J. Biochem. 83, 1305-1319 (1978)

Amino Acid Sequences of Two Ferredoxins from Pokeweed,

Sadao WAKABAYASHI,* Toshiharu HASE,* Keishiro WADA,* Hiroshi MATSUBARA,* Koichi SUZUKI,** and Shinichi T A K A I C H I * * 1 •Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560, and "Department of Biochemistry, Faculty of Science, Saitama University, Urawa, Saitama 338 Received for publication, November 22, 1977

The amino acid sequences of two ferredoxins isolated from pokeweed, Phytolacca americana, were determined. Tryptic peptides of maleyl-carboxymethyl-ferredoxin I and carboxymethyl-ferredoxin II were prepared and analyzed. The large peptides were further digested with staphylococcal protease and chymotrypsin. Ferredoxins I and II were composed of 96 and 98 amino acid residues, respectively. Though ferredoxin I lacks tryptophan and methionine, ferredoxin II contains both of them. In a comparison of the amino acid sequences with those of other higher plant ferredoxins, ferredoxin I is one residue shorter than others at the carboxyl-terminus and ferredoxin II one longer than others at the amino-terminus. Ferredoxins I and II differ in 23 sites from each other and in 27 to 37 sites from other higher plant ferredoxins. This suggests that duplication of the ferredoxin gene occurred after the divergence of pokeweed from other higher plants. A phylogenetic tree including all other ferredoxins was.constructed.

Chloroplast-type ferredoxins have been isolated from many plants, including blue-green algae, and shown to have homologous amino acid sequences. Their molecular evolution was discussed (1-4). Recently, the existence of two molecular species 1 Present address: Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113. Abbreviations: Cm-ferredoxin, S-carboxymethyl-ferredoxin; TPCK-, tosyl-phenylalaninechloromethylketone; N-terminal, amino-terminal; C-terminal, carboxylterminal; PTH, phenylthiohydantoin derivative; Cmc or Cm-cysteine, S-carboxymethylcysteine.

Vol. 83, No. 5, 1978

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of ferredoxin in one organism has been reported. Two such ferredoxins have been found in bluegreen algae, Aphanothece sacrum (5), Nostoc sp. MAC (6), Nostoc muscorum (7), and Nostoc verrucosum (8), in horsetails, Equisctum telmateia (9), and Equisetum arvense (10), and in higher plants, Pisum sativum (11,12), corn (13), and wheat (Akulova, E.A., personal communication, 1975). Three species of the genus Phytolacca, P. americana, P. esculenta, and P. japonica, were shown to have two or three molecular species of ferredoxin which could be separated on gel electrophoresis (14, 15). One component from each species of Photolacca showed an identical electrophoretic mobility, but

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Phytolacca americana

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S. WAKABAYASHI et al.

EXPERIMENTAL Materials—P. americana ferredoxins: P. americana ferredoxins were isolated as described previously (14). Two ferredoxins were separated on a DEAE-cellulose column with a linear gradient from 0.25 M to 0.45 M NaCl in 0.05 M Tris-HCI buffer, pH 7.5. The absorbance ratios, Ait0/At7t, were 0.68 and 0.47 for ferredoxins I and II, respectively. Enzymes and chemicals: All reagents and enzymes used in this experiment were as described in previous papers (16,17). Methods—Preparation of S-carboxymethylferredoxin: S-carboxymethyl (Cm)-ferredoxins w e r e prepared according to Crestfield et al. (18). Preparation and digestion with trypsin of maleyl-carboxymethyl-ferredoxin I and separation of pep tides: About 55 mg of Cm-ferredoxin I was dissolved in 2 ml of 0.2 M sodium bicarbonate buffer, pH 9.0, and 80 mg of finely powdered maleic anhydride (a 40-fold molar excess) was added to the solution with stirring over a period of 1 h at room temperature. The pH of the reaction mixture was maintained at about 9.0 with 1 M NaOH. Then a little maleic anhydride was added to the mixture to adjust the pH to 8.0 for digestion with trypsin. After 30 min, 1 mg of tosyl-phenylalanine chloromethyl ketone (TPCK)trypsin was added and digestion was carried out for 2 h at 40°C. The digest was chromatographed on a Bio-Gel P-10 column (2 x 180 cm) with 0.2 M NH 4 HCO,-NH 4 OH buffer, pH 9.0. Fractions of 2.8 ml were collected, monitoring the absorbancies at 220 and 280 nm. Demaleylation of peptides: Two peptides obtained from the chromatography on Bio-Gel P-10 were demaleylated, before second enzymatic

digestion, in 5 % C H 3 C 0 0 H and 1 % pyridine (v/v) for 32 h at 37°C (19). Since demaleylation was not complete under these conditions, Peptide MT-2, as described later, was demaleylated again in 30% CHjCOOH for 90 h at room temperature after the second digestion with staphylococcal protease. Digestion of Cm-ferredoxin II with trypsin and separation of peptides: About 25 mg of Cmferredoxin II was digested with 0.5 mg of TPCKtrypsin in 1.5 ml of 0.1 M Tris-HCI buffer, pH 8.0, for 2 h at 40°C. The digest was separated on a Bio-Gel P ^ column (2x180 cm) with 0.2 M NH 4 HCO,-NH 4 OH buffer, pH 9.0. Each fraction (2.2 ml) was monitored at 220 nm and 280 nm, and the fractions containing peptides were further purified by paper electrophoresis at pH 3.6 and/or 6.5 and paper chromatography. Further digestion of large peptides: Two peptides (2 ^mol each) obtained by tryptic digestion of maleyl-Cm-ferredoxin I were further digested separately with 0.15 mg of staphylococcal protease in 1 ml of 0.1 M Tris-HCI, pH 8.0, for 3 h at 40°C. Each digest was chromatographed on a Bio-Gel P-4 column and peptides were further purified as described above. The large peptides derived from tryptic digestion of Cm-ferredoxin II were further digested with chymotrypsin and the resulting peptides were purified by paper electrophoresis at pH 3.6. Amino acid composition and sequence analysis: The amino acid compositions of proteins and peptides were determined with an amino acid analyzer (Beckman, model 120B) after acid hydrolysis, as described previously (16, 17). A manual Edman degradation procedure was applied to Cm-ferredoxins and peptides (0.1 pmol) to determine the amino (N>terminal sequences. Phenylthiohydantoin (PTH) derivatives were identified by thin-layer chromatography on Merck silica gelglass plates using various solvent systems (20, 21). The carboxyl (C)-terminal sequences were determined by digestion with carboxypeptidase A and B (22). Nomenclature: MT- and T- refer to tryptic peptides of maleyl-Cm-ferredoxin and tryptic peptides of Cm-ferredoxin, respectively. C- and S- refer to the peptides derived by second digestion with chymotrypsin and staphylococcal protease, respectively. All values are expressed in moles

/ . Biochem.

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the other components all showed different mobilities (15). From the viewpoint of the molecular evolution of ferredoxin, it is interesting to compare the sequences of ferredoxins of these three species in relation to the two pairs of ferredoxin isolated from two species of horsetail (9, 10). This paper describes the amino acid sequences of two ferredoxins from P. americana and discusses the presence of two molecular species of ferredoxin in one plant. The molecular evolution of chloroplast-type ferredoxin is also discussed.

SEQUENCES OF P. americana FERREDOXINS

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per mole of peptide or protein. RESULTS

TABLE I. Amino acid compositions of carboxymethyl-ferredoxins I and II of P. americana. Ferredoxin I From acid hydrolysate* Lysine Histidine Arginine Cm-cysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Total residues

3.92 1.06 1.06 4.98 11.9 11.5 5.77 10.9 4.03 6.98 8.42 8.87 0.00 3.83 5.88 2.98 2.95

Ferredoxin II

From sequence study 4 1 1 5 12& 12 6 lib

c

4 7 8 9 0 4 6 3 3 0 96

From acid hydrolysate1 2.85 0.86 0.96 4.57 7.97 11.5 7.33 11.6 3.84 7.70 13.8 8.35 0.98 2.10 6.22 4.05 2.02

From sequence study 3 1 1 5 8>> 12 8 12b

c

4 7 13 8 1 2 6 4 2 1 98

* Acid hydrolyses were performed on carboxymethyl-ferredoxins for 24 and 72 h. The values of threonine and serine were obtained by extrapolations to zero time of hydrolysis. Values of valine and isoleucine were of 72 h hydrolysates. b Sum of acid and amide forms. c Not determined. Vol. 83, No. 5, 1978

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Amino Acid Compositions and N- and CTerminal Sequences of Cm-Ferredoxins I and II— The amino acid compositions of Cm-ferredoxins I and II are shown in Table I. For ferredoxin I, these values agreed with those deduced from the sequence study. However, for ferredoxin II, there were minor discrepancies, that is, glycine and alanine were overestimated by one residue each and serine was underestimated in the compositional analysis. From the absorption spectrum it appeared that ferredoxin II contained tryptophan {14), and this was confirmed as described later. The N-terminal sequences were established by

manual Edman degradation, using 0.1 (imo\ each of Cm-ferredoxins I and II, up to 20 residues without any ambiguity. The established sequences were as follows: ferredoxin I, Ala-Thr-Tyr-Lys-ValThr - Leu - Val - Thr - Pro - Ser - Gly - Thr -Gln-Thr-IleAsp-Cmc-Pro-Asp- and ferredoxin II, Ala-AlaSer-Tyr-Lys - Val - Thr - Phe - Val - Thr - Pro - Ser - GlyThr-Asn-Thr-Ile-Thr-Cmc-Pro-. The C-terminal sequences were studied using carboxypeptidase A. Carboxypeptidase A released from Cm-ferredoxin I only valine and isoleucine in equal amounts at any reaction time, and they were later sequenced as -De-Val. From ferredoxin n , carboxypeptidase A released only alanine at 20 min and alanine, threonine, and leucine at 1 h. They were sequenced as -Leu-Thr-Ala as described later. Sequence Studies of Maleyl-Cm-Ferredoxin I—

S. WAKABAYASHI el al.

1308

MT- -S-2

100 50 100 FRACTION NUMBER

FRACTION NUMBER

Fig. 1. Elution pattern of tryptic digest of maleyl-Cmferredoxin I of P. americana. The tryptic digest was chromatographed on a Bio-Gel P-10 (minus 400 mesh) column (2x180 cm) with 0.2 M NH4HCO,-NH4OH buffer, pH 9.0, at a flow rate of 1.5 ml per h. Fractions (2.8 ml each) were monitored by following the absorbances at 220 nm ( ) and 280 nm ( ).

TABLE II. Amino acid compositions of Peptides MT-1 and MT-2 of carboxymethyl-ferredoxin I.

Lysine Histidine Arginine Cm-cysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Total residues Yield (%)

MT-1

MT-2

0.80(1)

2.73(3) 1.06(1)

0.87(1) 2.07(2) 5.04(5) 5.54(6) 2.01(2) 3.17(3) 3.10(3) 2.44(2) 3.83(4) 2.76(3) 1.03(1) 3.74(4) 2.71(3)

3.06(3) 7.10(7) 5.88(6) 3.53(4) 8.16(8) 0.91(1) 5.10(5) 3.90(4) 6.03(6) 2.63(3) 2.13(2) 2.78(3)

40

56

75

67

ISO

Fig. 2. Elution pattern of staphylococcal protease digest of Peptide MT-1 of ferredoxin I. The staphylococcal protease digest was chromatographed on a BioGel P-4 column (2x180 cm) with 0.2 M NH 4 HCO,NH4OH buffer, pH 9.0, at a flow rate of 10 ml per h. Each fraction (2.2 ml) was monitored at 220 nm ( ) and 280 nm ( ).

To follow the experimental processes described below Fig. 4, summarizing the sequence studies, should be consulted. The elution pattern from a Bio-Gel P-10 column of peptides derived by tryptic digestion of maleyl-Cm-ferredoxin I is shown in Fig. 1. Two peptides, MT-1 and MT-2, were obtained without any further purification. The amino acid compositions of these peptides are shown in Table II. Since MT-1 contained one residue of arginine, it was concluded to be the N-terminal half of ferredoxin I. Peptide MT-1 (Residues 1-40)—Only six steps of Edman degradation were carried out. After demaleylation, staphylococcal protease digestion of this peptide (2 ftmol) produced 4 peptides, MT-1S-l to S-4. These peptides were separated by Bio-Gel P-4 column chromatography; the elution pattern is shown in Fig. 2. From the third fraction no peptides was isolated. This might be maleic acid produced by demaleylation, and was not studied further. Peptides MT-l-S-2, MT-1S-3, and MT-l-S-4 in the first and second fractions were further purified by paper electrophoresis. The amino acid compositions of these peptides are listed in Table III. Edman degradation of each J. Biochem.

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00 30

SEQUENCES OF P. amtricana FERREDOX1NS

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TABLE HI. Amino acid compositions of Peptides MT-l-S-1 to MT-l-S-4 of carboxymethyl-ferredoxin-I. MT-l-S-1

Total residues Yield (%) Color reaction Purification

MT-l-S-3

MT-l-S-4

1.03 (1) 1.09 4.03 4.68 0.89 1.03 1.93 1.09

(1) (4) (5) (1) (1) (2) (1)

3.05 1.04 1.87 0.89

(3) (1) (2) (1)

0.95 (1)

1.04 (1)

0.99 (1)

1.04 (1) 1.07 (1) 1.00 (1) 0.92 (1) 2.02 (2)

1.98 (2)

4 25 P

0.99 (1) 1.05 (1) 1.02 (1)

1.96 (2) 0.95 (1)

22 51 P

4 40

PEi

PE,

10 49 P,S PE,

P: Pauli reaction positive. S: Sakaguchi reaction positive. PE t and PE,: Paper electrophoresis at pH 3.6 and 6.5, respectively.

peptide (Peptide MT-l-S-1, 2 steps; MT-l-S-2, 22 steps; MT-l-S-3, 4 steps; and MT-l-S-4, 10 steps) was carried out. Peptide MT-l-S-1, assumed to be the N-terminal region of the original peptide judging from its amino acid composition and Nterminal sequence, seemed to be derived by an unexpected tryptic activity causing cleavage at the C-side of lysine. This was probably due to contamination by trypsin for some reason. From the sequence study of Peptide MT-1, Peptide MT-l-S-2 was concluded to follow Peptide MT-l-S-1. Peptide MT-l-S-4 containing arginine, was thought to be the C-terminal portion of the original peptide, and so it was possible to place Peptide MT-l-S-3 between Peptides MT-l-S-2 and MT-l-S-4. These studies established the sequence of Peptide MT-1.

Peptide MT-2 (Residues 41-96)— Edman degradation was carried out up to 22 steps without any ambiguity. After demaleylation, this peptide (2 ^mol) was digested with staphylococcal protease and the digest was subjected to Bio-Gel P-4 column Vol. 83, No. 5, 1978

chromatography; the elution pattern is shown in Fig. 3. As in the case of Peptide MT-1, there was a fraction containing no peptide. From the electrophoretic mobility, it appeared that the demaleylation was not complete, and so each fraction was demaleylated again as described in "EXPERIMENTAL." Four peptides, MT-2-S-1 to S-4, were purified by paper electrophoresis and chromatography. The amino acid compositions of these peptides are listed in Table IV. Edman degradation of each peptide (MT-2-S-1, 4 steps; MT-2-S-2, 11 steps; MT-2-S-3, 14 steps; MT-2-S-4, 8 steps) was carried out. Edman degradation of Peptide MT-2-S-3 was performed up to 14 steps, but after the 12th step the procedure was not successful because of the high hydrophobicity of the middle portion of the peptide. The sequence of this portion was determined with a tryptic peptide of Cm-ferredoxin I, purified by paper electrophoresis. The amino acid composition of this peptide was as follows after hydrolysis for

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Lysinc Arginine Cm-cysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isolcucine Leucine Tyrosine

MT-l-S-2

S. WAKABAYASHI et al.

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TABLE IV. Amino acid compositions of Peptides MT-2-S-1 to MT-2-S-4 of carboxymethyl-ferredoxin I. MT-2-S-1

Aspartic acid Threonine Scrine Glutamic acid Proline Glycine AJanine Valine Isoleucine Leucine Phenylalanine

MT-2-S-3

MT-2-S-4

0.80 (1)

0.91 (1)

0.93 (1) 0.93 (1)

1.78 (2) 1.14 (1) 2.94 (3)

3.81 (4)

0.74 (1) 1.01 (1) 1.71 (2)

2.66 (3) 2.26 (2)

1.16 (1) 3.27 (3)

2.96 (3) 2.06 (2) 1.94 (2) 0.95 (1) 0.91 (1) 0.95 (1)

Total residues Yield (%) Color reaction Purification

1.11 1.03 1.89 1.93 3.08 0.98 1.01 1.90

(1) (1) (2) (2) (3) (1) (1) (2)

1.10 (1) 0.93 (1) 2.22 (2)

0.80 (1) 0.60 (1)

19

11

18

8

11

49

22

20

PE,, BPAW

PE,

PE,, BPAW

PE., BPAW

P

See footnote to Table III for notation, except for BPAW (butanol: pyridine: acetic acid: water, 30 : 20 : 6 : 24, (v/v)).

8.0 6.0 4.0

!\

MT-2-SH MT-2-S2 MT-2-S-3 MT-2-S-4

E 2.0 c O

Si i.o

0.0

o.o 50 100 FRACTION NUMBER

150

Fig. 3. Elution pattern of staphylococcal protease digest of Peptide MT-2 of ferredoxin I. The digest was chromatographed and monitored as described in Fig. 2.

24 h: Lys, 0.86X0; His, 1.21(1); Asp, 2.13(2); Thr, 2.05(2); Glu, 3.24(3); Gly, 1.00(1); Val, 1.81(2); lie, 1.69(2). The amino acid sequence of this peptide was determined completely by Edman degradation (13 steps) and detection of free valine on the amino acid analyzer after the 13th step, and overlapped Peptides MT-2-S-3 and MT-2-S-4; this peptide was assumed to be the C-terminal region of Cm-ferredoxin I, judging from the results of C-terminal analysis with carboxypeptidase A. The 12th residue of Peptide MT-2-S-3 was determined to be lysine from its amino acid composition and tryptic specificity. These studies completed the sequence of Peptide MT-2. Complete Amino Acid Sequence of Cm-Ferredoxin I—Figure 4 summarizes the results of sequence study of pokeweed ferredoxin I. Sequence Studies of Cm-Ferredoxin II—The tryptic digest of Cm-ferredoxin II was separated on a Bio-Gel P-4 column as shown in Fig. 5. From the first peak two peptides, T-2 and T-4, / . Biochem.

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Lysine Histidine Cm-cysteine

MT-2-S-2

m O m Z

20

Z

P

HT_!

, MT-l-S-l

1

g MT-l-S-2

30 40 50 Asp-Ala-Ala-Glu-Glu-Ala-Gly-Leu-Asp-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ser-Cya-Ser-Ser-Cys-Thr-Gly-LysMT-1

1

MT-l-S-3

1

1

MT-l-S-4

MT-2

1

— MT-2-S-1

60

fn

70

£

Val-Thr-Ala-Gly-Thr-Val-Asp-Gln-Glu-Asp-Gln-Ser-Phe-Leu-Asp-Asp-Asp-Gln-Ile-Glu-Ala-Gly-Phe-Val-LeuMT-2 MT-2-S-1

1

MT-2-S-2

1

MT-2-S-3

80 90 96 Thr-Cys-Val-Ala-Phe-Pro-Lys-Gly-Asp-Val-Thr-Ile-Glu-Thr-His-Lys-Glu-Glu-Asp-rU-Val MT-2 MT-2-S-4

Fig. 4. Summary of the results of sequence studies of P. americana ferredoxin I. MT- and S- refer to peptides derived by tryptic digestion of maleyl-Cm-ferredoxin and staphylococcal protease digestion of tryptic peptides, respectively. Peptide T was a tryptic peptide of Cm-ferredoxin. Arrows, ( - » ) and (•

Amino acid sequences of two ferredoxins from pokeweed, Phytolacca americana.

J. Biochem. 83, 1305-1319 (1978) Amino Acid Sequences of Two Ferredoxins from Pokeweed, Sadao WAKABAYASHI,* Toshiharu HASE,* Keishiro WADA,* Hiroshi...
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