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The C- and N-terminal amino acid sequences of Clostridium pasteurianum ferredoxin
AHCHIVES
OF
BIOCHEMISTRY
The
AND
C- and
BIOPHYSICS
N-Terminal
Clostridium M. TANAKA, From
the
Department
Biochemistry
(196-k)
Amino
Acid
pasteurianom
T. NAKASHIMA, of
570-574
106,
Received
Biophysics, December
of
Ferredoxin’
H. F. MOWER and
Sequences
AND K. T. YASUNOBU
University
of
Hawaii,
Honolulu,
Hawaii
12, 1963
Clostridium pasteurianum ferredoxin is a protein consisting of a single polypeptide chain. The amino-terminal amino acid sequences have been determined to be AlaTyr-Lysfrom the tryptic and chymotryptic peptides, and alanine was shown to be the N-terminal amino acid by dinitrophenylation. The carboxyl-terminal amino acid sequences have been shown to be -Val-Ser-Glu by the action of carboxypeptidase A on performic acid oxidized ferredoxin. The results indicate that the ferredoxin polypeptide contains one tvrosine, lysine, and phenylalanine with a molecular weight of about _ 5800.
Ferredoxin was isolated from Clostridium pasteurianum by Mortenson et al. (I), and similar ferredoxins were obtained from other anaerobic microorganisms by Valentine (2)) Buchanan (3), and Arnon (4). The material from Cl. pasteurianum was shown to be a low molecular weight protein (58OG6000) and to contain five nonheme iron atoms and an equal number of labile sulfide ions (5). The substance functions as a redox electron carrier of unusually low potential (-413 mV.) (5) and plays a key role in a diverse number of oxidation-reduction reactions in bacterial extracts. The bacterial ferredoxins are related to photosynthetic pyridine nucleotide reductase as studied by Hill (6), San Pietro (7), and Arnon (4). The two substancesare interchangeable in certain enzyme reactions and each possessequal molar amounts of iron and sulfide atoms. These substances differ in such properties as molecular weight, amino acid composition, amount of iron and 1 A preliminary report of this work was presented at the Symposium on Ferredoxins and Other Non-Heme Iron Chelates held in association with the Pacific Slope Biochemical Conference; August 1963, Honolulu, Hawaii. Similar results were also reported at that time by A. Tsugita, University of Osaka.
sulfide atoms, and in spectra. They represent a new type of iron redox chelate of unknown structure. We report here our first results in the determination of the complete amino acid sequence of Cl. pasteurianum ferredoxin. MATERIALS
ISOLATION
METHODS
Clostridium pasteurianum was grown according to the method of Carnahan and Castle (9) except that ammonium sulfate (1 g. per liter) was added as a mtrogen source. The cells were dried and stored at 0°C. (8). In a typical preparation2 2 kg. of dried cells were autolyzed in 200 g. batches by suspension in 2.5 liters of water at room temperature. The suspension was stirred vigorously for 1 hour and cooled to 5”; an equal volume of cold acetone was then added and the mixture was centrifuged at 30009 for 20 minutes. The supernatant was then stirred for 15 minutes at 5” with 25 g. of DEAE-cellulose which had previously been equilibrated with 1.0 M tris buffer pH 7.3 and washed free of tris with water. The suspension was filtered and the yellow ferredoxin free filtrate discarded. The process was repeated ten times. After the 7th extraction, it was necessary to treat the filtrate with a second 5-gm. quantity of DEAE-cellulose. * This suggested (personal
570
AND
OF FERREDOXIN~
method of preparation is based upon procedure devised by L. E. Mortenson communication).
a
TERMINAL
AMINO
SCID
The two batches of DEAE-cellulose were combined and washed with cold water (500 ml.). Ferredoxin was eluted by suspending the DEAE in 300 ml. of 0.1 M tris buffer pH 7.3, 0.4 Af N&l. The suspension was filtered. A total of three extractions was necessary. ilmmonium sulfate was added to the combined filtrates to 66:/, saturation and the misture centrifuged. The precipitate was saved for recovery of ferredoxin. Ammonium sulfate was added to the supernatant to 90yc saturation and the mixture centrifuged. The supernatant was discarded and the residue dissolved in 600 ml. of water. The ferredoxin was then absorbed on a series of DEAE columns. Each column, 12 X 2.3 cm., was loaded until the dark band of ferredoxin extended 5, of the length of the column. Three such columns were required. Each column was washed with 500 ml. water, and 1.0 liter 0.1 M pH i.3 tris butfer. The ferredoxin was recovered from the column by gradient elution; the reservoir contained 2 lit,ers of 0.1 &I tris pH 7.3,0.2 M SaCl, and the mixing chamber contained 500 ml. 0.1 M buffer pH 7.3. Twenty-ml. fractions were collected. The purity of ferredoxin was determined by the ratio of ultraviolet absorption at 390 and 280 mM. Samples with a ratio of 0.79 or greater were pooled and dialyzed. Ferredoxin was recovered from the 6652 ammonium sulfate precipitate by an identical procedure. In this case, the nlaterial from the DEAE column had a 390/280 ratio of 0.76a.Z and was dialyzed and reapplied to a second group of columns to raise the ratio to 0.79-0.82. The conbined yield equaled 1.4 g. about 60:; recovery of the ferredosin from the dried cells. This material had a 390/280 ratio of 0.80 and was judged to be about 90”; pure.
I’IZEPAK.~TION
OF 1~0s SULFIDE-FREE FERREDOXIK
In order to remove iron and sulfide ions from intact ferredoxin, trichloroacetic acid treatment was used. For this experiment, 903 mg. of ferredoxin was dissolved in 40 ml. of water. Then, 20 ml. of 157; trichloroacetic acid was added and the mixture was allowed to stand for 1 hour at -1”. The mixture was then centrifuged, and the precipitate was washed with water and centrifuged again to obtain the precipitated ferredosin. The precipitate was then washed with 40 ml. of ethanol and 40 ml. of ether several times and dried in WKUO.
P~{EPARSTION
OF hRF0~541c FERREDOXIS
ACID-OXIDIZED
Due to the possibility of the formation of disulfide bonds from the oxidation of the sulfhydryl groups after the removal of iron and sulfide, the ferredoxin was oxidized with performic acid. For
SEQIJENCEO
571
FERREDOXIN
this step, 750 mg. of iron-sulfide-free ferredoxin was dissolved in 25 ml. of formic acid. To this solution was added performic acid produced by mixing 45 ml. of formic acid with 5 ml. of 3OojG hydrogen peroxide. The mixture was allowed to react for four hours at -10” and then lyophlilized. RESI-LTS
AMINO-TERMINAL
AMINO
ACID ANALYSIS
This determination was carried out according to the procedure described by Frankel-Conrat (9). For this experiment, 0.4 pnloles of the intact ferredoxin was utilized. After the usual dinitrophenylation, acid hydrolysis, and chronlatography steps, the nature of the Dn’P-amino acid was investigated by the usual two-dimensional chronlatographic procedure. The only DNPamino acid present in measurable amounts was DNP-alanine. After correction for destruction, 0.2 residue of DNP-alanine was present per mole of ferredoxin. The results are so low that it was difficult to determine whether alanine was the amino-terminal amino acid of ferredoxin. H~DROLYSIH OF IRON SULFIDE-FREE, OXIDIZED FERREDOXIN BY TRYPSIP~
In order to obtain a peptide fragment from the amino-terminal portion of the molecule, trypsin was used to hydrolyze the iron sulfide-free oxidized ferredoxin. Icor the experiment, 3.1 JIoles of the TCA-treated, oxidized ferredoxin was dissolved in 1.8 ml. of 0.2 ~11K-ethyl morpholine-HCl buffer, pH 8.0. Then, 0.36 lug. of trypsin which had been treated with 0.06 N HCl at 27” for 24 hours was added, and the reaction was allowed to proceed for 24 hours at 27”. At the end of the experinlent, the pH of the digest was lowered to 3.1 by t’he addition of HCl and the hydrolyzate was evaporated to dryness. The powder mas then dissolved in 1.0 ml. of 0.2 11 pyridine-acetic acid buffer pH 3.1. The sample was applied to a column (1 X 40 cm) of Dowex 50 X-2 which had been equilibrated with the starting buffer. Gradient elution was used and the reservoir contained 500 1111. of 2.0 JU pyridine-acetic acid buffer, pH 5.0, and the mixing chamber contained 500 ml. of 0.2 M pyridine-acetic acid buffer, pH 3.1. The chromatogram ob-
572
TANAKA,
NAKASHIMA,
MOWER
tained from the experiment is shown in Fig. 1. Two peptides in equimolar amounts were obtained. This suggests the presence of only one lysine in the peptide chain. After combining the various appropriate fractions, peptides T-l and T-2 were then analyzed. After hydrolysis with 6 N HCI for 24 hours, the peptides were analyzed quantitatively in the automatic amino acid analyzer (10). Peptide T-l showed that it contained the major fraction of the amino acids of ferredoxin. Peptide T-2 showed that it contained alanine, tyrosine, and lysine in a 1: 1: 1 ratio. The sequence of the peptide was investigated with the subtractive Edman procedure (11). In step 1 of this procedure, alanine disappeared quantitatively, and the residue after acid hydrolysis indicated the presence of tyrosine and alanine in a 1: 1 ratio. In the second step, tyrosine disappeared and the residue after acid hydrolysis proved to be lysine. Thus, the sequence of the peptide is Ala-Tyr-Lys. The Rf of the peptide was 0.19 when chromatographed in the solvent system butanol-acetic acid-water (4: 1: 5). The peptide was basic when checked by high voltage electrophoresis using pyridine-acetic acid buffer, pH 6.5. Since there is I
1
FRACTION
NUMBER
1. Chromatography of tryptic digest. 3.1 moles of digest was applied to a 1 X 40 cm. column of Dowex 50 X-S. Gradient elution with pyridineacetic acid buffer (see text) was used. FIG.
AND
YASUNOBU
only one lysine residue in the protein, obviously this peptide was obtained from the amino-terminal portion of ferredoxin. HYDROLYSIS OF TCA-TREATED, OXIDIZED FERREDOXIN BY CHYMOTRYPSIN For the experiment, 34.5 mg. of oxidized ferredoxin was dissolved in 20 ml. of 0.2 M N-ethyl morpholine-HCl buffer, pH 7.8. Then 4 mg. of chymotrypsin was added at 0, 8, and 24 hours, during which times the temperature was maintained at 29”, and the reaction was allowed to proceed for 35 hours. The pH of the reaction mixture was then adjusted to pH 3.0 by the addition of HCl, and lyophilized. The sample was dissolved in 2.0 ml of 0.2 M pyridine-acetic acid buffer, pH 3.1, and applied to a column (1.1 X 56.5 cm.) of Dowex 50 X-2 which had been equilibrated with the same buffer. Forty-two fractions of 2.0 ml. were collected with the starting buffer, and gradient elution was used thereafter. In the latter step, the reservoir contained 500 ml. of 2.0 N pyridine-acetic acid buffer, pH 5, while the mixing chamber contained 500 ml. of 0.2 N pyridine-acetic acid buffer, pH 3.1. The chromatogram that was obtained is shown in Fig. 2. Peptide C-2, which was obtained in 90 % yield, was hydrolyzed and analyzed in the automatic amino acid analyzer. The amino acids alanine and tyrosine were present in a 1: 1 ratio. In order to determine the amino acid sequence of the peptide, the subtractive Edman procedure was utilized. In the first step, alanine disappeared and the residue after acid hydrolysis when analyzed in the automatic amino acid analyzer showed the presence of tyrosine only. Furthermore, the Rf of the peptide in butanol: HOAc : water (4: 1: 5) was 0.79. The peptide behaved as a neutral peptide when analyzed with high-voltage paper electrophoresis with pyridine-acetic acid buffer, pH 6.5. Thus C-2 is the peptide from the amino-terminus of the molecule; this cleavage is expected since chymotrypsin hydrolyzes proteins on the carboxyl side of tyrosine. The C-l peptide could be separated by high-voltage paper electrophoresis into two acidic peptides. These peptides contain the remaining amino acids of the ferredoxin peptide.
TERMIN4L
AMINO
ACID
FRACTION
SEQUENCE0
573
FERREDOXIN
NO.
FIG. 2. Chromatography of chymotryptic digest. 50 pmoles of digest was applied to a 1.1 X 56.5 cm. column of Dowex 50 X-8. Stepwise and gradient elution with pyridineacetic acid buffer (see text) were used as indicated in figure. C-TERMIEAL
AMINO ACID ANALYSIS CARBOXYPEPTIDASE
WITH
In order to complete our proof that ferredoxin is a single polypeptide chain, a Cterminal amino acid analysis was made with carboxypeptidase. For the experiment, 6.0 mg. of TCA-treated, oxidized ferredoxin was dissolved in 3.0 ml. of 0.2 M N-ethyl morpholine buffer pH 8.0 and was incubated with 0.12 mg. of carboxypeptidase A. Aliquots of O.l-PMole were removed at the tinle intervals shown in Fig. 3. They were then absorbed to 50 mg. of Dowex 50 X-8 in the acid fonn. The resin was washed thoroughly and the amino acids were eluted by the addition of 0.5 ml. of 4 N PL’H,OH. After filtration the supernatant was evaporated to dryness, redissolved in water, and again evaporated to dryness. This operation was repeated several times in order to remove ammonia. The entire sample was then analyzed in the automatic amino acid analyzer; the results obtained are summarized in Fig. 3. Since serine, glutamine, and asparagine are eluted at the samepoint in the chromatogram, it was necessary to carry out additional experiments to clarify the nature of the pentultimate amino acid. For this purpose, acid hydrolysis with 6 N HCl was carried out for 24 hours at 10.5’. The sample
SER.
0
5 TIME
1 IO
(Hours)
FIG. 3. Kinetics of hydrolysis by carboxypeptidase. 1.0 moles of TCA-oxidized ferredoxin and 0.12 mg. carboxypeptidase in a total volume of 3.0 ml. of 0.2 M N-ethylmorpholine-HCl buffer was allowed to react at 28°C. 0.1 PM aliquots were removed at the time intervals indicated. The aliquots were absorbed and eluted from Dowex 50 X-8 and analyzed in the automatic amino acid analyzer.
was then analyzed in the automatic amino acid analyzer. The results clearly demonstrated that serine is the pentultimate amino acid.
574
TANAKA,
NAKASHIMA,
DISCUSSION
The amino acid composition of bacterial ferredoxins has been recently published (4). In these studies it was assumed that the molecular weight was lO,OOO-12,000. Additional studies (6) have shown that a reevaluation of the molecular weight indicates that the molecular weight is about 5800. In the present study, the amino acid sequences in the vicinity of the N-terminal amino acids have been determined. The presence of only one lysine, tyrosine and phenylalanine in the peptide chain could be demonstrated in our work. This indicates that ferredoxin has a molecular weight and amino acid composition one-half of that originally reported by Buchanan et al. (4). Due to the presence of iron and cysteine residues in the protein moiety, it was necessary to remove the iron by trichloroacetic acid treatment and to oxidize the cysteine residues by performic acid. Determination of the N-terminal amino acid residue by the dinitrofluorobenzene method (9) on intact ferredoxin gave low yields of DNP-ala which made it difficult to determine the source of this amino acid. However, trypsin digestion of the TCAtreated, oxidized ferredoxin followed by Dowex 50 chromatography yielded AlaTyr-Lys, and chymotrypsin digestion followed by Dowex 50 chromatography yielded Ala-Tyr. The amino acid sequences were determined on these peptides and showed alanine to be N-terminal amino acid. The C-terminal amino acids were determined to be Val-Ser-GluCOOH by the use of carboxypeptidase. The carboxypeptidase experiments with the iron-removed, performic acid-oxidized ferredoxin and intact ferredoxin (12) gave the same results, suggesting that the C-terminal portion of the molecule is susceptible to hydrolysis in both forms of ferredoxin. The enzymes chymotrypsin and trypsin, which have been so useful for protein structural studies, have limited use in the case of ferredoxin due to the lack of linkages susceptible to these enzymes. Studies are in progress to determine the complete amino acid sequence, and the present results have fixed the position of six of the fifty (4) amino acid residues in ferre-
MOWER
AND
YASUNOBU
doxin. The results also confirm the fact that ferredoxin consists of a single polypeptide chain of about 5800 molecular weight. Note added in proof. Further studies have shown that the C-terminal sequence is -ValGlu-GluCOOH. The previous result appears to be due to a small amount of cleavage of the protein by acid during the preparation of iron-free ferredoxin. ACKNOWLEDGMENTS We wish to acknowledge the excellent technical assistance of Miss Ann Benson, This work was supported in part by National Science Foundation grants GB-1363, GB-884, and G-19085. REFERENCES 1. MORTENSON, L. E., VALENTINE, R. C., AND CARNAHAN, J. E., Biochem. Biophys. Res. Commun. 7, 448 (1962). 2. VALENTINE, R. C., JACKSON, R. L., AND WOLFE, R. S., Biochem. Biophys. Res. Commun. 7, 453 (1962). 3. VALENTINE, R. C., BRILL, W. J., AND SAYERS, R. D., Biochem. Biophys. Res. Commun. 12, 315 (1963). 4. BUCHANAN, R. B., LOVENBERG, W., AND RABINOWITZ, J. C., Proc. Natl. Acad. Sci. 49, 345 (1963). 5. TAGAWA, K., AND ARNON, D. I., Nature 196, 537 (1962). 6. BUCHANAN, R. B., AND S~HACHMAN, H., unpublished experiments. 7. SAN PIETRO, A., MCELROY, W. D., AND GLASS, B., Eds., “Light and Life,” p. 631, Johns Hopkins University Press, Baltimore, Maryland, 1961. 8. CARNAHAN, J. E., AND CASTLE, J. E., J. Bacterial. 76, 121 (1958). 9. FRANKEL-CONRAT, H., HARRIS, J. I., AND LEVY, A. L., in “Methods of Biochemical Analysis” (D. Glick, ed.), Vol. II, p. 359. Wiley (Interscience), New York, 1958. 10. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem. 30, 1190 (1958). 11. KONIGSBERG, W., AND HILL, R. J., J. Biol. Chem., 237, 2547 (1962). A., unpublished experiments re12. TSUGITA, ported at the Symposium on Ferredoxins and Other Non-Heme Iron Chelates sponsored by the National Science Foundation and held in conjunction with the Pacific Slope Biochemical Conference, August 1963, Honolulu, Hawaii.
OF
BIOCHEMISTRY
The
AND
C- and
BIOPHYSICS
N-Terminal
Clostridium M. TANAKA, From
the
Department
Biochemistry
(196-k)
Amino
Acid
pasteurianom
T. NAKASHIMA, of
570-574
106,
Received
Biophysics, December
of
Ferredoxin’
H. F. MOWER and
Sequences
AND K. T. YASUNOBU
University
of
Hawaii,
Honolulu,
Hawaii
12, 1963
Clostridium pasteurianum ferredoxin is a protein consisting of a single polypeptide chain. The amino-terminal amino acid sequences have been determined to be AlaTyr-Lysfrom the tryptic and chymotryptic peptides, and alanine was shown to be the N-terminal amino acid by dinitrophenylation. The carboxyl-terminal amino acid sequences have been shown to be -Val-Ser-Glu by the action of carboxypeptidase A on performic acid oxidized ferredoxin. The results indicate that the ferredoxin polypeptide contains one tvrosine, lysine, and phenylalanine with a molecular weight of about _ 5800.
Ferredoxin was isolated from Clostridium pasteurianum by Mortenson et al. (I), and similar ferredoxins were obtained from other anaerobic microorganisms by Valentine (2)) Buchanan (3), and Arnon (4). The material from Cl. pasteurianum was shown to be a low molecular weight protein (58OG6000) and to contain five nonheme iron atoms and an equal number of labile sulfide ions (5). The substance functions as a redox electron carrier of unusually low potential (-413 mV.) (5) and plays a key role in a diverse number of oxidation-reduction reactions in bacterial extracts. The bacterial ferredoxins are related to photosynthetic pyridine nucleotide reductase as studied by Hill (6), San Pietro (7), and Arnon (4). The two substancesare interchangeable in certain enzyme reactions and each possessequal molar amounts of iron and sulfide atoms. These substances differ in such properties as molecular weight, amino acid composition, amount of iron and 1 A preliminary report of this work was presented at the Symposium on Ferredoxins and Other Non-Heme Iron Chelates held in association with the Pacific Slope Biochemical Conference; August 1963, Honolulu, Hawaii. Similar results were also reported at that time by A. Tsugita, University of Osaka.
sulfide atoms, and in spectra. They represent a new type of iron redox chelate of unknown structure. We report here our first results in the determination of the complete amino acid sequence of Cl. pasteurianum ferredoxin. MATERIALS
ISOLATION
METHODS
Clostridium pasteurianum was grown according to the method of Carnahan and Castle (9) except that ammonium sulfate (1 g. per liter) was added as a mtrogen source. The cells were dried and stored at 0°C. (8). In a typical preparation2 2 kg. of dried cells were autolyzed in 200 g. batches by suspension in 2.5 liters of water at room temperature. The suspension was stirred vigorously for 1 hour and cooled to 5”; an equal volume of cold acetone was then added and the mixture was centrifuged at 30009 for 20 minutes. The supernatant was then stirred for 15 minutes at 5” with 25 g. of DEAE-cellulose which had previously been equilibrated with 1.0 M tris buffer pH 7.3 and washed free of tris with water. The suspension was filtered and the yellow ferredoxin free filtrate discarded. The process was repeated ten times. After the 7th extraction, it was necessary to treat the filtrate with a second 5-gm. quantity of DEAE-cellulose. * This suggested (personal
570
AND
OF FERREDOXIN~
method of preparation is based upon procedure devised by L. E. Mortenson communication).
a
TERMINAL
AMINO
SCID
The two batches of DEAE-cellulose were combined and washed with cold water (500 ml.). Ferredoxin was eluted by suspending the DEAE in 300 ml. of 0.1 M tris buffer pH 7.3, 0.4 Af N&l. The suspension was filtered. A total of three extractions was necessary. ilmmonium sulfate was added to the combined filtrates to 66:/, saturation and the misture centrifuged. The precipitate was saved for recovery of ferredoxin. Ammonium sulfate was added to the supernatant to 90yc saturation and the mixture centrifuged. The supernatant was discarded and the residue dissolved in 600 ml. of water. The ferredoxin was then absorbed on a series of DEAE columns. Each column, 12 X 2.3 cm., was loaded until the dark band of ferredoxin extended 5, of the length of the column. Three such columns were required. Each column was washed with 500 ml. water, and 1.0 liter 0.1 M pH i.3 tris butfer. The ferredoxin was recovered from the column by gradient elution; the reservoir contained 2 lit,ers of 0.1 &I tris pH 7.3,0.2 M SaCl, and the mixing chamber contained 500 ml. 0.1 M buffer pH 7.3. Twenty-ml. fractions were collected. The purity of ferredoxin was determined by the ratio of ultraviolet absorption at 390 and 280 mM. Samples with a ratio of 0.79 or greater were pooled and dialyzed. Ferredoxin was recovered from the 6652 ammonium sulfate precipitate by an identical procedure. In this case, the nlaterial from the DEAE column had a 390/280 ratio of 0.76a.Z and was dialyzed and reapplied to a second group of columns to raise the ratio to 0.79-0.82. The conbined yield equaled 1.4 g. about 60:; recovery of the ferredosin from the dried cells. This material had a 390/280 ratio of 0.80 and was judged to be about 90”; pure.
I’IZEPAK.~TION
OF 1~0s SULFIDE-FREE FERREDOXIK
In order to remove iron and sulfide ions from intact ferredoxin, trichloroacetic acid treatment was used. For this experiment, 903 mg. of ferredoxin was dissolved in 40 ml. of water. Then, 20 ml. of 157; trichloroacetic acid was added and the mixture was allowed to stand for 1 hour at -1”. The mixture was then centrifuged, and the precipitate was washed with water and centrifuged again to obtain the precipitated ferredosin. The precipitate was then washed with 40 ml. of ethanol and 40 ml. of ether several times and dried in WKUO.
P~{EPARSTION
OF hRF0~541c FERREDOXIS
ACID-OXIDIZED
Due to the possibility of the formation of disulfide bonds from the oxidation of the sulfhydryl groups after the removal of iron and sulfide, the ferredoxin was oxidized with performic acid. For
SEQIJENCEO
571
FERREDOXIN
this step, 750 mg. of iron-sulfide-free ferredoxin was dissolved in 25 ml. of formic acid. To this solution was added performic acid produced by mixing 45 ml. of formic acid with 5 ml. of 3OojG hydrogen peroxide. The mixture was allowed to react for four hours at -10” and then lyophlilized. RESI-LTS
AMINO-TERMINAL
AMINO
ACID ANALYSIS
This determination was carried out according to the procedure described by Frankel-Conrat (9). For this experiment, 0.4 pnloles of the intact ferredoxin was utilized. After the usual dinitrophenylation, acid hydrolysis, and chronlatography steps, the nature of the Dn’P-amino acid was investigated by the usual two-dimensional chronlatographic procedure. The only DNPamino acid present in measurable amounts was DNP-alanine. After correction for destruction, 0.2 residue of DNP-alanine was present per mole of ferredoxin. The results are so low that it was difficult to determine whether alanine was the amino-terminal amino acid of ferredoxin. H~DROLYSIH OF IRON SULFIDE-FREE, OXIDIZED FERREDOXIN BY TRYPSIP~
In order to obtain a peptide fragment from the amino-terminal portion of the molecule, trypsin was used to hydrolyze the iron sulfide-free oxidized ferredoxin. Icor the experiment, 3.1 JIoles of the TCA-treated, oxidized ferredoxin was dissolved in 1.8 ml. of 0.2 ~11K-ethyl morpholine-HCl buffer, pH 8.0. Then, 0.36 lug. of trypsin which had been treated with 0.06 N HCl at 27” for 24 hours was added, and the reaction was allowed to proceed for 24 hours at 27”. At the end of the experinlent, the pH of the digest was lowered to 3.1 by t’he addition of HCl and the hydrolyzate was evaporated to dryness. The powder mas then dissolved in 1.0 ml. of 0.2 11 pyridine-acetic acid buffer pH 3.1. The sample was applied to a column (1 X 40 cm) of Dowex 50 X-2 which had been equilibrated with the starting buffer. Gradient elution was used and the reservoir contained 500 1111. of 2.0 JU pyridine-acetic acid buffer, pH 5.0, and the mixing chamber contained 500 ml. of 0.2 M pyridine-acetic acid buffer, pH 3.1. The chromatogram ob-
572
TANAKA,
NAKASHIMA,
MOWER
tained from the experiment is shown in Fig. 1. Two peptides in equimolar amounts were obtained. This suggests the presence of only one lysine in the peptide chain. After combining the various appropriate fractions, peptides T-l and T-2 were then analyzed. After hydrolysis with 6 N HCI for 24 hours, the peptides were analyzed quantitatively in the automatic amino acid analyzer (10). Peptide T-l showed that it contained the major fraction of the amino acids of ferredoxin. Peptide T-2 showed that it contained alanine, tyrosine, and lysine in a 1: 1: 1 ratio. The sequence of the peptide was investigated with the subtractive Edman procedure (11). In step 1 of this procedure, alanine disappeared quantitatively, and the residue after acid hydrolysis indicated the presence of tyrosine and alanine in a 1: 1 ratio. In the second step, tyrosine disappeared and the residue after acid hydrolysis proved to be lysine. Thus, the sequence of the peptide is Ala-Tyr-Lys. The Rf of the peptide was 0.19 when chromatographed in the solvent system butanol-acetic acid-water (4: 1: 5). The peptide was basic when checked by high voltage electrophoresis using pyridine-acetic acid buffer, pH 6.5. Since there is I
1
FRACTION
NUMBER
1. Chromatography of tryptic digest. 3.1 moles of digest was applied to a 1 X 40 cm. column of Dowex 50 X-S. Gradient elution with pyridineacetic acid buffer (see text) was used. FIG.
AND
YASUNOBU
only one lysine residue in the protein, obviously this peptide was obtained from the amino-terminal portion of ferredoxin. HYDROLYSIS OF TCA-TREATED, OXIDIZED FERREDOXIN BY CHYMOTRYPSIN For the experiment, 34.5 mg. of oxidized ferredoxin was dissolved in 20 ml. of 0.2 M N-ethyl morpholine-HCl buffer, pH 7.8. Then 4 mg. of chymotrypsin was added at 0, 8, and 24 hours, during which times the temperature was maintained at 29”, and the reaction was allowed to proceed for 35 hours. The pH of the reaction mixture was then adjusted to pH 3.0 by the addition of HCl, and lyophilized. The sample was dissolved in 2.0 ml of 0.2 M pyridine-acetic acid buffer, pH 3.1, and applied to a column (1.1 X 56.5 cm.) of Dowex 50 X-2 which had been equilibrated with the same buffer. Forty-two fractions of 2.0 ml. were collected with the starting buffer, and gradient elution was used thereafter. In the latter step, the reservoir contained 500 ml. of 2.0 N pyridine-acetic acid buffer, pH 5, while the mixing chamber contained 500 ml. of 0.2 N pyridine-acetic acid buffer, pH 3.1. The chromatogram that was obtained is shown in Fig. 2. Peptide C-2, which was obtained in 90 % yield, was hydrolyzed and analyzed in the automatic amino acid analyzer. The amino acids alanine and tyrosine were present in a 1: 1 ratio. In order to determine the amino acid sequence of the peptide, the subtractive Edman procedure was utilized. In the first step, alanine disappeared and the residue after acid hydrolysis when analyzed in the automatic amino acid analyzer showed the presence of tyrosine only. Furthermore, the Rf of the peptide in butanol: HOAc : water (4: 1: 5) was 0.79. The peptide behaved as a neutral peptide when analyzed with high-voltage paper electrophoresis with pyridine-acetic acid buffer, pH 6.5. Thus C-2 is the peptide from the amino-terminus of the molecule; this cleavage is expected since chymotrypsin hydrolyzes proteins on the carboxyl side of tyrosine. The C-l peptide could be separated by high-voltage paper electrophoresis into two acidic peptides. These peptides contain the remaining amino acids of the ferredoxin peptide.
TERMIN4L
AMINO
ACID
FRACTION
SEQUENCE0
573
FERREDOXIN
NO.
FIG. 2. Chromatography of chymotryptic digest. 50 pmoles of digest was applied to a 1.1 X 56.5 cm. column of Dowex 50 X-8. Stepwise and gradient elution with pyridineacetic acid buffer (see text) were used as indicated in figure. C-TERMIEAL
AMINO ACID ANALYSIS CARBOXYPEPTIDASE
WITH
In order to complete our proof that ferredoxin is a single polypeptide chain, a Cterminal amino acid analysis was made with carboxypeptidase. For the experiment, 6.0 mg. of TCA-treated, oxidized ferredoxin was dissolved in 3.0 ml. of 0.2 M N-ethyl morpholine buffer pH 8.0 and was incubated with 0.12 mg. of carboxypeptidase A. Aliquots of O.l-PMole were removed at the tinle intervals shown in Fig. 3. They were then absorbed to 50 mg. of Dowex 50 X-8 in the acid fonn. The resin was washed thoroughly and the amino acids were eluted by the addition of 0.5 ml. of 4 N PL’H,OH. After filtration the supernatant was evaporated to dryness, redissolved in water, and again evaporated to dryness. This operation was repeated several times in order to remove ammonia. The entire sample was then analyzed in the automatic amino acid analyzer; the results obtained are summarized in Fig. 3. Since serine, glutamine, and asparagine are eluted at the samepoint in the chromatogram, it was necessary to carry out additional experiments to clarify the nature of the pentultimate amino acid. For this purpose, acid hydrolysis with 6 N HCl was carried out for 24 hours at 10.5’. The sample
SER.
0
5 TIME
1 IO
(Hours)
FIG. 3. Kinetics of hydrolysis by carboxypeptidase. 1.0 moles of TCA-oxidized ferredoxin and 0.12 mg. carboxypeptidase in a total volume of 3.0 ml. of 0.2 M N-ethylmorpholine-HCl buffer was allowed to react at 28°C. 0.1 PM aliquots were removed at the time intervals indicated. The aliquots were absorbed and eluted from Dowex 50 X-8 and analyzed in the automatic amino acid analyzer.
was then analyzed in the automatic amino acid analyzer. The results clearly demonstrated that serine is the pentultimate amino acid.
574
TANAKA,
NAKASHIMA,
DISCUSSION
The amino acid composition of bacterial ferredoxins has been recently published (4). In these studies it was assumed that the molecular weight was lO,OOO-12,000. Additional studies (6) have shown that a reevaluation of the molecular weight indicates that the molecular weight is about 5800. In the present study, the amino acid sequences in the vicinity of the N-terminal amino acids have been determined. The presence of only one lysine, tyrosine and phenylalanine in the peptide chain could be demonstrated in our work. This indicates that ferredoxin has a molecular weight and amino acid composition one-half of that originally reported by Buchanan et al. (4). Due to the presence of iron and cysteine residues in the protein moiety, it was necessary to remove the iron by trichloroacetic acid treatment and to oxidize the cysteine residues by performic acid. Determination of the N-terminal amino acid residue by the dinitrofluorobenzene method (9) on intact ferredoxin gave low yields of DNP-ala which made it difficult to determine the source of this amino acid. However, trypsin digestion of the TCAtreated, oxidized ferredoxin followed by Dowex 50 chromatography yielded AlaTyr-Lys, and chymotrypsin digestion followed by Dowex 50 chromatography yielded Ala-Tyr. The amino acid sequences were determined on these peptides and showed alanine to be N-terminal amino acid. The C-terminal amino acids were determined to be Val-Ser-GluCOOH by the use of carboxypeptidase. The carboxypeptidase experiments with the iron-removed, performic acid-oxidized ferredoxin and intact ferredoxin (12) gave the same results, suggesting that the C-terminal portion of the molecule is susceptible to hydrolysis in both forms of ferredoxin. The enzymes chymotrypsin and trypsin, which have been so useful for protein structural studies, have limited use in the case of ferredoxin due to the lack of linkages susceptible to these enzymes. Studies are in progress to determine the complete amino acid sequence, and the present results have fixed the position of six of the fifty (4) amino acid residues in ferre-
MOWER
AND
YASUNOBU
doxin. The results also confirm the fact that ferredoxin consists of a single polypeptide chain of about 5800 molecular weight. Note added in proof. Further studies have shown that the C-terminal sequence is -ValGlu-GluCOOH. The previous result appears to be due to a small amount of cleavage of the protein by acid during the preparation of iron-free ferredoxin. ACKNOWLEDGMENTS We wish to acknowledge the excellent technical assistance of Miss Ann Benson, This work was supported in part by National Science Foundation grants GB-1363, GB-884, and G-19085. REFERENCES 1. MORTENSON, L. E., VALENTINE, R. C., AND CARNAHAN, J. E., Biochem. Biophys. Res. Commun. 7, 448 (1962). 2. VALENTINE, R. C., JACKSON, R. L., AND WOLFE, R. S., Biochem. Biophys. Res. Commun. 7, 453 (1962). 3. VALENTINE, R. C., BRILL, W. J., AND SAYERS, R. D., Biochem. Biophys. Res. Commun. 12, 315 (1963). 4. BUCHANAN, R. B., LOVENBERG, W., AND RABINOWITZ, J. C., Proc. Natl. Acad. Sci. 49, 345 (1963). 5. TAGAWA, K., AND ARNON, D. I., Nature 196, 537 (1962). 6. BUCHANAN, R. B., AND S~HACHMAN, H., unpublished experiments. 7. SAN PIETRO, A., MCELROY, W. D., AND GLASS, B., Eds., “Light and Life,” p. 631, Johns Hopkins University Press, Baltimore, Maryland, 1961. 8. CARNAHAN, J. E., AND CASTLE, J. E., J. Bacterial. 76, 121 (1958). 9. FRANKEL-CONRAT, H., HARRIS, J. I., AND LEVY, A. L., in “Methods of Biochemical Analysis” (D. Glick, ed.), Vol. II, p. 359. Wiley (Interscience), New York, 1958. 10. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem. 30, 1190 (1958). 11. KONIGSBERG, W., AND HILL, R. J., J. Biol. Chem., 237, 2547 (1962). A., unpublished experiments re12. TSUGITA, ported at the Symposium on Ferredoxins and Other Non-Heme Iron Chelates sponsored by the National Science Foundation and held in conjunction with the Pacific Slope Biochemical Conference, August 1963, Honolulu, Hawaii.