. E. Castle,J. Bacteriol. 75, i21 (1958).
[38]
CLOSTR1D1ALFERREDOXINS
433
Biotin, 100/zg/ml, 0.1 ml p-Aminobenzoic acid, 100/zg/ml, 0.1 ml
N a2SO4, 7.1%, 2.0 ml NH4C1, 20%, 16 ml This solution is diluted to 1 liter, and 250 ml is added to each of the 750-ml flasks. The flasks are plugged with nonabsorbent cotton and autoclaved for 15 minutes at 121 °. While the flasks are hot, 10 ml of a sterile solution of 5% KH2PO4 + 9.2% K2HPO4"3H20 is added. When the flasks have cooled to 30 °, nitrogen gas that has been sterilized by passage through a cotton filter is bubbled through them. Just before inoculation of each flask, 2 ml of 5% FeC13"6H20 (in absolute alcohol) is added with a sterile pipette. Each Erlenmeyer flask is inoculated with 20-25 ml of the culture grown on the potato medium and bubbling with sterile nitrogen is continued. After growth for 16-20 hours at 30 °, the A660 is 11-12. The doubling time is 1.2-1.8 hours. Three or four of these flasks are then used to inoculate 40 liters of the synthetic medium modified as follows and contained in a large carboy. The calcium carbonate concentration is decreased to 1 g per liter, and the phosphate concentration is doubled. Cells are usually harvested in late log phase when the turbidity as determined by the A660 is between 3 and 4. The yield of wet cells from a 40-liter culture is approximately 200-250 g. A portion of the freshly harvested cells is used to prepare the clastic enzymes as described below and the remainder is stored in the deep freeze for use in the preparation of ferredoxin.
Preparation of DEAE-Cellulose The DEAE-cellulose used in all steps described in this contribution is prepared in the following manner: Newly purchased DEAE-cellulose is suspended in a large volume of 0.5 N NaOH in a large vessel for about 12 hours. The liquid is decanted with suction, and the cellulose derivative is washed with water 3 times. It is then suspended in 0.5 N HC1 for 12 hours, and washed with water by decantation 3 times. The wash with 0.5 N NaOH is repeated once again, and the material is washed with water by decantation. The washed DEAE-cellulose is suspended in water, and the suspension is adjusted to pH 6.5 with KH2PO4 and diluted to give a final concentration of phosphate of 0.3-1.0 M. This material is used for preparation of the columns for a particlar step, and it is washed with about l0 column volumes of water before use. The DEAEcellulose recovered from the column after it has been used in a chromatographic step is regenerated by suspending it in 0.5 N NaOH once, washing it with water, and equilibrating it with potassium phosphate at pH 6.5 as described above.
434
NITROGEN FIXATION
[38]
Preparation of Clastic Enzymes The ferredoxin-free clastic enzyme system is prepared as follows: 100 g of freshly harvested cells of C. pasteurianum is suspended in water to give a final volume of 200 ml and broken by sonication of 60-ml aliquots for 1.5 minutes with a Branson Model W185D cell disruptor. The suspension is centrifuged for 1 hour at 40,000 g. The supernatant solution is then passed over a column of DEAE-cellulose (2.2 × 20 cm). The pass-through (about 30 mg of protein per milliliter), containing the clastic enzymes, free of ferredoxin, is immediately frozen in 5-ml aliquots and stored in liquid nitrogen.
Assay Methods Reagents Potassium phosphate buffer, 0.25 M, pH 6.5 Sodium pyruvate, 0.5 M Coenzyme A, 10 mg/ml Hydroxylamine, 14%, neutralized 11 Ferric chloride reagent. Mix 100 ml of 10% FeCI3 in 0.2 N HCI, 200 ml of 12% trichloroacetic acid, and 200 ml of 3 N HCI and dilute to 600 ml with water Procedure. The following solutions are added to test tubes: 0.10 ml of phosphate buffer, 0.02 ml of pyruvate, 0.005 ml of CoA, 0.3 ml of the clastic enzymes (about 8-10 mg of protein), 0.2-1.0/zg of ferredoxin, and water to make the volume 1 ml. The tubes are incubated at 37 °. After 10 minutes, 1.5 ml of the ferric chloride reagent is added. The solutions are filtered through Whatman No. 1 filter paper by gravity (centrifugation is not satisfactory) and the absorbancy of the filtrate at 540 nm is determined using as a blank the control tube incubated in the absence of ferredoxin. There is an optimum level of clastic enzyme preparation which should be used in the assay so that the amount of reaction is maximal with a particular level of a d d e d ferredoxin. This is illustrated in Fig. 1. Since the activities of the clastic enzyme preparations vary, an experiment similar to that shown in Fig. 1 should be done with each enzyme preparation to determine the amount of protein to be used for the ferredoxin assay. A unit of activity in this assay is defined as the amount needed to give a change of 1.0 in absorbance at 540 nm.
I'F. Lipmannand L. C. Tuttle,J. Biol. Chem. 159, 21 (1945).
[38]
CLOSTRIDIALVEkkV.DOXlYS I
[
I
~35
I
E c
o
0.30 0.20
c
0.10 o
I
I
I
I
4
8
12
16
Protein Per ml Assay Mixture
FIG. 1. D e p e n d e n c e o f pyruvate "clastic" reaction on enzyme concentration. (See text for details.)
Purification of Ferredoxin from C. acidiourici
Step 1. Extract. Frozen cells of Clostridium acidi-urici (Vol. VI [97], 350 g, are thawed overnight at 4 ° and suspended in 0.05 M potassium phosphate buffer, pH 7.8, to a final volume of 900 ml. DNase (0.1 ml of a solution containing 5 mg/ml, Worthington) is added to facilitate suspension of the cells and reduce the viscosity of the mixture. Aliquots (60 ml) are sonicated for 1.5-minute intervals in a Branson Model W183D cell disruptor. The cell debris and unbroken cells are removed by centrifugation at 40,000 g for 1 hour. RNase (4 mg) is added to the supernatant solution. Step 2. DEAE-CeUulose-1. This crude extract (650 ml) is applied to a DEAE-cellulose column (3.0 × 30 cm). The pass-through (650 ml) is collected and used for the purification of formyhetrahydrofolate synthetase (Vol. VI [51]). The column is then washed with 300 ml of water. The ferredoxin is eluted with a linear salt gradient in the absence of buffer obtained by adding 1.8 liters of 0.7 M NaC1 to 1.8 liter of 0.1 M NaC1 contained in a mixing chamber, and 10-ml fractions are collected. A typical elution pattern is shown in Fig. 2A. Ferredoxin is recognized from the Aag0 measurement of individual fractions. It is eluted with 1000-1460 ml of eluent. These fractions are pooled and diluted 4-fold with distilled water to give a volume of 2100 ml. Step 3. DEAE-Cellulose-2. The diluted ferredoxin is reapplied to a second DEAE-cellulose column (3.0 × 23 cm). After application of the sample, the column is washed with 500 ml of 0.15 M Tris.chloride buffer, pH 7.4. The ferredoxin is eluted with a salt gradient obtained by adding 1.3 liters of 0.27 M NaCl in 0.15 M Tris.chloride buffer, pH 7.4, to 1.5 liters of 0.07 M NaC1 in 0.15 M Tris.chloride buffer, pH 7.4, in a mixing chamber, and 10-ml fractions are collected. It is usually possible to allow this chromatographic step to proceed overnight. The ferredoxin is eluted with 900-1600 ml of eluent (Fig. 2B).
436
NITROGEN FIXATION
[38]
Step 4. DEAE-Cellulose-3. T o c o n c e n t r a t e the f e r r e d o x i n the fractions are p o o l e d a n d d i l u t e d 3-fold with distilled water a n d r e a d s o r b e d o n a t h i r d DEAE-cellulose c o l u m n (3.0 × 8 cm), a n d e l u t e d with a small v o l u m e o f 0.15 M T r i s . c h l o r i d e buffer, p H 7.4, c o n t a i n i n g 0.5 M NaCi. Step 5. Sephadex G-75. T h e c o n c e n t r a t e d solution (26 ml) is t h e n a p p l i e d to a c o l u m n o f S e p h a d e x G-75 (3.0 × 90 cm) which has b e e n
I
)
]
I
I
I
I
16.0
I
A
~ 8.o
~
°
i
4'0i ~~ ~ 0
0
400
I
I ~-,
800
1200
1600
2000
ml Effluent
8.0-
B
w
v
8
4.0-~
2.o-
i
o
[,.~
o
I
400
800
1200
1600
!000
ml Effluent
Fie.. 2. Purification of ferredoxin from Clostridiumacidi-urici. (See text for details.)
[38]
CLOSTRIDIALFERREDOX1NS
437
equilibrated with 0.05 M Tris.chloride buffer, pH 7.4, and 5-ml fractions are collected. It is usual to allow this step to proceed overnight. The ferredoxin is eluted with 250-340 ml of eluent (Fig. 2C). Step 6. Crystallization. The fractions containing the ferredoxin are combined, and the solution is made 70% in ammonium sulfate. After 1 hour at 0°, the precipitated ferredoxin is collected by centrifugation for 20 minutes at 39,000 g. It is then resuspended in 40 ml of 0.15 M Tris.chloride buffer, pH 7.4, and stored in a tube under vacuum at 4 °. The purification of the protein from C, acidi-urici is summarized in Table I.
Purification of Ferredoxin from C. pasteurianum The procedure used is very similar to that described for the isolation of ferredoxin from C. acidi-urici; however, some differences in results and in the procedure may be noted because the ferredoxin content of C. pasteurianum is significantly lower than that of the purine-fermenting organism, and relatively higher concentrations of ammonium sulfate are required for its precipitation. Step 1. Extract. Frozen cells of C. pasteurianum (450 g) grown as described in a previous section, are thawed overnight at 4 °. The extract is prepared as described for cells of C. acidi-urici. Step 2. DEAE-Cellulose-1. The extract is applied to a DEAE-cellulose column (4.5 X 30 cm) and is washed with 400 ml of water. The ferre-
"//
I
I
I
I
I
C
30.0
I
E 20.0
c
10.0
I
150
200
250
300
ml Effluent
Fig. 2c
350
400
438
NITROGEN FIXATION
[38]
TABLE I PURIFICATION OF FERREDOXIN FROM Clostridium acidi-urici
Fraction I. Extract c II. DEAE-cellulose-1
III. IV. V. V1.
DEAE-cellulose-2 DEAE-cellulose-3 Sephadex G-75 Crystals
Volume (ml)
Protein a (mg)
650 2100 2040 33 93 50
11,626 1,196 494 411 352 354
Ferredoxin o Units Mg 119,600 111,090 90,168 88,440 83,700 84,000
As~o/A28o
345 321 261 255 242 243
0.046 0.322 0.726 0.728 0.775 0.789
aDetermined by a modification of the Lowry method, with crystalline bovine serum albumin as standa.r,d [J. C. Rabinowitz and W. E. Pricer, Jr., J. Biol. Chem. 237, 2898 (1962)]. bThe ferredoxin was determined by its activity in the enzymatic assay with the Clostridium pasteurianum "clastic system." A unit of activity causes a net increase of 1.0 at 540 nm in the assay. A standard solution of ferredoxin is included in each assay, and the unit activity of ferredoxin varies from day to day. T h e milligrams of ferredoxin recovered are based on this enzymatic test. cPrepared from 350 g of wet cells in the absence of 2-mercaptoethanol.
doxin is eluted with a linear salt gradient in the absence of buffer obtained by adding 1.8 liters of 0.7 M NaC1 to 1.8 liter of 0.1 M NaCI, and 10-ml fractions are collected. The elution pattern obtained is shown in Fig. 3A. The ferredoxin is eluted with 1560-1800 ml of eluent. 4.0
I
I
I
l
I
I
i
I
3.0-
•
16.0
- 12.0 T
E e-
E c
Q O~
O
2.0-
8.0
f-
N c .D
~, - 4 . 0
1.0-
400 FIG. 3.
800 1200 ml Effluent
1600
<
2000
Purification of ferredoxin from Clostridium pasteurianum. (See text for details.)
[38]
CLOSTRIDIALFERREDOXINS
439
The fractions containing ferredoxin are combined, diluted to 900 ml, and stored overnight under reduced pressure. Step 3. DEAE-Cellulose-2. The ferredoxin is readsorbed on a second DEAE-cellulose column (4.5 × 23 cm). The column is washed with 500 ml of 0.15 M Tris.chloride buffer, pH 7.4, and the ferredoxin is eluted with a linear salt gradient obtained by adding 1.5 liter of 0.27 M NaC1 in 0.15 M Tris.chloride buffer, pH 7.4, to a mixing chamber containing I
I
I
I
I
I
I
I
]
B
3.0
T 2.0 8
1.0
--J
0
,
400
,
I
800
1200
I
1600
2000
ml Effluent I
i
I
I
I
I
I
I
12.0
I
C
T v
E c
0 O~
E
8.0
0 ¢0 k"N
8
r,,) C
c
4.0
I 0
I
100
.,P'-.-J
- -
200
300
ml Effluent FIG. 3-B-C
400
500
440
NITROGEN F1XATION
[38]
1.5 liters of 0.07 M NaC1 in 0.15 M Tris.chloride buffer, pH 7.4. Tenmilliliter fractions are collected, and the elution pattern obtained is shown in Fig. 3B. The ferredoxin is eluted with 1300-1600 ml of eluent. The combined fractions are diluted 3-fold and stored overnight in an evacuated flask. Step 4. DEAE-CeUulose-3. In order to concentrate the ferredoxin, the material is adsorbed on a third DEAE-cellulose column (3.0 × 8 cm) and is eluted with a small volume of 0.5 M NaC1 in 0.15 M Tris.chloride buffer, pH 7.4. Step 5. Sephadex G-75. This material, 20 ml, is applied to a column of Sephadex G-75 equilibrated with 0.05 M Tris.chloride buffer, pH 7.4. The ferredoxin is obtained in the eluate after 310-370 ml of eluate have been collected (Fig. 3C). Step 6. Crystallization. This solution is brought to 60% saturation with ammonium sulfate, and the small amount of precipitate formed is removed by centrifugation. The supernatant solution is brought to 85% saturation with ammonium sulfate and allowed to stand at 5° overnight. The precipitate is collected by centrifugation and redissolved in 0.15 M Tris.chloride buffer, pH 7.4. The protein is recrystallized by the addition of ammonium sulfate to 80% saturation. Crystals form immediately. The purification of the protein from C. pasteurianum is summarized in Table II.
Preparation of Apoferredoxins ApoJerredoxind. The iron and acid-labile sulfide are removed from ferredoxin by treatment of the protein with a mercurial. 7 Ferredoxin (15 mg) in 2.7 ml of 0.1 M Tris.chloride buffer, pH 7.4, is treated with 1.8 ml of 0.05 M sodium mersalyl (Sigma Chemical Co.) in the same buffer at room temperature for 2 minutes. The small amount of precipitate T A B L E II PURIfiCATION OV FERREDOXIN FROM Clostridium
Fraction I. II. III. IV. V. VI.
Extract c DEAE-cellulose- 1 DEAE-cellulose-2 DEAE-cellulose-3 S e p h a d e x G-75 Crystals
pasteurianurn
Volume (ml)
Protein" (mg)
Ferredoxin b (units)
A39o/Azso
520 900 1100 20 80 17
4992 549 319 197 142 112
79,560 73,980 62,150 56,000 46,000 45,220
0.0535 0.366 0.625 0.635 0.79 0.83
aSee Table I. bSee Table I. c p r e p a r e d f r o m 400 g o f wet cells in the absence o f 2 - m e r c a p t o e t h a n o l .
[38]
CLOSTRIDIALFERREDOX1NS
441
that forms is removed by centrifugation. The solution is then passed over a column of Chelex 100 (Bio-Rad Laboratories, Berkeley, California) (2 x 17 cm) equilibrated with water. The protein is eluted with water at room temperature and 1.5 ml fractions are collected. The protein is eluted in a single peak with 18-24 ml of eluent. These fractions are pooled, lyophilized, and redissolved in distilled water to give a concentration of 4.0 mg of protein per milliliter. This solution is then passed over a column of Sephadex G-25 equilibrated with water at room temperature. The column is developed with water at room temperature, and 0.5-ml fractions are collected. The absorbancy at 280 nm and the protein content of the fractions are determined. A single proteincontaining component is obtained with 2-6 ml of eluent. A component with absorbancy at 280 nm is eluted afterward, but this fraction contains no protein. The fractions containing apoferredoxin-I are lyophilized. Apoferredoxin-II. The iron and acid-labile sulfide are removed from ferredoxin by treatment of the protein with acid under aerobic conditions, s Ferredoxin (14 mg) in 11 ml of 0.15 M Tris.chloride buffer, pH 7.4, is made 4-5% in trichloroacetic acid with a concentrated solution (30%) of the acid. This mixture is allowed to stand for 1 hour at 0 °. Evolution of H2S gas occurs, and the brown color of ferredoxin is completely bleached at the end of the period. The white suspension is centrifuged at 35,000 g for 10 minutes, and the supernatant solution is discarded. The precipitate is washed with either 3 ml of 0.023 M formic acid or 5% trichloroacetic acid, allowed to stand for 30 minutes at 0 °, and centrifuged. The precipitate is finally taken up in 4 ml of 0.1 M Tris.chloride buffer, pH 9.0, and the insoluble material is removed by centrifugation. The almost colorless supernatant solution is then passed over a Sephadex G-25 column (1.2 × 38 cm) previously equilibrated with 0.02 M Tris.chloride buffer, pH 8.5, and the protein is eluted with the same buffer. Alternatively, the solution is dialyzed extensively against 0.01 M sodium acetate and then distilled water and lyophilized. The apoprotein is obtained as a white solid. Apoferredoxin-III. This preparation s is similar to that of apoferredoxinI, but all solutions are 0.05 M with respect to 2-mercaptoethanol. Alternatively, apoferredoxin-III is prepared from apoferredoxin-II as follows: Apoferredoxin-II (54 mg) in 10 ml of 0.1 M Tris.chloride buffer, pH 8.5, containing 8 M urea, 2.5 mM EDTA, and a small amount of antifoam A is flushed at room temperature with prepurified nitrogen for 10 minutes. The mixture is made 0.1 M with respect to 2mercaptoethanol and flushed with nitrogen for 4 hours. At the end of the period the product, apoferredoxin-III, is reisolated free of urea in 0.02 M Tris.chloride buffer, pH 8.5, containing 0.1 M 2-mercapto-
442
NITROGEN FIXATION
[38]
ethanol by gel filtration on Sephadex G-25 (1.8 × 40 cm) which had been equilibrated with the buffer.
Reconstitution of Ferredoxin from Apoferredoxin
Reconstitutionfrom dpoferredoxin-U Apoferredoxin-I, 1 rag, is dissolved in 1.0 ml of 0.1 M Tris.chloride buffer, pH 7.4, 0.05 M in 2-mercaptoethanol. Then, 30/~1 of 0.1 M Fe2(NH4)2SO4 and 30/~1 of 0.1 M Na2S are added and the solution is incubated at 37 ° for 10 minutes. The protein is reisolated by chromatography on DEAE-cellulose (0.5 x 8 cm). After applying the protein, the column is washed with water and 0.15 M NaC1 to remove excess reagents. The protein is eluted with 0.80 M NaCI. Reconstitution from Apoferredoxin-II. s Apoferredoxin-II, 1.8 rag, is incubated for 4 hours at room temperature in 4 ml of 0.1 M Tris.chloride buffer, pH 8.5, containing 8 M urea, 0.07 M 2-mercaptoethanol and a small amount of antifoam while being flushed with nitrogen gas. Then 0.078 ml of 0.1 M FeSO4(NH4hSO4, 0.078 ml of 0.1 M Na2S, and 140 ~moles of 2-mercaptoethanol are added while still flushing with nitrogen gas. The reaction mixture is then diluted 3-fold with deaerated 0.1 M Tris.chloride buffer, pH 8.5, and incubated for 15 minutes at 37 °. The reconstituted ferredoxin is reisolated by chromatography on a DEAEcellulose column (0.8 × 8 cm). The reaction mixture is applied, then the column is washed successively with 10 ml of 0.15 M Tris.chloride buffer, pH 7.4, and 20 ml of 0.23 M NaCI in 0.005 M Tris.chloride buffer, pH 7.4, to remove excess reagents. The ferredoxin is finally eluted with 0.58 M NaCI in 0.005 M Tris.chloride buffer, pH 7.4. Reconstitution from Apoferredoxin-III. s Apoferredoxin-III, 1.8 mg in 1.6 ml of 0.1 M Tris.chloride buffer, pH 8.5, and 0.05 M 2-mercaptoethanol is incubated for 15 minutes at 37 ° with 0.078 ml of 0.1 M Fe~(NH4)~SO4 and 0.078 ml of 0.1 M Na2S. The reconstituted ferredoxin is isolated as described in the previous paragraph. Radioactive Ferredoxins. The reconstitution procedures have been described here with unlabeled iron and sulfide salts. However, labeled ferredoxins may be obtained by using SSFe2SO4(NH4)~SO4 or [35S]sodium sulfide. The labeled sodium sulfide obtained from commercial sources is purified by diffusion. For this purpose, 1.5 ml of 0.05 M [zSS]sodium sulfide is acidified with 0.5 ml of 1 N HC1 in an all-glass bubbling train and bubbled with prepurified nitrogen. The HzS evolved is trapped in a receiving tube containing 2 ml of 0.02 N NaOH. The labeled Na2S solution is mixed with 3 ml of 0.15 M unlabeled Na2S, and the concentration of sulfide is determined by iodometric titration.
[38]
CLOSTRIDIAL FERREDOXINS
443
Properties
Ferredoxin from different clostridial species is obtained in different crystalline forms? The ultraviolet spectra of ferredoxin and apoferredoxins prepared from C. acidi-urici are shown in Fig. 4. The absorption spectra of other clostridial ferredoxins are similar although not identical to this spectrum. The molar absorption of C. acidi-urici ferredoxin at 280 nm is 38,900 and at 390 nm is 30,600. 6The value A390.'A2s0for C. acidi-urici ferredoxin is 0.787. The value for this ratio for C. pasteurianum ferredoxin is 0.83. This value is a very useful index of the purity of the protein and can be used to detect deterioration of the crystalline preparation. The molecular weight of C. acidi-urici ferredoxin has been determined by sedimentation velocity5 and differential sedimentation equilibrium, '~ and values of 5600 and 5820, respectively, have been obtained. The molecular weight calculated from the amino acid sequence plus 8 atoms each of iron and sulfur is 6230. The sedimentation coefficient is 1.4, and the partial specific volume of this protein as determined using density gradient columns s''3 or by the differential sedimeni
4O
A
0 x
B. APOFERREDOXIN - I \
,,p
"
A. NATIVE FERREDOXIN
\,
35
C. APOFERREDOXIN - II
\ \~,L.
30
F~ ' ~
\
I--
z
25
\
8Z 20
o
\
\
\
\
\
\
5
\
\
\
\
\
o "\ %.
300
400
500
600
WAVELENGTH, nm
FIG. 4. doxins.
T h e absorption spectrum o f CIostridium acidi-urici ferredoxin and apoferre-
'~S. J. Edelstein and H. K. Schachman, J, Biol. Chem. 242, 306 (1967). '~A. Hvidt, G. J o h a n s e n , K. Linderstrom-Lang, and F. Vaslos, C. R. Trav. Lab. Carlsberg, Ser. Chim., 29, 129 (1954).
444
NIT~O(,rN FIXATION
[38]
ration equilibrium analysis TM is 0.63 and 0.61, respectively. Clostridium acidi-urici f e r r e d o x i n contains 8 atoms o f iron p e r mole o f protein as det e r m i n e d by atomic absorption spectroscopy a n d 8 atoms of acid-labile sulfur d e t e r m i n e d by the specific activity o f f e r r e d o x i n reconstituted with [zSS]sulfide o f k n o w n specific activity 6 or t h r o u g h use o f the colorimetric assay o f Fogo a n d Popowsky TM as described previously. ~ Clostridial f e r r e d o x i n accepts two electrons w h e n titrated with s o d i u m dithionite, 15 or on reduction with a c r u d e h y d r o g e n a s e p r e p a r a tion f r o m C. pasteurianum, TM spinach chloroplasts, Ir or a highly purified p y r u v a t e : f e r r e d o x i n o x i d o r e d u c t a s e f r o m C. acidi-urici. TM T h e b e h a v i o r o f clostridial f e r r e d o x i n s a n d a p o f e r r e d o x i n s in the Lowry protein assay using the p h e n o l r e a g e n t are s u m m a r i z e d in T a b l e I I I . n F e r r e d o x i n gives an unusually high color equivalent in this test c o m p a r e d to bovine s e r u m albumin, despite the fact that it contains relatively few aromatic a m i n o acids. T h e a m i n o acid sequences o f f o u r clostridial-type f e r r e d o x i n s are given in T a b l e IV. Clostridial f e r r e d o x i n s contain only a single basic a m i n o acid residue or none. T h e isoelectric point o f this protein is p H 3.7. 5 All the clostridial-type f e r r e d o x i n s that have b e e n e x a m i n e d contain 8 cysteine residues, a n d these are in similar positions relative to one another. T h e r e is also a significant s y m m e t r y in the molecule in that the sequence f r o m residues 30 to 55 is very similar to the sequence that occurs in the first half o f the molecule, if two deletions are introduced. A n u m b e r o f a m i n o acids are completely lacking in these materials. For e x a m p l e , C. acidi-urici f e r r e d o x i n lacks phenylalanine, histidine, tryptoTABLE IIl PWCSlCALCONSTANTSor Clostridium acidi-uriz'i FERREDOXIN AND DERIVATIVES IN PROTEIN DETERMINATIONS
Apoferredoxin Molecular weight A3~/mg/ml E390
Folin protein assay Aee0/mg ferredoxin A~0/mg albumin c660 x 10 -4
Ferredoxin
I
II
6,230 5.00 30,600
7,390 0.0 0.0
5,530 0.0 0.0
1.78 19.7
1.10 14.8
1.12 11.3
14j. K. Fogo and M. Popowsky, Anal. Chem. 21,732 (1949). lsS. G. Mayhew, D. Petering, G. Palmer, and G. P. Foust, J. Biol. Chem. 244, 2830 (1969). lnB. E. Sobel and W. Lovenberg, Biochemistry 5, 6 (1966). 17M. C. W. Evans, D. O. Hall, H. Bothe, and F. R. Whatley, Biochem.J. 110, 485 (1968). lSK. Uyeda and J. C. Rabinowitz,J. Biol. Chem. 246, 3111 (1971).
[38]
CLOSTRID1ALFERREDOXINS
445
TABLE I V AMINO ACID SEQUENCE OF CLOSTRIDIAL-TYPE FERREDOXINS
Residue No. 1e 2 3 4 5 6 7 8 9 10 11 12 13
14 15 16 17
18 19 20 21 22 23 24 25 26 27 28 29
~,
Organism b
c
d
No.
Ala Tyr Val Ile Asn Glu Ala Cys Ile Ser Cys Gly Ala Cys Asp Pro Glu Cys Pro Val Asp Ala Ile Ser Glu Gly Asp Ser Arg
Ala Tyr Lys lle Ala Asp Ser Cys Val Ser Cys Glu Ala Cys Ala Ser Glu Cys Pro Val Asn Ala Ile Ser Glu Gly Asp Ser Ile
Ala Phe Val Ile Asn Asp Ser Cys Val Ser Cys Gly Ala Cys Ala Gly Glu Cys Pro Val Ser Ala Ile Thr Gin Gly Asp Thr Gin
Ala Tyr Val Ile Asn Asp Ser Cys Ile Ala Cys Gly AIa Cys Lys Pro Glu Cys Pro Val Asn _r Ser Gin Gin Gly _r Ser lie
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
Residue
Organism ,,
b
c
d
Tyr Val Ile Asp Ala Asp Thr Cys lie Asp Cys Gly Ala Cys Ala Gly Val Cys Pro Val Asp Ala Pro Val Gin Ala
Phe Val lie Asp Ala Asp Thr Cys Ile Asp Cys Gly Asn Cys Ala Asn Val Cys Pro Val Gly Ala Pro Val Gin Glu
Phe Val Ile Asp Ala Asp Thr Cys Ile Asp Cys Gly Asn Cys Ala Asn Val Cys Pro Val Gly Ala Pro Asn Gln Glu
Tyr Ala lie Asp Ala Asp Ser Cys Ile Asp Cys Gly Ser Cys Ala Ser Val Cys Pro Val Gly Ala Pro Asn Pro Glu Asp
"Clostridium acidi-urici. S. C. Rail, R. E. Bolinger, and R. D. Cole,Biochemistry 8, 2486 (1969). bClostridium pasteurianum. M. Tanaka, T. Nakashima, A. Benson, H. Mower, and K. T. Yasunobu, Biochemistry 5, 1666 (1966). CClostr~dium butyricum. A. M. Benson, H. F. Mower, and K. T. Yasunobu, Arch. Biochem. Biophys. 121,563 (1967). aMicrococcus aerogenes. J. N. Tsunoda, K. T. Yasunobu, and H. R. Whiteley, J. Biol. Chem. 243, 6262 (1968). eThe sequence is given from the amino-terminal end at position 1 to the carboxy-terminal end at position 55. ¢The residue n u m b e r has been shifted to emphasize the similarity of the sequence to that of the other clostridial ferredoxins. This ferredoxin contains a total of 54 amino acid residues.
phan, leucine, methionine, and lysine. Other clostridial ferredoxins also lack several amino acids, although these may differ from those mentioned above.
446
NITROGEN FIXATION
[39]
The reaction of clostridia! ferredoxin with iron chelating agents and with sulfhydryl reagents has been investigated? 9 Clostridium acidi-urici ferredoxin reacts slowly with the ferrous chelating agent 0-phenanthroline under anaerobic conditions. After either chemical or enzymatic reduction of the protein, there is a rapid reaction of one of the 8 iron atoms with the chelating agent, but the reaction of the remaining iron is inhibited. In the presence of high concentrations of urea or guanidine hydrochloride and aerobic conditions, all the iron in the protein reacts with o-phenanthroline. Only 65-80% of the iron reacts under anaerobic conditions in the presence of these denaturants. Ferredoxin undergoes gradual degradation in the presence of 6.4 M urea or 4 M guanidine hydrochloride under aerobic conditions, but the protein is relatively stable in these denaturants under anaerobic conditions. Native, enzymatically reduced, or chemically reduced ferredoxin does not react with the ferric chelating agent Tiron. In 4 M guanidine hydrochloride under aerobic conditions, 8 moles of iron per mole of protein react with Tiron. Under anaerobic conditions, however, only approximately half the iron in the protein reacts with Tiron. Native ferredoxin reacts with the mercurial sulfhydryl reagent CMB in neutral buffered solution. 5 Slightly more than the theoretical value of 24 moles of CMB reacts per mole of protein. However, native ferredoxin does not react with the sulfhydryl reagent DTNB under these conditions, and there is only a slight reaction in the presence of 6.4 M urea. In 4 M guanidine hydrochloride, under either anaerobic or aerobic conditions, approximately 14 moles of DTNB react per mole of ferredoxin. This reaction is due exclusively to the inorganic sulfide in ferredoxin. The reaction of both the inorganic sulfide and the cysteine residues in ferredoxin with DTNB occurs in the presence of 4 M guanidine hydrochloride and EDTA under anaerobic conditions. 19R.Malkin and J. C. Rabinowitz,Biochemistry6, 3880 (1967).
[39] Purification of Nitrogenase from
Clostridium pasteurianum By LEONAaDE. MORTENSON Nitrogenase from Clostridium pasteurianum is an anionic protein complex, and because of this the first successful purification involved adsorption on cationic materials, la The two most successful steps were selective 1L.E. Mortenson, Biochim.Bioplqs.Acta 127, 18 (1966). 2L. E. Mortenson, J. A. Morris, and D. Y. Jeng, Biochim. Biophys. Acta 141, 516 (1967).
CLOSTRIDIALFERREDOXINS
431
upon spectrophotometric measurement of the oxidation of Na2S204 (315 nm peak) while it serves as the reductant for nitrogenase or hydrogenase. The method is particularly useful for kinetic studies of these enzyme systems.
[38] P r e p a r a t i o n
and Properties
of Clostridial
Ferredoxins By JESSE RABINOWlTZ Ferredoxin was first isolated from Clostridiumpasteurianumby Mortenson, Valentine, and Carnahan. 1 The protein functions as an electron transfer factor in the enzymatic formation of acetyl phosphate and hydrogen from pyruvate by extracts of that organism treated with DEAE-cellulose and in a variety of other enzymatic reactions subsequently described. 2 It was characterized as an iron-sulfur protein when the presence of acid-labile sulfur was recognized. 3 Ferredoxin has been found in all clostridial species that have been examined. Procedures for the purification of clostridial ferredoxin have been based on the acidic nature of the protein and its resultant high affinity for DEAEcellulose, 3 its small size relative to other proteins and resultant solubility in acetone, 4 and the relative ease of crystallization of the protein with ammonium sulfate. Clostridial ferredoxin has a molecular weight of about 6000 and is composed of a single polypeptide chain containing 55 or fewer amino acid residues, 8 of which are cysteine, s The protein also contains 8 iron atoms and 8 atoms of acid-labile sulfur. 6 The biological activity of this protein is associated with its unusually low redox potential. Although the amino acid sequence of the protein isolated from several clostridial species is known (Table IV), the molecular structure with respect to the iron and acid-labile sulfur atoms is not known at this time. na The iron and sulfur atoms can be removed by 1L. E. Mortenson, R. C. Valentine, and J. E. Carnahan, Biochem. Biophys. Res. Commun. 7, 448 (1962). 2R. C. Valentine, Bacteriol. Rev. 28, 497 (1964). 3B. B. Buchanan, W. Lovenberg, and J. C. Rabinowitz, Proc. Nat. Acad. Sci. U.S. 49, 345 ( 1963). 4L. E. Mortenson, Biochim. Biophys. Acta 81, 71 (1964). 5W. Lovenberg, B. B. Buchanan, and J. c. Rabinowitz, J. Biol. Chem. 238, 3899 (1963). 6J.-S. Hong, Ph.D. Thesis, Univ. of California, Berkeley (1969). eaNote added in proof: This is no longer true. The structure of the iron-sulfur complex of the ferredoxin isolated from Micrococcus aerogenes, which is a clostridial-type ferredoxin, has been determined: L. C. Sieker, E. Adman, and L. H. Jensen, Nature 235, 40 (1972).
432
NITROGEN FIXATION
[38]
t r e a t m e n t o f f e r r e d o x i n with mercurial sulfhydryl reagents, 7 by treatm e n t with acid, s or by heat. ° T h e a p o f e r r e d o x i n f o r m e d by these treatments can be isolated, and f e r r e d o x i n can be reconstituted f r o m this material p r e p a r e d in this m a n n e r by using 5°Fe-labeled salts or [aSS]sodium sulfide in the reconstitution reactions. Growth of Clostridium pasteurianum Potato Tube Cultures. Lyophilized cultures o f C. pasteurianum (American T y p e Culture Collection No. 6013) or spore stocks o f this organism in soil are most easily revived by growth on "potato m e d i u m . " This is p r e p a r e d in the following manner: Solid CaCO3 is a d d e d to cover the bottom o f a test tube (18 × 150 mm). Fresh potatoes, peeled, washed, and finely diced, are a d d e d to the test tube to give a layer about 3 cm high, and 10-12 ml o f a 2% sucrose solution in tap water is a d d e d to the tubes. T h e tubes are plugged with n o n a b s o r b e n t cotton and autoclaved for 20 minutes at 120 °. After cooling, the tubes are sealed by adding a plug o f absorbent cotton and 2 d r o p s o f 2 N K2COz followed by 2 drops o f 4 M pyrogallol. T h e tube is immediately closed with a r u b b e r stopper and may be stored for 2 weeks. T o inoculate, the absorbent cotton is discarded; it is replaced with a fresh seal after inoculation. T h e inoculated tubes are incubated at 30 ° . It may take 30-40 hours for a p o t a t o - m e d i u m culture to start growing when inoculated f r o m soil or lyophilized spore stocks, and about 15 hours when an inoculum grown on the p o t a t o - m e d i u m culture is used. Since there is a vigorous evolution o f h y d r o g e n d u r i n g growth, tubes should not be s t o p p e r e d too tightly. T h e fully grown p o t a t o - m e d i u m culture may be stored in the r e f r i g e r a t o r u p to a m o n t h and used for subsequent inoculations into liquid media. Growth on Synthetic Medium. In o r d e r to grow a 40-liter culture o f the organism, f o u r 500-ml cultures are used as an inoculum. This m e d i u m 1° is p r e p a r e d in the following m a n n e r : Solid CaCO3 (1.5 g) and 250 ml o f tap water are a d d e d to each o f four 750-ml E r l e n m e y e r flasks. T h e following c o m p o n e n t s are dissolved in 900 ml o f tap water:
Sucrose, 40 g MgCI2.6H20, 7.4%, 4 ml NaCI, 20%, 1 ml Na~MoO4.2H20, 10%, 0.2 ml 7Richard Malkin and J. C. Rabinowitz, Biochem. Biophys. Res. Commun. 23, 822 (1966), 8J.-S. Hong and J. C. Rabinowitz, Biochem. Biophys. Res. Commun. 29, 246 (1967). 9T. Devanathan, J. M. Akagi, R. T. Hersh, and R. H. Himes,J. Biol. Chem.244, 2846 (1969). 10j. E. Carnahan and J. E. Castle,J. Bacteriol. 75, i21 (1958).
[38]
CLOSTR1D1ALFERREDOXINS
433
Biotin, 100/zg/ml, 0.1 ml p-Aminobenzoic acid, 100/zg/ml, 0.1 ml
N a2SO4, 7.1%, 2.0 ml NH4C1, 20%, 16 ml This solution is diluted to 1 liter, and 250 ml is added to each of the 750-ml flasks. The flasks are plugged with nonabsorbent cotton and autoclaved for 15 minutes at 121 °. While the flasks are hot, 10 ml of a sterile solution of 5% KH2PO4 + 9.2% K2HPO4"3H20 is added. When the flasks have cooled to 30 °, nitrogen gas that has been sterilized by passage through a cotton filter is bubbled through them. Just before inoculation of each flask, 2 ml of 5% FeC13"6H20 (in absolute alcohol) is added with a sterile pipette. Each Erlenmeyer flask is inoculated with 20-25 ml of the culture grown on the potato medium and bubbling with sterile nitrogen is continued. After growth for 16-20 hours at 30 °, the A660 is 11-12. The doubling time is 1.2-1.8 hours. Three or four of these flasks are then used to inoculate 40 liters of the synthetic medium modified as follows and contained in a large carboy. The calcium carbonate concentration is decreased to 1 g per liter, and the phosphate concentration is doubled. Cells are usually harvested in late log phase when the turbidity as determined by the A660 is between 3 and 4. The yield of wet cells from a 40-liter culture is approximately 200-250 g. A portion of the freshly harvested cells is used to prepare the clastic enzymes as described below and the remainder is stored in the deep freeze for use in the preparation of ferredoxin.
Preparation of DEAE-Cellulose The DEAE-cellulose used in all steps described in this contribution is prepared in the following manner: Newly purchased DEAE-cellulose is suspended in a large volume of 0.5 N NaOH in a large vessel for about 12 hours. The liquid is decanted with suction, and the cellulose derivative is washed with water 3 times. It is then suspended in 0.5 N HC1 for 12 hours, and washed with water by decantation 3 times. The wash with 0.5 N NaOH is repeated once again, and the material is washed with water by decantation. The washed DEAE-cellulose is suspended in water, and the suspension is adjusted to pH 6.5 with KH2PO4 and diluted to give a final concentration of phosphate of 0.3-1.0 M. This material is used for preparation of the columns for a particlar step, and it is washed with about l0 column volumes of water before use. The DEAEcellulose recovered from the column after it has been used in a chromatographic step is regenerated by suspending it in 0.5 N NaOH once, washing it with water, and equilibrating it with potassium phosphate at pH 6.5 as described above.
434
NITROGEN FIXATION
[38]
Preparation of Clastic Enzymes The ferredoxin-free clastic enzyme system is prepared as follows: 100 g of freshly harvested cells of C. pasteurianum is suspended in water to give a final volume of 200 ml and broken by sonication of 60-ml aliquots for 1.5 minutes with a Branson Model W185D cell disruptor. The suspension is centrifuged for 1 hour at 40,000 g. The supernatant solution is then passed over a column of DEAE-cellulose (2.2 × 20 cm). The pass-through (about 30 mg of protein per milliliter), containing the clastic enzymes, free of ferredoxin, is immediately frozen in 5-ml aliquots and stored in liquid nitrogen.
Assay Methods Reagents Potassium phosphate buffer, 0.25 M, pH 6.5 Sodium pyruvate, 0.5 M Coenzyme A, 10 mg/ml Hydroxylamine, 14%, neutralized 11 Ferric chloride reagent. Mix 100 ml of 10% FeCI3 in 0.2 N HCI, 200 ml of 12% trichloroacetic acid, and 200 ml of 3 N HCI and dilute to 600 ml with water Procedure. The following solutions are added to test tubes: 0.10 ml of phosphate buffer, 0.02 ml of pyruvate, 0.005 ml of CoA, 0.3 ml of the clastic enzymes (about 8-10 mg of protein), 0.2-1.0/zg of ferredoxin, and water to make the volume 1 ml. The tubes are incubated at 37 °. After 10 minutes, 1.5 ml of the ferric chloride reagent is added. The solutions are filtered through Whatman No. 1 filter paper by gravity (centrifugation is not satisfactory) and the absorbancy of the filtrate at 540 nm is determined using as a blank the control tube incubated in the absence of ferredoxin. There is an optimum level of clastic enzyme preparation which should be used in the assay so that the amount of reaction is maximal with a particular level of a d d e d ferredoxin. This is illustrated in Fig. 1. Since the activities of the clastic enzyme preparations vary, an experiment similar to that shown in Fig. 1 should be done with each enzyme preparation to determine the amount of protein to be used for the ferredoxin assay. A unit of activity in this assay is defined as the amount needed to give a change of 1.0 in absorbance at 540 nm.
I'F. Lipmannand L. C. Tuttle,J. Biol. Chem. 159, 21 (1945).
[38]
CLOSTRIDIALVEkkV.DOXlYS I
[
I
~35
I
E c
o
0.30 0.20
c
0.10 o
I
I
I
I
4
8
12
16
Protein Per ml Assay Mixture
FIG. 1. D e p e n d e n c e o f pyruvate "clastic" reaction on enzyme concentration. (See text for details.)
Purification of Ferredoxin from C. acidiourici
Step 1. Extract. Frozen cells of Clostridium acidi-urici (Vol. VI [97], 350 g, are thawed overnight at 4 ° and suspended in 0.05 M potassium phosphate buffer, pH 7.8, to a final volume of 900 ml. DNase (0.1 ml of a solution containing 5 mg/ml, Worthington) is added to facilitate suspension of the cells and reduce the viscosity of the mixture. Aliquots (60 ml) are sonicated for 1.5-minute intervals in a Branson Model W183D cell disruptor. The cell debris and unbroken cells are removed by centrifugation at 40,000 g for 1 hour. RNase (4 mg) is added to the supernatant solution. Step 2. DEAE-CeUulose-1. This crude extract (650 ml) is applied to a DEAE-cellulose column (3.0 × 30 cm). The pass-through (650 ml) is collected and used for the purification of formyhetrahydrofolate synthetase (Vol. VI [51]). The column is then washed with 300 ml of water. The ferredoxin is eluted with a linear salt gradient in the absence of buffer obtained by adding 1.8 liters of 0.7 M NaC1 to 1.8 liter of 0.1 M NaC1 contained in a mixing chamber, and 10-ml fractions are collected. A typical elution pattern is shown in Fig. 2A. Ferredoxin is recognized from the Aag0 measurement of individual fractions. It is eluted with 1000-1460 ml of eluent. These fractions are pooled and diluted 4-fold with distilled water to give a volume of 2100 ml. Step 3. DEAE-Cellulose-2. The diluted ferredoxin is reapplied to a second DEAE-cellulose column (3.0 × 23 cm). After application of the sample, the column is washed with 500 ml of 0.15 M Tris.chloride buffer, pH 7.4. The ferredoxin is eluted with a salt gradient obtained by adding 1.3 liters of 0.27 M NaCl in 0.15 M Tris.chloride buffer, pH 7.4, to 1.5 liters of 0.07 M NaC1 in 0.15 M Tris.chloride buffer, pH 7.4, in a mixing chamber, and 10-ml fractions are collected. It is usually possible to allow this chromatographic step to proceed overnight. The ferredoxin is eluted with 900-1600 ml of eluent (Fig. 2B).
436
NITROGEN FIXATION
[38]
Step 4. DEAE-Cellulose-3. T o c o n c e n t r a t e the f e r r e d o x i n the fractions are p o o l e d a n d d i l u t e d 3-fold with distilled water a n d r e a d s o r b e d o n a t h i r d DEAE-cellulose c o l u m n (3.0 × 8 cm), a n d e l u t e d with a small v o l u m e o f 0.15 M T r i s . c h l o r i d e buffer, p H 7.4, c o n t a i n i n g 0.5 M NaCi. Step 5. Sephadex G-75. T h e c o n c e n t r a t e d solution (26 ml) is t h e n a p p l i e d to a c o l u m n o f S e p h a d e x G-75 (3.0 × 90 cm) which has b e e n
I
)
]
I
I
I
I
16.0
I
A
~ 8.o
~
°
i
4'0i ~~ ~ 0
0
400
I
I ~-,
800
1200
1600
2000
ml Effluent
8.0-
B
w
v
8
4.0-~
2.o-
i
o
[,.~
o
I
400
800
1200
1600
!000
ml Effluent
Fie.. 2. Purification of ferredoxin from Clostridiumacidi-urici. (See text for details.)
[38]
CLOSTRIDIALFERREDOX1NS
437
equilibrated with 0.05 M Tris.chloride buffer, pH 7.4, and 5-ml fractions are collected. It is usual to allow this step to proceed overnight. The ferredoxin is eluted with 250-340 ml of eluent (Fig. 2C). Step 6. Crystallization. The fractions containing the ferredoxin are combined, and the solution is made 70% in ammonium sulfate. After 1 hour at 0°, the precipitated ferredoxin is collected by centrifugation for 20 minutes at 39,000 g. It is then resuspended in 40 ml of 0.15 M Tris.chloride buffer, pH 7.4, and stored in a tube under vacuum at 4 °. The purification of the protein from C, acidi-urici is summarized in Table I.
Purification of Ferredoxin from C. pasteurianum The procedure used is very similar to that described for the isolation of ferredoxin from C. acidi-urici; however, some differences in results and in the procedure may be noted because the ferredoxin content of C. pasteurianum is significantly lower than that of the purine-fermenting organism, and relatively higher concentrations of ammonium sulfate are required for its precipitation. Step 1. Extract. Frozen cells of C. pasteurianum (450 g) grown as described in a previous section, are thawed overnight at 4 °. The extract is prepared as described for cells of C. acidi-urici. Step 2. DEAE-Cellulose-1. The extract is applied to a DEAE-cellulose column (4.5 X 30 cm) and is washed with 400 ml of water. The ferre-
"//
I
I
I
I
I
C
30.0
I
E 20.0
c
10.0
I
150
200
250
300
ml Effluent
Fig. 2c
350
400
438
NITROGEN FIXATION
[38]
TABLE I PURIFICATION OF FERREDOXIN FROM Clostridium acidi-urici
Fraction I. Extract c II. DEAE-cellulose-1
III. IV. V. V1.
DEAE-cellulose-2 DEAE-cellulose-3 Sephadex G-75 Crystals
Volume (ml)
Protein a (mg)
650 2100 2040 33 93 50
11,626 1,196 494 411 352 354
Ferredoxin o Units Mg 119,600 111,090 90,168 88,440 83,700 84,000
As~o/A28o
345 321 261 255 242 243
0.046 0.322 0.726 0.728 0.775 0.789
aDetermined by a modification of the Lowry method, with crystalline bovine serum albumin as standa.r,d [J. C. Rabinowitz and W. E. Pricer, Jr., J. Biol. Chem. 237, 2898 (1962)]. bThe ferredoxin was determined by its activity in the enzymatic assay with the Clostridium pasteurianum "clastic system." A unit of activity causes a net increase of 1.0 at 540 nm in the assay. A standard solution of ferredoxin is included in each assay, and the unit activity of ferredoxin varies from day to day. T h e milligrams of ferredoxin recovered are based on this enzymatic test. cPrepared from 350 g of wet cells in the absence of 2-mercaptoethanol.
doxin is eluted with a linear salt gradient in the absence of buffer obtained by adding 1.8 liters of 0.7 M NaC1 to 1.8 liter of 0.1 M NaCI, and 10-ml fractions are collected. The elution pattern obtained is shown in Fig. 3A. The ferredoxin is eluted with 1560-1800 ml of eluent. 4.0
I
I
I
l
I
I
i
I
3.0-
•
16.0
- 12.0 T
E e-
E c
Q O~
O
2.0-
8.0
f-
N c .D
~, - 4 . 0
1.0-
400 FIG. 3.
800 1200 ml Effluent
1600
<
2000
Purification of ferredoxin from Clostridium pasteurianum. (See text for details.)
[38]
CLOSTRIDIALFERREDOXINS
439
The fractions containing ferredoxin are combined, diluted to 900 ml, and stored overnight under reduced pressure. Step 3. DEAE-Cellulose-2. The ferredoxin is readsorbed on a second DEAE-cellulose column (4.5 × 23 cm). The column is washed with 500 ml of 0.15 M Tris.chloride buffer, pH 7.4, and the ferredoxin is eluted with a linear salt gradient obtained by adding 1.5 liter of 0.27 M NaC1 in 0.15 M Tris.chloride buffer, pH 7.4, to a mixing chamber containing I
I
I
I
I
I
I
I
]
B
3.0
T 2.0 8
1.0
--J
0
,
400
,
I
800
1200
I
1600
2000
ml Effluent I
i
I
I
I
I
I
I
12.0
I
C
T v
E c
0 O~
E
8.0
0 ¢0 k"N
8
r,,) C
c
4.0
I 0
I
100
.,P'-.-J
- -
200
300
ml Effluent FIG. 3-B-C
400
500
440
NITROGEN F1XATION
[38]
1.5 liters of 0.07 M NaC1 in 0.15 M Tris.chloride buffer, pH 7.4. Tenmilliliter fractions are collected, and the elution pattern obtained is shown in Fig. 3B. The ferredoxin is eluted with 1300-1600 ml of eluent. The combined fractions are diluted 3-fold and stored overnight in an evacuated flask. Step 4. DEAE-CeUulose-3. In order to concentrate the ferredoxin, the material is adsorbed on a third DEAE-cellulose column (3.0 × 8 cm) and is eluted with a small volume of 0.5 M NaC1 in 0.15 M Tris.chloride buffer, pH 7.4. Step 5. Sephadex G-75. This material, 20 ml, is applied to a column of Sephadex G-75 equilibrated with 0.05 M Tris.chloride buffer, pH 7.4. The ferredoxin is obtained in the eluate after 310-370 ml of eluate have been collected (Fig. 3C). Step 6. Crystallization. This solution is brought to 60% saturation with ammonium sulfate, and the small amount of precipitate formed is removed by centrifugation. The supernatant solution is brought to 85% saturation with ammonium sulfate and allowed to stand at 5° overnight. The precipitate is collected by centrifugation and redissolved in 0.15 M Tris.chloride buffer, pH 7.4. The protein is recrystallized by the addition of ammonium sulfate to 80% saturation. Crystals form immediately. The purification of the protein from C. pasteurianum is summarized in Table II.
Preparation of Apoferredoxins ApoJerredoxind. The iron and acid-labile sulfide are removed from ferredoxin by treatment of the protein with a mercurial. 7 Ferredoxin (15 mg) in 2.7 ml of 0.1 M Tris.chloride buffer, pH 7.4, is treated with 1.8 ml of 0.05 M sodium mersalyl (Sigma Chemical Co.) in the same buffer at room temperature for 2 minutes. The small amount of precipitate T A B L E II PURIfiCATION OV FERREDOXIN FROM Clostridium
Fraction I. II. III. IV. V. VI.
Extract c DEAE-cellulose- 1 DEAE-cellulose-2 DEAE-cellulose-3 S e p h a d e x G-75 Crystals
pasteurianurn
Volume (ml)
Protein" (mg)
Ferredoxin b (units)
A39o/Azso
520 900 1100 20 80 17
4992 549 319 197 142 112
79,560 73,980 62,150 56,000 46,000 45,220
0.0535 0.366 0.625 0.635 0.79 0.83
aSee Table I. bSee Table I. c p r e p a r e d f r o m 400 g o f wet cells in the absence o f 2 - m e r c a p t o e t h a n o l .
[38]
CLOSTRIDIALFERREDOX1NS
441
that forms is removed by centrifugation. The solution is then passed over a column of Chelex 100 (Bio-Rad Laboratories, Berkeley, California) (2 x 17 cm) equilibrated with water. The protein is eluted with water at room temperature and 1.5 ml fractions are collected. The protein is eluted in a single peak with 18-24 ml of eluent. These fractions are pooled, lyophilized, and redissolved in distilled water to give a concentration of 4.0 mg of protein per milliliter. This solution is then passed over a column of Sephadex G-25 equilibrated with water at room temperature. The column is developed with water at room temperature, and 0.5-ml fractions are collected. The absorbancy at 280 nm and the protein content of the fractions are determined. A single proteincontaining component is obtained with 2-6 ml of eluent. A component with absorbancy at 280 nm is eluted afterward, but this fraction contains no protein. The fractions containing apoferredoxin-I are lyophilized. Apoferredoxin-II. The iron and acid-labile sulfide are removed from ferredoxin by treatment of the protein with acid under aerobic conditions, s Ferredoxin (14 mg) in 11 ml of 0.15 M Tris.chloride buffer, pH 7.4, is made 4-5% in trichloroacetic acid with a concentrated solution (30%) of the acid. This mixture is allowed to stand for 1 hour at 0 °. Evolution of H2S gas occurs, and the brown color of ferredoxin is completely bleached at the end of the period. The white suspension is centrifuged at 35,000 g for 10 minutes, and the supernatant solution is discarded. The precipitate is washed with either 3 ml of 0.023 M formic acid or 5% trichloroacetic acid, allowed to stand for 30 minutes at 0 °, and centrifuged. The precipitate is finally taken up in 4 ml of 0.1 M Tris.chloride buffer, pH 9.0, and the insoluble material is removed by centrifugation. The almost colorless supernatant solution is then passed over a Sephadex G-25 column (1.2 × 38 cm) previously equilibrated with 0.02 M Tris.chloride buffer, pH 8.5, and the protein is eluted with the same buffer. Alternatively, the solution is dialyzed extensively against 0.01 M sodium acetate and then distilled water and lyophilized. The apoprotein is obtained as a white solid. Apoferredoxin-III. This preparation s is similar to that of apoferredoxinI, but all solutions are 0.05 M with respect to 2-mercaptoethanol. Alternatively, apoferredoxin-III is prepared from apoferredoxin-II as follows: Apoferredoxin-II (54 mg) in 10 ml of 0.1 M Tris.chloride buffer, pH 8.5, containing 8 M urea, 2.5 mM EDTA, and a small amount of antifoam A is flushed at room temperature with prepurified nitrogen for 10 minutes. The mixture is made 0.1 M with respect to 2mercaptoethanol and flushed with nitrogen for 4 hours. At the end of the period the product, apoferredoxin-III, is reisolated free of urea in 0.02 M Tris.chloride buffer, pH 8.5, containing 0.1 M 2-mercapto-
442
NITROGEN FIXATION
[38]
ethanol by gel filtration on Sephadex G-25 (1.8 × 40 cm) which had been equilibrated with the buffer.
Reconstitution of Ferredoxin from Apoferredoxin
Reconstitutionfrom dpoferredoxin-U Apoferredoxin-I, 1 rag, is dissolved in 1.0 ml of 0.1 M Tris.chloride buffer, pH 7.4, 0.05 M in 2-mercaptoethanol. Then, 30/~1 of 0.1 M Fe2(NH4)2SO4 and 30/~1 of 0.1 M Na2S are added and the solution is incubated at 37 ° for 10 minutes. The protein is reisolated by chromatography on DEAE-cellulose (0.5 x 8 cm). After applying the protein, the column is washed with water and 0.15 M NaC1 to remove excess reagents. The protein is eluted with 0.80 M NaCI. Reconstitution from Apoferredoxin-II. s Apoferredoxin-II, 1.8 rag, is incubated for 4 hours at room temperature in 4 ml of 0.1 M Tris.chloride buffer, pH 8.5, containing 8 M urea, 0.07 M 2-mercaptoethanol and a small amount of antifoam while being flushed with nitrogen gas. Then 0.078 ml of 0.1 M FeSO4(NH4hSO4, 0.078 ml of 0.1 M Na2S, and 140 ~moles of 2-mercaptoethanol are added while still flushing with nitrogen gas. The reaction mixture is then diluted 3-fold with deaerated 0.1 M Tris.chloride buffer, pH 8.5, and incubated for 15 minutes at 37 °. The reconstituted ferredoxin is reisolated by chromatography on a DEAEcellulose column (0.8 × 8 cm). The reaction mixture is applied, then the column is washed successively with 10 ml of 0.15 M Tris.chloride buffer, pH 7.4, and 20 ml of 0.23 M NaCI in 0.005 M Tris.chloride buffer, pH 7.4, to remove excess reagents. The ferredoxin is finally eluted with 0.58 M NaCI in 0.005 M Tris.chloride buffer, pH 7.4. Reconstitution from Apoferredoxin-III. s Apoferredoxin-III, 1.8 mg in 1.6 ml of 0.1 M Tris.chloride buffer, pH 8.5, and 0.05 M 2-mercaptoethanol is incubated for 15 minutes at 37 ° with 0.078 ml of 0.1 M Fe~(NH4)~SO4 and 0.078 ml of 0.1 M Na2S. The reconstituted ferredoxin is isolated as described in the previous paragraph. Radioactive Ferredoxins. The reconstitution procedures have been described here with unlabeled iron and sulfide salts. However, labeled ferredoxins may be obtained by using SSFe2SO4(NH4)~SO4 or [35S]sodium sulfide. The labeled sodium sulfide obtained from commercial sources is purified by diffusion. For this purpose, 1.5 ml of 0.05 M [zSS]sodium sulfide is acidified with 0.5 ml of 1 N HC1 in an all-glass bubbling train and bubbled with prepurified nitrogen. The HzS evolved is trapped in a receiving tube containing 2 ml of 0.02 N NaOH. The labeled Na2S solution is mixed with 3 ml of 0.15 M unlabeled Na2S, and the concentration of sulfide is determined by iodometric titration.
[38]
CLOSTRIDIAL FERREDOXINS
443
Properties
Ferredoxin from different clostridial species is obtained in different crystalline forms? The ultraviolet spectra of ferredoxin and apoferredoxins prepared from C. acidi-urici are shown in Fig. 4. The absorption spectra of other clostridial ferredoxins are similar although not identical to this spectrum. The molar absorption of C. acidi-urici ferredoxin at 280 nm is 38,900 and at 390 nm is 30,600. 6The value A390.'A2s0for C. acidi-urici ferredoxin is 0.787. The value for this ratio for C. pasteurianum ferredoxin is 0.83. This value is a very useful index of the purity of the protein and can be used to detect deterioration of the crystalline preparation. The molecular weight of C. acidi-urici ferredoxin has been determined by sedimentation velocity5 and differential sedimentation equilibrium, '~ and values of 5600 and 5820, respectively, have been obtained. The molecular weight calculated from the amino acid sequence plus 8 atoms each of iron and sulfur is 6230. The sedimentation coefficient is 1.4, and the partial specific volume of this protein as determined using density gradient columns s''3 or by the differential sedimeni
4O
A
0 x
B. APOFERREDOXIN - I \
,,p
"
A. NATIVE FERREDOXIN
\,
35
C. APOFERREDOXIN - II
\ \~,L.
30
F~ ' ~
\
I--
z
25
\
8Z 20
o
\
\
\
\
\
\
5
\
\
\
\
\
o "\ %.
300
400
500
600
WAVELENGTH, nm
FIG. 4. doxins.
T h e absorption spectrum o f CIostridium acidi-urici ferredoxin and apoferre-
'~S. J. Edelstein and H. K. Schachman, J, Biol. Chem. 242, 306 (1967). '~A. Hvidt, G. J o h a n s e n , K. Linderstrom-Lang, and F. Vaslos, C. R. Trav. Lab. Carlsberg, Ser. Chim., 29, 129 (1954).
444
NIT~O(,rN FIXATION
[38]
ration equilibrium analysis TM is 0.63 and 0.61, respectively. Clostridium acidi-urici f e r r e d o x i n contains 8 atoms o f iron p e r mole o f protein as det e r m i n e d by atomic absorption spectroscopy a n d 8 atoms of acid-labile sulfur d e t e r m i n e d by the specific activity o f f e r r e d o x i n reconstituted with [zSS]sulfide o f k n o w n specific activity 6 or t h r o u g h use o f the colorimetric assay o f Fogo a n d Popowsky TM as described previously. ~ Clostridial f e r r e d o x i n accepts two electrons w h e n titrated with s o d i u m dithionite, 15 or on reduction with a c r u d e h y d r o g e n a s e p r e p a r a tion f r o m C. pasteurianum, TM spinach chloroplasts, Ir or a highly purified p y r u v a t e : f e r r e d o x i n o x i d o r e d u c t a s e f r o m C. acidi-urici. TM T h e b e h a v i o r o f clostridial f e r r e d o x i n s a n d a p o f e r r e d o x i n s in the Lowry protein assay using the p h e n o l r e a g e n t are s u m m a r i z e d in T a b l e I I I . n F e r r e d o x i n gives an unusually high color equivalent in this test c o m p a r e d to bovine s e r u m albumin, despite the fact that it contains relatively few aromatic a m i n o acids. T h e a m i n o acid sequences o f f o u r clostridial-type f e r r e d o x i n s are given in T a b l e IV. Clostridial f e r r e d o x i n s contain only a single basic a m i n o acid residue or none. T h e isoelectric point o f this protein is p H 3.7. 5 All the clostridial-type f e r r e d o x i n s that have b e e n e x a m i n e d contain 8 cysteine residues, a n d these are in similar positions relative to one another. T h e r e is also a significant s y m m e t r y in the molecule in that the sequence f r o m residues 30 to 55 is very similar to the sequence that occurs in the first half o f the molecule, if two deletions are introduced. A n u m b e r o f a m i n o acids are completely lacking in these materials. For e x a m p l e , C. acidi-urici f e r r e d o x i n lacks phenylalanine, histidine, tryptoTABLE IIl PWCSlCALCONSTANTSor Clostridium acidi-uriz'i FERREDOXIN AND DERIVATIVES IN PROTEIN DETERMINATIONS
Apoferredoxin Molecular weight A3~/mg/ml E390
Folin protein assay Aee0/mg ferredoxin A~0/mg albumin c660 x 10 -4
Ferredoxin
I
II
6,230 5.00 30,600
7,390 0.0 0.0
5,530 0.0 0.0
1.78 19.7
1.10 14.8
1.12 11.3
14j. K. Fogo and M. Popowsky, Anal. Chem. 21,732 (1949). lsS. G. Mayhew, D. Petering, G. Palmer, and G. P. Foust, J. Biol. Chem. 244, 2830 (1969). lnB. E. Sobel and W. Lovenberg, Biochemistry 5, 6 (1966). 17M. C. W. Evans, D. O. Hall, H. Bothe, and F. R. Whatley, Biochem.J. 110, 485 (1968). lSK. Uyeda and J. C. Rabinowitz,J. Biol. Chem. 246, 3111 (1971).
[38]
CLOSTRID1ALFERREDOXINS
445
TABLE I V AMINO ACID SEQUENCE OF CLOSTRIDIAL-TYPE FERREDOXINS
Residue No. 1e 2 3 4 5 6 7 8 9 10 11 12 13
14 15 16 17
18 19 20 21 22 23 24 25 26 27 28 29
~,
Organism b
c
d
No.
Ala Tyr Val Ile Asn Glu Ala Cys Ile Ser Cys Gly Ala Cys Asp Pro Glu Cys Pro Val Asp Ala Ile Ser Glu Gly Asp Ser Arg
Ala Tyr Lys lle Ala Asp Ser Cys Val Ser Cys Glu Ala Cys Ala Ser Glu Cys Pro Val Asn Ala Ile Ser Glu Gly Asp Ser Ile
Ala Phe Val Ile Asn Asp Ser Cys Val Ser Cys Gly Ala Cys Ala Gly Glu Cys Pro Val Ser Ala Ile Thr Gin Gly Asp Thr Gin
Ala Tyr Val Ile Asn Asp Ser Cys Ile Ala Cys Gly AIa Cys Lys Pro Glu Cys Pro Val Asn _r Ser Gin Gin Gly _r Ser lie
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
Residue
Organism ,,
b
c
d
Tyr Val Ile Asp Ala Asp Thr Cys lie Asp Cys Gly Ala Cys Ala Gly Val Cys Pro Val Asp Ala Pro Val Gin Ala
Phe Val lie Asp Ala Asp Thr Cys Ile Asp Cys Gly Asn Cys Ala Asn Val Cys Pro Val Gly Ala Pro Val Gin Glu
Phe Val Ile Asp Ala Asp Thr Cys Ile Asp Cys Gly Asn Cys Ala Asn Val Cys Pro Val Gly Ala Pro Asn Gln Glu
Tyr Ala lie Asp Ala Asp Ser Cys Ile Asp Cys Gly Ser Cys Ala Ser Val Cys Pro Val Gly Ala Pro Asn Pro Glu Asp
"Clostridium acidi-urici. S. C. Rail, R. E. Bolinger, and R. D. Cole,Biochemistry 8, 2486 (1969). bClostridium pasteurianum. M. Tanaka, T. Nakashima, A. Benson, H. Mower, and K. T. Yasunobu, Biochemistry 5, 1666 (1966). CClostr~dium butyricum. A. M. Benson, H. F. Mower, and K. T. Yasunobu, Arch. Biochem. Biophys. 121,563 (1967). aMicrococcus aerogenes. J. N. Tsunoda, K. T. Yasunobu, and H. R. Whiteley, J. Biol. Chem. 243, 6262 (1968). eThe sequence is given from the amino-terminal end at position 1 to the carboxy-terminal end at position 55. ¢The residue n u m b e r has been shifted to emphasize the similarity of the sequence to that of the other clostridial ferredoxins. This ferredoxin contains a total of 54 amino acid residues.
phan, leucine, methionine, and lysine. Other clostridial ferredoxins also lack several amino acids, although these may differ from those mentioned above.
446
NITROGEN FIXATION
[39]
The reaction of clostridia! ferredoxin with iron chelating agents and with sulfhydryl reagents has been investigated? 9 Clostridium acidi-urici ferredoxin reacts slowly with the ferrous chelating agent 0-phenanthroline under anaerobic conditions. After either chemical or enzymatic reduction of the protein, there is a rapid reaction of one of the 8 iron atoms with the chelating agent, but the reaction of the remaining iron is inhibited. In the presence of high concentrations of urea or guanidine hydrochloride and aerobic conditions, all the iron in the protein reacts with o-phenanthroline. Only 65-80% of the iron reacts under anaerobic conditions in the presence of these denaturants. Ferredoxin undergoes gradual degradation in the presence of 6.4 M urea or 4 M guanidine hydrochloride under aerobic conditions, but the protein is relatively stable in these denaturants under anaerobic conditions. Native, enzymatically reduced, or chemically reduced ferredoxin does not react with the ferric chelating agent Tiron. In 4 M guanidine hydrochloride under aerobic conditions, 8 moles of iron per mole of protein react with Tiron. Under anaerobic conditions, however, only approximately half the iron in the protein reacts with Tiron. Native ferredoxin reacts with the mercurial sulfhydryl reagent CMB in neutral buffered solution. 5 Slightly more than the theoretical value of 24 moles of CMB reacts per mole of protein. However, native ferredoxin does not react with the sulfhydryl reagent DTNB under these conditions, and there is only a slight reaction in the presence of 6.4 M urea. In 4 M guanidine hydrochloride, under either anaerobic or aerobic conditions, approximately 14 moles of DTNB react per mole of ferredoxin. This reaction is due exclusively to the inorganic sulfide in ferredoxin. The reaction of both the inorganic sulfide and the cysteine residues in ferredoxin with DTNB occurs in the presence of 4 M guanidine hydrochloride and EDTA under anaerobic conditions. 19R.Malkin and J. C. Rabinowitz,Biochemistry6, 3880 (1967).
[39] Purification of Nitrogenase from
Clostridium pasteurianum By LEONAaDE. MORTENSON Nitrogenase from Clostridium pasteurianum is an anionic protein complex, and because of this the first successful purification involved adsorption on cationic materials, la The two most successful steps were selective 1L.E. Mortenson, Biochim.Bioplqs.Acta 127, 18 (1966). 2L. E. Mortenson, J. A. Morris, and D. Y. Jeng, Biochim. Biophys. Acta 141, 516 (1967).