Download The Amino Acid Sequence of Chlorella fusca Plastocyanin

164
BIOCHEMICAL SOCIETY TRANSACTIONS
The sequences proposed in Fig. 1 for the Ps. aerirgirioJaand Ps.strrtzeri proteins have
been confirmed by the isolation and characterization of the expected peptides from
tryptic digests of the haeni-free proteins. From each of these two proteins a second tryptic
peptide was isolated that contained two residues of cysteine and a residue of histidine.
The amino acid composition was such that the peptide could not have been derived from
the region shown in Fig. 1.
The N-terminal heptapeptide sequence of the cytochromes c4 is very similar to the
N-terminus of Rhodospirillirm rubrum cytochrome c2 (Dus et a/., 1968). A similar N terminal sequence, Tyr-A?p-Ala-Ala-Ala-Gly-Lys-,
has also been detected in another
monohaem cytochrome c from a photosynthetic bacterium, the cytochrome 12-555 of
Chlorobium thiostilfatophilurn (Gibson, 1961 ; R. P. Ambler & T. Meyer, unpublished
work).
The very close similarity between the cytochromes c4 of A. vinelandii and the pseudonomads was unexpected. The organisms are very different in metabolism and in subcellular structure, and in the current classification (Breed et al., 1957) are placed in
different orders. However, they all have G I C contents of their DNA in the range
56-66% (Hill, 1966).
The deiiitrifying pseudomonads contain a wide range of haem-c-containing proteins.
In addition to cytochromes c-551, c4 and c5 they contain cytochrome c peroxidase
(Kodama & Mori, 1969; Ellfolk & Soininen, 1970) and cytochrome c oxidase (Horio
et al., 1961). The interrelation of these components is still obscure (Horio & Kamen,
1970).
We thank Dr. T. Kodama for the gift of Ps. stutzeri cytochrome c4 [‘cytochrome c-552 (II)’]
and Dr. T. Meyer for the Rsp. rubrum cytochrome c2. The sequencer was purchased by the Medical Research Council, who support this part of the work.
Breed, R. S., Murray, E. G. D. &Smith, N. R. (1957) Bergey’J Mar7rtalofDeterrizinarit.e Bacteriology, 7th edn., p p . 89, 281, Livingston, Edinburgh
Dent, C. E. (1947) Biochem. J. 41,240-253
Dus, K., Sletten, K. & Kamen, M. D. (1968) J. Bid. Chern. 243, 5507-5518
Edman, P. & Begg, G. (1967) Eur. J. Biochem. 1, 80-91
Ellfolk, N. & Soininen, R. (1970) Acta Chem. Scand. 24, 2126-2136
Gibson, J. (1961) Biochetti. J. 79, 151-158
Hill, L. R. (1966) J. Gen. Microbiol. 44,419-437
Horio, T. & Kamen, M. D. (1970) Annu. Reu. Microbiol. 24,399-428
Horio, T., Higashi, T., Yamanaka, T., Matsubara, H. &Okunuki, J. (1961)J. Bid. Chem. 236,
944-951
Kodarna, T. & Mori, T. (1969) J. Biochem. (Tokyo) 65, 621-628
Kodama, T. & Shidara, S. (1969) J . Biochem. (Tokyo) 65, 351-360
Neumann, N. P. & Burris, R. H. (1959) J . Biol. Chenz. 234, 3286-3290
Swank, R. T. & Burris, R. H. (1969) Biochim. Biophys. Acta 180, 473-489
Tissieres, A. (1956) Biorheni. J . 64, 582-589
The Amino Acid Sequence of Chlorella fusca Plastocyanin
JANICE KELLY and R. P. AMBLER
Department of Molecirlur B;olog3>,Unii7ersityof Editibnrgh, Edinbrrrgh EH 9 3JR, U.K .
Plastocyanin is a blue copper protein of low molecular weight. It was first found by
Katoh (1960) in Chlorella ellipsoida, and has since been shown to be present in the
chloroplasts of green algae and higher plants, where it functions in photosynthetic electron transport (see, e.g., Arnon e t a / . , 1970).
For the present study the protein was isolated from spray-dried cells of Chlorellaji~sca,
by a development of the method of Gorman & Levine (1966), in yields of about 0.7mg of
pure protein/g of dry cells. The amino acid sequence of the protein was investigated by
1973
531 st MEETING, LANCASTER
108 109 110 1 1 1
Azurins
165
112
W-
80
81
132
114
115
116 117
- ~ ~ ~ - P h e - P r ~ - G l y -.H.(1.7-14
i-. r e n i d n e c ) CO2Ii
-Tgr-XY:.-F1le-Php-Cy,
79
113
~
85
84
83
C.firrcnplastocyanin -Tyr-Gly-Tyr-PI~?-Cys
-
all1
-
i'rn
86
-
I{<?.
. .(12
rPzid~ier)
-C@*H
Fig. 2. Amino acidsequences around the single thiolgroups of azurins (Ambler, 1971) and
C. jirsca plastocyariiii
XXXrepresents a site that is filled by five different amino acids in the nine azurins studied.
isolation and characterization of the peptides obtained from chymotrypsin and thermolysin digests of the protein. Since the protein contains no arginine and little lysine
(4 residues) and has many bonds sensitive to cleavage by pseudotrypsin (Keil-Dlouha
et a/., 1971), tryptic digestion has proved to be of little use. The N-terminal sequence of
the molecule was checked by using an automatic Sequenator (Beckman model 890;
Edman & Begg, 1967); results were obtained as far as residue 36, and were in complete
agreement with the sequence deduced by conventional methods. The structure deduced
was a single polypeptide chain of 98 residues (Fig. 1).
Preliminary analyses indicate the presence of small amounts of carbohydrate, and
electrophoretic mobilities suggest that a labile group may be associated with residue
9 or 10.
Incomplete experiments with plastocyanin from spinach (Brussica oleracea), C/ilorella
pyrenoidosa and Scenedesmus obliquus have shown very considerable similarities in
sequence. The sequence of the plastocyanin from French bean (Pliaseohrs vulgaris) is
currently under investigation by Milne & Wells (1970).
Katoh et al. (1961) suggested that the thiol groups of plastocyanin contributed to the
binding of the copper. The proposed sequence contains only a single cysteine residue
(position 83), clustered around by several aromatic residues. In the bacterial azurins,
which are copper proteins that are believed to be of structure and function comparable
with those of plastocyanin (Malkin & Malmstrom, 1970), there is a similar concentration
of aromatic residues in the vicinity of the single thiol group (Fig. 2) (Ambler & Brown,
1967; Ambler, 1971). The sequence similarities are not sufficient to provide convincing
evidence for homology between the two classes of proteins, but are striking enough to
suggest at least a functional similarity. In both cases the residues concerned are close to
the C-terminus of the molecule. Further studies [e.g. of the plastocyanin reported to be
present in the blue-green alga Anahaena variahilis (Lightbody & Krognian, 1967)]
may clarify the relationship.
We are very grateful to Dr. B. Prokes and the staff of the Department of Applied Algology,
Trebon, Czechoslovakia, for their supply of the algal cells and for their interest in the work.
Vol. 1
166
BTOCH F M TCAL SOCIETY TR ANSACTJONS
Ambler, R . P. ( I 971) in Rrccrit Dere/optncnfs in thc Clreiiiic~ulStudy of Proteiir Structures
(Previcro, A,, Pechcre, J.-I-'. & Coletti-Previero,M.-A., eds.), pp. 289-305, Inserm, Paris
Ambler, R. P. & Brown, L. H. (1967) Biochctn. J. 104, 784-825
Arnon, D. I., Chain, R. K., McSwain, B. D., Tsujimoto, H. Y . & Knaff, D. B. (1970) Proc.
Not. Acud. Sci. U.S. 67, 1404-1409
Edrnan, P. & Begg, G. (1967) Errr. J . Biocheni. 1, 80-91
Gorman, D. S. & Levine, R. P. (1966) Plant Plrysiol. 41, 1637-1642
Katoh, S. (1960) Nature (London) 186, 533-534
kdtoh, S.,Suga, I.,Shiratori, I. &Takaniiya,A.(1961) Arch. Bioclienr. Biophys. 94,136-141
Keil-Dlouha,V., Zylber, N. & Keil, B. (1971) in Recent Decelopnients in the Chemical Study of
Protein Structures (Previero, A,, Pechere, J.-F. & Coletti-Previero,M.-A., eds.), pp. 133-144,
Inserm Paris
Lightbody, J. J. & Krogman, D. W. (1967) Biochim. Biophys. Actu 131, 508-515
Malkin, R. & Malmstrom, B. G. ( I 970) Adcan. Enzyinol. Relat. Areas Mo/.Bid. 33,177-244
Milne, P. R. &Wells, J. R. E. (1970)J. Biol. Cheni. 245, 1566-IS74
Amino Acid Sequence of Cytochrome c5 from Pseudomonas mendocina
R. P. AMBLER and ELIZABETH TAYLOR
Department of Molecular Biology, University of Ediiibiirgh, Edinbnrgh EH9 3JK, U.K .
Many bacteria contain several soluble c-type cytochromes. Both pseudomonads (Horio,
1958) and Azotobacter (cytochrome cs; Tissieres, 1956) contain proteins with a-band
maxima at about 555nm. In studies of other cytochromes from pseudomonads, we have
frequently met with cytochromes of the cs type, but chroniatographic properties and
yields have been very erratic. All purifications used have involved exposure to low pH
values (pH4-5; Ambler, 1963a), and Swank & Burris (1969) have shown instability of
Azotobacter cytochrome cs at acid pH values. However, it has been found possible to
isolate an adequately homogeneous cytochrome of this type from Pseudomonas mendocina CH-110 (Palleroni et a/., 1970), in good yield.
The growth of the organism and the general procedures for the isolation of the protein
were similar to those that have been described for Pseudomonas cytochrome c-551
(Ambler, 1963~).The major cytochrome present (c-551) was eluted from CM-cellulose
at pH4.45, and a mixture of the cytochrome c5 and cytochronie c4 (Ambler & Murray,
1973) was eluted at pH4.75. The latter components were then separated by chromatography on DEAE-cellulose (at p H 8 . 5 in 12m~-tris-HCl,with an NaCl gradient; cytochrome cs was eluted at about 32ni~-NaCl,ahead ofcytochrome c4), and further purified
by (NH&SO, precipitation (7G95 % saturation) and gel filtration through Sephadex
G-75 (superfine grade; in 50m~-ammoniumacetate, pH5.1). The yield of cytochrome
cs was about 2.2pmo1/100g of acetone-dried cells (compared with about 8pmol of cytochrome c-551 and 0.12pmol of cytochrome c4).
This cytochrome cs was homogeneous by gel electrophoresis, and had a haerii content
(from visible spectrum) of about I residue/10000 daltons of amino acids. The behaviour
of the protein on Sephadex (3-75 was indistinguishable from that of the 9000-dalton
cytochronie c-551. Amino acid analysis showed the complete absence of tyrosine and
phenylalaiiine (<0.03 renidue/haem group), and a valine content of 1.1 residue/haem
group. Nevertheless N-terminal group analysis by the dansyl method showed both
alanine and serine.
After removal of the haem (Hg2Cl2-0.l M-HC1-8M-urea, 3 7 T , 24h), the apoprotein
was digested with trypsin or chymotrypsin, and the peptides formed were isolated and
characterized by standard methods (Ambler & Brown, 1967; Gray, 1972). The peptides
could all be fitted together to form the unique sequence shown in Fig. 1 , which agrees
very well with the amino acid analysis results for the whole protein, and with the results
of C-terminal analysis with carboxypeptidase A. However, at least four different tryptic
peptides were derived from the N-terminal region of the molecule, and all the available
1973