The amino acid sequence of cytochrome c553 from Microcystis aeruginosa

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 270, No. 1, April, pp. ZlS-226,198s

The Amino Acid Sequence of Cytochrome CATHLEEN

c 553from Microcystis

L. COHN, MARK A. HERMODSON,

AND DAVID

aeruginosa

W. KROGMANN’

Department of Biochemistry, Purdue University, West Lqfavette, Indiana 47907 Received August 22,1988, and in revised form November 14,1988

Cytochrome cs3 is an electron donor to P700 in the photosynthetic electron transfer chain of cyanobacteria and eukaryotic algae. We have purified this cytochrome from the cyanobacterium Microcystis aerugirwsa and determined its amino acid sequence. When the amino acid sequence of this protein is compared to sequences of cytochromes c5% from other organisms, one sees that the evolution of net charge is more pronounced than the evolution of overall structure, further documenting a pronounced shift in the isoelectric point of this protein during the evolution of cyanobacteria. Cyanobacteria and algae also contain cytochrome cSo (M, 15,500) which is quite different from cytochrome c553(M, 10,500). When the amino acid sequence of cytochrome c553is compared to that of cytochrome c550,two regions of similar sequence are recognized. o 1989 Academic Press, Inc.

Cytochrome cm3 transfers electrons from cytochrome f to P700, replacing the copper protein plastocyanin which catalyzes this reaction in higher plant chloroplasts. This replacement occurs in cyanobacteria and in some eukaryotic algae that are experiencing copper deprivation (1). Natural waters often become deficient in copper and these oxygenic photosynthetic organisms sustain their activities by substituting the cytochrome for the copper containing plastocyanin. Earlier work from this laboratory has shown that these two functionally interchangeable proteins show parallel variations in their isoelectric points depending on the genus from which the proteins have been isolated (2). The isoelectric points of cytochrome c553 and plastocyanin in eukaryotic algae and the simpler forms of cyanobacteria are, like the plastocyanins of higher plants, acidic (p13.8 to 6.5) while those of the more complex cyanobacteria are basic (PI 7.5 to 9.5). It is of interest to compare the structures of the acidic and basic cytochromes in order to see what regions are responsi1 To whom correspondence should be addressed. 219

ble for the change in isoelectric point and to recognize features important to function and to evolutionary history. An important shift from basic to acidic residues in the region between residues 67 and 77 (as in Fig. 2) in cytochrome c553was noted earlier (2) and is confirmed in the sequence reported here. Cytochrome c5%is a member of the Class I c type cytochromes (3). These cytochromes have low-spin iron, His + Met heme coordination, heme near the N-terminus, and lengths of 80 to 120 amino acids. Mitochondrial respiratory cytochrome c is the prominent member of this class with many other members found in bacteria. All are related by similarity in amino acid sequence. The cytochrome c5% described in this paper is seen to contain regions of sequence that show striking similarity to regions of a cytochrome c550 from the same source. This latter cytochrome, whose sequence is described in an accompanying paper (4), is different from all of the Class I c cytochromes in size, redox potential, and heme binding. EXPERIMENTAL PROCEDURES Materials. Micmcgstis aeruginosa was collected from the Potomac River at Pohick Bay in 1983 as has 0003-9861/89 $3.00 Copyright All rights

0 1989 by Academic Press. Inc. of reproduction in any form reserved.

220

COHN,

HERMODSON,

AND

KROGMANN

10 20 Asp Gly Ala Ser Ile Phe Ser Ala Asn Cys Ala Ser Cys His Met Gly Gly Lys Asn Val N terminus CB-1 GB-2-I I-

30 40 Val Asn Ala Ala Lys Thr Leu Lys Lys Glu Asp Leu Val Lys Tyr Gly Lys Asp Ser Val CB-2

50

Glu Ala Ile I---

60

Val Thr Gln Val Thr Lys Gly Met Gly Ala Met Pro Ala Phe Gly Gly Arg CB-2 +---CB-3 1 V8-2

JO

Leu Ser Ala Glu Asp Ile -V8-2,-i

80

Glu Ala Val Ala Asn Tyr Val Leu Ala Gln Ala Glu Lys Gly Trp CB-3

FIG. 1. The amino acid sequence of cytochrome c 653. The fragments labeled CB-1, CB-2, and CB-3 were obtained by cyanogen bromide cleavage and the fragment VS-2 was isolated from a digest done with Staphylococcus aurem VS protease.

been described (5). The cells were broken by freezing and thawing three times. The broken cells were easily separated from the soluble extract by filtration through cloth. The cell mass was extracted repeatedly to remove nearly all of the blue pigment which served as a convenient indicator for removal of the soluble proteins (6). The aqueous extract was filtered through paper (Schleicher & Schull, No. 595) to remove small, green membrane fragments and centrifuged at 27,OOOg in a continuous flow Sorvall centrifuge at a flow rate of 200 ml/min to remove phycobilisome fragments. The soluble proteins were pumped through a Pellicon Millipore No. PTHK00005 filter (M, 100,000 cutoff) which removed 95% of the phycobiliprotein. Next the extract was concentrated on a Millipore PTGA00005 filter (I& 10,000 cutoff) to reduce the volume. The concentrated extract was brought to 50% saturation with (NH&SO, and centrifuged at 10,OOOg for 10 min to remove the last

tracesofphycocyanin.The dialyzed against fuged as above,

supernatantsolutionwas

5 mM Tris buffer, pH 7.8, and centrithen loaded on a DE52 column (35

X 250 mm) equilibrated with 5 mM Tris buffer, pH 7.8. The individual proteins were eluted as previously described (7). The cytochrome cm fraction was loaded on a DEAE Sephadex A-25 column (25 X 150 mm) equilibrated with 5 mM Tris buffer, pH 9, and was eluted with 0.05 M NaCl. The protein was then passed through a Sephadex G-50 gel filtration column equilibrated with 50 mM ammonium bicarbonate. A final purification was achieved by HPLC, using a Synchropak RP-R C-18 reverse phase column (250 X 1.4 mm) eluted with a 0 to 60% acetonitrile gradient in 0.1% trifluoroacetic acid. The cytochrome eluted as a symmetrical peak at 42% acetonitrile. The same column was used for peptide separations. Elution was monitored at 230 nm at a flow rate of 1 ml/min. Solvents for HPLC were from Burdick & Jackson. Cyanogen bromide was from Pierce; all other chemicals were reagent grade. Staphylococcus aureus V8 protease was obtained from Pierce and endoproteinase Lys-C from Boehringer. Enzymatic cleavages. Cleavage with endoproteinase Lys-C was performed using a modification of the

Microcgstis

aerugirwsa

CYTOCHROME

TABLE AMINO

ACID COMPOSITIONS

OF C~TOCHROYE CB-1

Amino

acid

Whole protein

Aspartic acid Asparagine Threonine Serine Glutamic acid Glutamine Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Cysteine Methionine Tryptophan

7.8 (4) (4) 3.2 (3) 4.6 (5) 7.0 (5) 1.4 (1) 8.6 (9) 12.1(13) 6.7 (8) 2.7 (3) 4.4 (4) 1.5 (2) 2.1(2) 0.9 (1) 7.9 (8) 1.3 (1) 2.0 (2) 2.8 (3) -0)

Total

81

221

csh?

I

cm AND PEPTIDES

USED IN SEQUENCE

CB-2

CB-3

Amino

ANALYSIS V8-2

acid residues

1-15

16-51

55-81

42-66

2.1(l) (1) 0.2 (0) 2.8 (3) 0.0 (0)

4.2 (2)

2.0 (1)

0.9 (1)

(2)

(2) 2.6 (3) 1.5 (1) 3.3 (2)

(1)

(0)

0.2 (0) 0.9 (1) 3.5 (3)

1.6 (2) 0.9 (1) 2.4 (1)

(0)

0)

(1)

0.0 (0) 1.5 (1) 3.5 (3) 0.4 (0) 1.0 (1) 0.0 (0) 0.0 (0) 0.9 (1) 1.3 (1) 0.4 (0) 0.0 (0) 1.6 (2) 0.1(l) -(O)

0.0 (0) 4.5 (4) 4.0 (3) 5.1(6) 1.0 (1) 2.0 (2) 0.5 (1) 0.2 (0) 0.2 (0) 7.0 (7) 0.0 (0) 0.0 (0) 0.4 (1) -CO)

0.9 (1) 3.0 (3) 5.6 (6) 2.1(2) 0.8 (1) 1.4 (2) 0.4 (1) 0.7 (1) 0.0 (0) 1.3 (1) 0.7 (1) 0.0 (0) 0.0 (0) -Cl)

15

procedure from the manufacturer. Denatured protein (20-25 mg) was suspended in 2 ml of 0.1 M ammonium bicarbonate and stirred at 37°C for 8 h. Endoproteinase Lys-C was added in equal amounts at 0,3, and 6 h to a final enzyme substrate ratio of l/40 (w/w). The reaction was stopped by adding 44% formic acid. S. uureus V8 protease was employed at 37°C for 12 h on a 5- to lo-mg sample of cytochrome cm dissolved in 0.1 M ammonium bicarbonate as described by Allen (8). Chemical cleouage. Cyanogen bromide cleavage was done according to Gross (9). Before digestion, the cytochrome was treated with B-mercaptoethanol to reduce any methionine sulfoxides. Cytochrome (20-25 mg) was dissolved in 2 ml of 70% formic acid. Cyanogen bromide crystals (l-2 mg) were added and the reaction was carried out in darkness at room temperature for 20 h. The mixture was dried with nitrogen and dissolved in 8.8% formic acid. All peptide fragments were purified by reverse-phase HPLC (10). Amino acid analysis and sequence analysis All the procedures have been described (11). All sequence determinations except that of peptide V8-2 were done with a Beckman 890 amino acid sequencer. Peptide

36

27

(1) 0.9 4.2 3.9 2.1 1.5 1.0 0.0 0.8 0.0 1.2 1.2 0.0 1.4 0.0

(1) (4) (4) (2) (2) (1) (0) (1) (0) (1) (1) (0) (2) (0)

25

VS-2 was sequenced using an Applied Biosystems 470A protein sequencer. The cyanogen bromide digest of cytochrome css3 was resolved into three peptides by reverse-phase HPLC (Fig. 3). The peak fractions were rechromatographed before sequence analyses and determination of the amino acid compositions. S aureus V8 gave many peaks on the reverse-phase HPLC system (Fig. 4). After rechromatography, an aliquot of each peak was subjected to amino acid composition analysis. Peak V8-2 contained a proline residue known from sequencing to be at the N-terminus of CB-3. V8-2 also contained a methionine which must precede the proline and so must provide the overlap between CB-2 and CB-3. This cytochrome proved resistant to carboxypeptidase digestion. Peptide CB-3 ended cleanly in the sequenator and the composition and sequence analyses gave reasonable agreement indicating a tryptophan at the carboxy terminus. Samples of the intact protein were subjected to digestion with endoproteinase Lys-C to cleave on the carboxy-terminal side of lysine at position 79. The digest was lyophilized and analyzed in a Finnigan 4000 mass spectrometer with

COHN,

I II

Porphyra Alaria

III

teneria

(4)

esculenta

(4)

fascia

(5)

Petalonia

IV

Bumilleriopsis

filiformis

V Porphorydium VI VII VIII IX X XI XII

Anacystis

cruentum nidulans

Synechococcus

sp.

Spirulina

reinhardtii

gracilis

aeruginosa

40

50

60

70

80

SO

--SMNT---IDAI

III

LEEN--EMNN---IKSI

IV

LEAN--GMNA---VSAI

V

LEAN--GMNS---VSAI

VI

LDEY--GMAS---IEAI

VII

LGKY--NMYs---AKAII

VIII

LEQF--GMNS---ADAI

XI

(8)

(4)

ALADN--KMVS---VNAI

X

(7)

(7)

II

IX

(6)

maxima (4)

Chlamydomonas

30

(4)

6312

boryanum

Wicrocystis

I

KROGMANN

(4)

flos-aquae

Plectonema

AND

(6)

Aphanizomenon

Euglena

XIII

HERMODSON,

LTAN--GKDT---WA1 DLAKYL-KGFDDDAVAAV LEQYLDGGFK---VESSI

XII

AIEEYLDGGYT---KEAIE

XIII

DLVKY--GKDS---VF.AIV

FIG. 2. Comparison of amino acid sequences of some cytochromes c5%. The conserved residues common to all sequences are boxed. These sequences are described by references as follows: I, II, IV, VI, X, and XII, Dickerson (12); III, Sugimura et al (13); V and VII, Sprinkle et al. (14); VIII and IX, Aitkin (15); XI, Merchant and Bogorad (16).

Microcgtis I

I

I

aeruginosa

I

00

70

//

60 w 50

E I e

40: z 30

CYTOCHROME

223

cW

culated molecular weight of 9861 to which a heme residue and two hydrogens should be added for a total molecular weight of 10,478. Figure 2 shows an alignment of the sequences of cytochromes c5% published to date. There is a highly conserved region at residue 6 through 21 which includes the heme binding site. Residues 29 to 33 are highly conserved. Another conserved region is found at residue 56 through 65 which includes methionine-64 which is the sixth ligand to the iron. The conserved tryptophan at the carboxyl terminus is noteworthy since tryptophan is rarely seen in “c”-type cytochromes. A matrix of differences among the cytochrome sequences described in Fig. 2 was

20

&I I 1

I IO

FIG. 3. Reverse-phase of the cyanogen bromide

I 20 MINUTES

I 30

IO

0

40

HPLC separation of peptides digest of cytochrome cm.

0.75

a Data General Nova/4 using a solid probe at 250°C and 70 eV. The samples gave clear evidence of m/z 262 and a larger peak of m/z 244 on EI analysis. The same peaks were obtained from an authentic Gly-Trp purchased from Sigma Chemical Co., corresponding to the mass of the dipeptide ion and the mass of the dehydrated form. A sample of the digest was introduced into a Finnigan TSQ tandem mass spectrometer and a daughter ion spectrum of m/z 244 (M + HH,O)+ ion was obtained. This was identical to the spectrum obtained from the authentic Gly-Trp dipeptide and gave further confirmation of the carboxy terminus. 0.25

RESULTS

AND

DISCUSSION

The amino acid sequence of M. amuginosa cytochrome c5= is shown in Fig. 1. The amino acid composition calculated for the complete sequence is in agreement with the composition determined by direct amino acid analysis after HCl hydrolysis of the protein (Table I). The yields for the amino acid sequence determinations are shown in Table II. Cytochrome cS3 is a polypeptide chain of 81 residues with a cal-

J

0;

0

-0

IO

20 MINUTES

30

FIG. 4. Reverse-phase HPLC separation of the S. aureus VS digest of cytochrome

40

of peptides csss.

Asp

GUY Ala Ser Ile Phe Ser Ala Asn CYS Ala Ser CYS His Met

GUY GUY LYS Asn Val Val Asn Ala Ala

2 3 4 5 6 7 8 9 10 11 12 13 14 15

16 17 18 19 20 21 22 23 24

Residue

1

Position

( 7, Spot test 1

( 4, Spot test 6

3 11 8 4 6 9 4

(14) 24

( 9) 11

CB-1 (nmol) 20 49 40 11 28 24 12 30

yield

CB-2 (nmol) 52 57 52 26 49 53 24 41 45

ACID SEQUENCE

26 26 10 25 15 8 20

N-terminus (nmol) 13

Peptide

AMINO

II

Pro Ala Phe GUY GUY Arg Leu Ser

61 62

GUY Ala Met

52 53 54 55 56 57 58 59 60

Ala Ile Val Thr Gln Val Thr LYS GUY Met

Glu

Residue

cm

42 43 44 45 46 47 48 49 50 51

41

Position

DATA FOR CYTOCHROME

TABLE

13 13 14 3 6 11 2 12 3 Small peak

CB-2 (nmol) 10

Peptide

yield

1,248 93

1,360 1,778 2,327 1,550 1,235 467

2,532 3,668 2,180

(pm00 17,383 16,184 17,096 1,420 6,882 10,372 1,016 953 379 6,211

VS-2

Spot test 24 15

CB-3 (nmol) 37 56 45 57 63

k

E

Micrmystis

aeruginosa

CYTOCHROME

~553

225

constructed. The six genera VI, VII, VIII, IX, X, and XIII are all cyanobacteria and the variations among sequences are too small to provide precise insights into the evolution of cyanobacteria. The overall similarity of these sequences directs attention to earlier observations on the evolution of charged residues in these cytochromes. The isoelectric point of cytochrome c553of A. jlos-aquae (VII in Fig. 2) is 9.3, and that of M aeruginosa (XIII in Fig. 2) is 5.5 (2). The parallel shift in p1 of plastocyanin suggests that the two genes for these proteins were driven to evolve a net charge that accommodates the evolution of another structure with which these proteins must interact. An accompanying paper (4) describes the amino acid sequence of cytochrome cm isolated from the same extracts of M aeruginosa used in this work. Two regions of sequence similarity are shared by these two very different cytochromes. The sequences of these two cytochromes from the same species indicates that cytochrome cSo has evolved from a restructuring of fragments of the cytochrome c553gene. ACKNOWLEDGMENTS The authors are grateful to Dr. K. Wood of the Purdue Mass Spectrometer Center for his help in identifying the C-terminal dipeptide as described in Table II. Financial support for this work was provided by Grant DMB-850912 from the Molecular Biology Program of the National Science Foundation. This is Journal Paper No. 11,665 from the Purdue University Agricultural Experiment Station. REFERENCES 1. WOOD, P. (1978) Eur. J. Biochem. 87,9-19. 2. Ho, K. K., AND KROGMANN, D. W. (1934) Biochim Biophys.

Acta 766,310-316.

3. AMBLER, R. (1982) in From Cyclotrons to Cytochromes (Kaplan, N. O., and Robinson, A. B., Eds.), pp. 263-279, Academic Press, London/ New York. 4. COHN, C. L., SPRINKLE, J. R., ALAM, J., HERMODSON, M., MEYER, T., AND KROGMANN, D. W. (1989) Arch Biochem Biophys., 270,227-235. 5. KROGMANN, D. W., BUTALLA, R., AND SPRINKLE, J. (1986) Plant Physid 80,667-671. 6. PADGETT, M. P., AND KROGMANN, D. W. (1987) Phdosyntk Rex 11,225-235.

226

COHN,

HERMODSON,

7. Ho, K. K., ULRICH, E. L., KROGMANN, D. W., AND GOMEZ-LOJERO, C. (1979) Biochim. Biu.&s. Acta 545,236-F&S. 8. ALLEN, G. (1981) Sequencing of Protein and Peptides, pp. 58-59, Elsevier Science, New York. 9. GROSS, E. (1967) in Methods in Enzymology (Hirs, C. H. W., Ed.), Vol. 11, pp. 238-255, Academic Press, New York. 10. PEARSON, J. D., MAHONEY, W. C., HERMODSON, M. A., AND REGNIER, F. E. (1981) J. Chrmtogr. 207,325-332.

AND

KROGMANN

11. MAHONEY, W. C., HOGG, R. W., AND HERMODSON, M. A. (1981) J. Biol Chem. 256,4350-4356. 12. DICKERSON, E. W. (1980) Sci. Amer. 243(3), 137153. 13. SUGIMURA, Y., HASE, T., MATSUBARA, H., AND SHIMOKURIYANA, M. (1981) J. Biochmn Tokyo 90,1213-1219. 14. SPRINKLE, J. R., HERMODSON, M., AND KROGMANN, D. W. (1986) Photos&h Res. 16.63-73. 15. AITKEN, A. (1974) Eur. J. Biochem 101,297-208. 16. MERCHANT, S., AND BOGORAD, L. (1987) J. Bid Chem 262,9062-9067.