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The amino acid sequence of low-potential cytochrome c550 from the cyanobacterium Microcystis aeruginosa
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 270, No. 1, April, pp. 227-235, 1989
The Amino Acid Sequence of Low-Potential Cyanobacterium Microcystis
Cytochrome aeruginosa
c550 from the
CATHLEEN L. COHN,* JAMES R. SPRINKLE,* JAWED ALAM,* MARK HERMODSON,* TERRY MEYER,? AND DAVID W. KROGMANN*pl *Department of Biodwmistry, Purdue University, West I.&aye&e, Indiana hY9OY; and TDepartment Biochemistry, Universi@ of Arizona, Tucson, Arizona 85721 Received
August
22,1988,
and in revised
form
November
of
14,1988
The low-potential cytochrome c550has been purified from the cyanobacterium Microcystis aerugirwsa and its amino acid sequence has been determined. The protein contains 135 amino acid residues with the Cys-X-X-Cys-His heme binding site at residues 37 to 41. The sequence from residue 28 to 45 shows similarity to cytochrome cs3 residues 1 to 18 when the heme binding sites are aligned. Another region of similarity is in the carboxylterminal regions of these two proteins. The two aligning regions of cytochrome c5= correspond to helical segments in other related cytochromes. A partial sequence of cytochrome es0 from Aphanimmenmjlos-aquae was obtained and showed a 48% identity to the sequence of the M aemginosa cytochrome. The single methionine residue in cytochrome c550of M. aeruginosa occurs at position 119 but there is no methionine in this region in the A.jZos-aquae cytochrome, indicating that methionine is not the sixth ligand to the heme iron atom. Histidine 92 is a possible sixth ligand in ikl. aeruginosa cytochrome Cam. The far-uv circular dichroism spectrum indicates that this protein is approximately 17% (Yhelix, 42% p-pleated sheet, and 41% random coil. 0 1989 Academic Press, Inc.
Holton and Myers (1) first described an unusual low-potential, autooxidizable cytochrome c from Anacystis nidulans and later characterized this cytochrome in detail (2,3). Most c type cytochromes belong to Class I as defined by Ambler (4) and their redox potentials range from 0 to +500 mV (5). Cytochromes of Class III are distinguished by lower redox potential and by having several hemes per polypeptide. The cytochrome of Holton and Myers contained a single heme and had a redox potential of -260 mV. Similar low-potential cytochromes have been observed in a number of other cyanobacteria as well as in several eukaryotic algae-a red alga (6), a diatom (‘7), and a green alga (8). This cytochrome has not been found in higher plants. The presence of the cytochrome in 1 To whom
correspondence
should
cyanobacteria may depend on growth conditions (9). A functional role for cytochrome c550in the electron transport reactions supporting cyclic phosphorylation in Photosystem 1 has been postulated (10). Fractionation experiments indicate that the cytochrome may be associated with Photosystem 2 (11). We have published preliminary data on the partial amino acid sequence of this protein (12) and can now report its complete primary structure. This is the first amino acid sequence of a cytochrome of this type. EXPERIMENTAL
PROCEDURES
Materials. The collection and fractionation of Microcystis aeruginxxa and Aphanizomenon jos-aquue were described by Ho et al. (9). Cytochrome ~550 purification was as described by Alam et al. (12). A final purification of the cytochrome single symmetrical peak.
be addressed.
227
0003-9861189
by rpHPLC
yielded
$3.00
Copyright 0 1989 by Academic Presq Inc. All rights of reproduction in any form reserved.
a
COHN ET AL. TABLE I AMINO ACID COMPOSITIONS
OF CYTOCHROME
C~AND
USED IN SEQUENCE ANALYSIS
PEPTIDES
Peptides/residue
Aspartic acid Asparagine Threonine Serine Glutamic acid Glutamine Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Cysteine Methionine Tryptophan
Whole protein
34-66 T-l
106-118 T-2
62-135 KC-2
91-103 vs-2
99-109 AN-l
104-109 vs-3
110-12’7 vs-4
120-135 CB-2
21.4 (12) (9) 12.0 (12) 5.8 (6) 15.0 (12) (3) 7.0 (7) 8.0 (8) 9.1 (9) 7.1(7) 3.9 (4) 20.0 (20) 4.5 (5) 2.7 (3) 2.9 (3) 5.9 (6) 4.8 (5) 1.7 (2) 0.6 (1) (1)
5.2 (1) (4) 2.7 (3) 2.2 (1) 2.7 (1) (1) 2.0 (2) 3.2 (3) 2.6 (3) 2.0 (2) 0.6 (0) 3.7 (4) 0.4 (0) 0.3 (0) 0.9 (1) 3.6 (4) 0.7 (1) 1.0 (2) 0.1(O) (0)
4.0 (2)
13.0 (9)
3.0 (3) 2.8 (3) 7.2 (7) (0) 6.8 (7) 3.2 (3) 5.2 (5) 4.3 (4) 2.7 (3) 9.6 (10) 4.2 (5) 1.5 (2) 2.0 (2) 0.3 (0) 4.5 (5) 0.4 (0) 0.7 (1) (1)
1.9 (2) (1) 0.6 (1) 0.0 (0) 1.2 (2) (0) 0.7 (1) 0.0 (0) 0.3 (0) 0.0 (0) 0.6 (1) 2.0 (2) 0.0 (0) 0.4 (1) 0.0 (0) 0.0 (0) 0.7 (1) 0.0 (0) 0.0 (0) (0)
0.9 (0) (1)
1.0 (1) 0.2 (0) 1.0 (1) (0) 0.0 (0) 0.3 (0) 2.1(2) 1.9 (2) 0.1(O) 1.1 (1) 1.6 (2) 0.0 (0) 0.0 (0) 0.4 (0) 0.0 (0) 0.0 (0) 0.0 (0) (0)
2.1(l) (1) 0.0 (0) 0.9 (1) 1.1(l) (0) 2.9 (3) 0.5 (0) 0.0 (0) 1.0 (1) 1.0 (1) 1.1(l) 0.0 (0) 1.0 (1) 1.0 (1) 0.0 (0) 1.0 (1) 0.0 (0) 0.0 (0) (0)
1.2 (1) 0.0 (0) 0.9 (1) (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 2.0 (2) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.9 (1) 0.0 (0) 0.0 (0) (0)
4.2 (3) (1) 0.0 (0) 0.0 (0) 1.1 (1) (0) 1.0 (1) 0.1(O) 3.1(3) 3.0 (3) 0.0 (0) 2.1(2) 1.6 (2) 0.0 (0) 0.0 (0) 0.0 (0) 0.9 (1) 0.0 (0) 0.8 (1) (0)
1.3 (1) (0) 1.1(l) 0.3 (0) 1.3 (1) UN 1.1(l) 2.2 (2) 1.2 (1) 1.0 (1) 0.9 (1) 2.0 (2) 0.8 (1) 1.0 (1) 0.0 (0) 0.0 (0) 2.0 (2) 0.0 (0) 0.0 (0) (1)
134
33
13
74
13
11
6
18
15
Total
i4j
(2)
Both an Applied Biosystems microsequencer and a Beckman 890 sequenator were used. Sequencer reagents for the Applied Biosystems microsequencer
Val -
Ala
Pro A.-g W-4-(
Leu
numbers
Asp
61”
Ar‘g
Trp
130 Gly
My
T,w
Ile
Tyr
were obtained from Applied Biosystems, Inc. Reagents and procedures for the Beckman 890 sequenator are by Mahoney (13). Endoproteinase Arg-C was
Phe
t4 m-2
-I
-m-2-
FIG. 1. The amino acid sequence of cytochrome c= from M. aacginosa.
Microcgstis
aer&no.sa
CYTOCHROME
229
cm 7.0
10 1.
M.aeru@nosa
LELDEKTLTITLNDAGESVTLTSEQATEGQ
2.
A. flos-aqua8
LELPETIRTVPLhPKEGTYVLSLEPVKEE-
1.
40 KLFVANCTKCHLQGKTKTNNNVSLGLGDLAKAEPPRDN
1. 2.
70 20 LLALIDYLEHPTSYDGEDDLSELHPNVSRPDIFPELRN MKNPITXQEEEEISEI
1. 2.
110 120 LTEDDVYNVAAYMLVAP-RLDERWGGTIYF LrDEPLKAIAEHILLEELVVcTKgOEK
50
30
60
2. ELENYACAQCBAGEVIK~LQLPGLEPEALdGhLPNe 90
100 PSIKSANIE---EN 130
FIG.2. Comparison of cytochromes & from M. aeru@nosa and A. jbs-aquue. purchased from Sigma Chemical Co. and trypsin (TPCK’ treated) from U.S. Biochemicals Corp. Endoproteinase Lys-C, endoproteinase Arg-C, and endoproteinase Asp-N were from Boehringer-Mannheim Biochemicals. The other reagents and enzymes were the same as in Cohn et aL (14). Enzymatic cleavage. Trypsin digestion was performed as described by Mahoney and Nute (15). The protein was dissolved in 1 ml of 8 M urea and diluted with 4 ml of 0.1 M N)4HC03 to give a final pH of 8. A solution of TPCK-treated trypsin (1% by weight of the protein in 0.1 M NH,HCOs) was added and the reaction incubated at 3’7°C for 2 h. A second aliquot (la, w/w) of trypsin was added and the reaction incubated another 3 h. The reaction was terminated by lowering the pH to 3.3 with 1 N HCl and the digest was lyophilized. Endoproteinase Arg-C was used with a modification of the procedure of Schenkein et al. (16). Approximately 60 mg of the cytochrome was dissolved in 1 ml of 8 M ultrapure urea in a stirred reaction vessel at 37°C and 4 ml of 106 mM NHIHCOI was added. Endoproteinase Arg-C (5 U, or 0.02 mg in water) was added. A second, equal aliquot of enzyme was added
2 Abbreviations used: TPCK, L-1-ptosylamino-2phenylethyl chloromethyl ketone; CNBr, cyanogen bromide.
cytochrome
c5So
cytochrome
c55S
after 36 min and the stirring continued for 3 h. The reaction was stopped by addition of 1 N HCl. A brown, flocculent precipitate was removed and the supernatant was fractionated by rpHPLC. Endoproteinase Lys-C and endoproteinase Asp-N were used according to the instructions of the manufacturer. No detergents or urea were needed to dissolve the peptides. Other procedures for enzymatic and chemical cleavage, peptide isolation, and amino acid sequence analysis have been described (14). Peptide im.!ation and sequencing. Table I shows the composition of those peptides used in determining the sequence of the cytochrome. CNBr cleaved cytochrome cW to give two peptides which were separated by rpHPLC. Each peak was rechromatographed and aliquots were taken for sequence analyses and for determination of amino acid composition. Trypsin digestion gave peptides which were separated by rpHPLC. The two largest peaks were rechromatographed and sequenced. Digestion with endoproteinase Lys-C yielded the two peptides which were separated by rpHPLC. Each peak was chromatographed a second time and aliquots were taken for amino acid sequence and composition analysis. Peptide KC-2 of the endoproteinase Lys-C (8 mg) was hydrolyzed with Staphylococcus ou~eus V8 protease and gave 10 peptides. This digest produced 10 major peaks, three of which were used in the sequence determination.
28 45 E G Q K L P V A N C T K C H L Q G K”“““” 1 D G A S I
9s
18 E S & g C A S C fi M G G K"""""
130
PNVSRPDIFPELRNLTEDDVYNVAAYMLVAPRLDERU.'.' 55 EAFGG-------B-&SAE~IEA~&N~V~--AQAEKGbj".'
81
FIG.3. Alignment of similar regions of cytochrome cm with cytochrome cW from M. aerugima
230
COHN
200
300
FIG. 4. Absorption
200
spectra
FIG. 5. CD spectrum ruginosa
210 220 230 NANOMETERS of cytochrome
AL.
400 WAVELENGTH, of cytochrome
Another sample of peptide KC-2 (10 mg) was digested with endoproteinase Asp-N. The resolution of this digest by rpHPLC allowed the identification of peaks AN-l and AN-2 by their amino acid compositions and these were sequenced. The sequence of cytochrome cW was derived as follows. The N-terminal sequence of the intact protein and of fragment CB-1 from cyanogen bromide cleavage gave the identities of residues 1 through 38. Fragment CB-2 gave the carboxyl-terminal sequence from residue 120 to 135. Peak T-l from the trypsin digest gave residues 34 through 66. An N-terminal sequence of peptide KC-2 from the endoproteinase Lys-C digest gave residues 62 to 102. The KC-2 peptide served as the source of all but one (T-2) of the peptides used in the remaining sequence. The KC-2 peptide was digested with S. aureus V8 protease and this digest, on chromatographing, yielded peptides M-2 (residues 91 to 103), VS-3 (residues 104 to log), and V8-4 (residues 110 to 126). The overlap between V8-2 and V8-3 was provided by the peptide AN-l isolated from endoproteinase-Asp-N digest of EK-2. This peptide contained residues 99 to 109. The overlap between V8-4 and VS-
190
ET
240
cW from
500 nm cW from
600
700
M. aerugnosa
5 was provided by T-2 from the trypsin digest. The cyanogen bromide fragment CB-2 which contains residues 120 to 135 completed the sequence. All positions in the sequence were verified by sequencing peptides a second time or by sequencing other peptides from this protein. The carboxyl terminus proved resistant to carboxy-peptidase analysis. One more peptide was isolated from an endoproteinase Asp-N digest of the
I
I/
1’ I
-0 8 16 24 M I NUTES
250 M. aeFIG. 6. Final
purification
of cytochrome
cm.
MicrocyAs I
aerugkoaa
CEI-2 CB-1 1 ii
i!
60
3 i! I Ii I I !! I I !
I
I
,I II I : : I ,
, j 1 ! i 1 i i i i i i ! i ! i ! \ i 1 ! i I i, ,.2
I
0 FIG. chrome
7. Separation cbW.
231
cm
The sequence is compared to that of the cytochrome from M. aeruginosa (Fig. 2). The identity between the 118 sites of the A.jlosaquae cytochrome with those of M. aeruginosa cytochrome is 48%. The sequences of the cytochromes c5= from these two organisms are 6’7% identical (14). When the amino acid sequences of cytochrome c550and cytochrome c553of M. aeruginosa (14) are compared (Fig. 3), two regions of sequence similarity are evident. The heme binding amino-terminal region of cytochrome cg3, residues 1 through 18, is similar to the heme binding region of cytochrome Cam, residues 28 through 45. Of these 18 residues, 9 are identical and 4 others differ by a minimum base change per codon of 1. The region between residues 93
I
i! i! j!,’
CYTOCHROME
1 I I I
I .o
I
8 16 22 MI NUTES of *CNBr
fragments
of cyto-
I I 1 , 1 I I I I I I I
0.8
cytochrome which yielded residues 119 to 135 on sequence analysis. Both of the C-terminal peptides ended cleanly and the composition analyses in Table I are consistent with the phenylalanine tyrosine carboxy1 terminus.
-
c30
-I
30
70
/60
I / / 11
I
/
w rl E
I 50 g
I /
RESULTS
AND
DISCUSSION
The amino acid sequence of cytochrome c550from M aeruginosa is given in Fig. 1. The primary structure contains 135 amino acids. The amino acid composition calculated from the complete sequence is in agreement with the composition cletermined by amino acid analysis of the hydrolyzed protein which is shown in Table I. Cytochrome cm0 is a polypepticle chain of 135 residues with a calculated molecular weight of 14,917 to which a heme residue and two hydrogens should be added for a total molecular weight of 15,537. Cytochrome c550from A. JEos-aquae was available in modest amounts and a partial sequence for this molecule was obtained.
2 40 s I I
/ I
I
30
/
20
I I
0.2 /
IO
/ I / ~: I
I
I
16
32
48
0 64
MINUTES
FIG. 8. Separation chrome cm.
of trypsin
fragment
T-l
of cyto-
Residue
Leu Glu Leu Asp Glu LYS Thr Leu Thr Ile Thr Leu Asn Asp Ala GUY Glu Ser Val Thr Leu Thr Ser Glu Gln Ala Thr Glu GUY Gln LYS
Position
1 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 30 31
N-terminus CB-1 (nmol 36 32 33 25 31 44 15 28 18 32 17 28 17 13 21 24 18 7 23 10 12 5 6 12 7 13 5 11 13 8 12
Peptide
yield
-_-..-.-
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76
Position
Ile Asp 5r Leu
Lt?U
LYS Thr Asn Asn Asn Val Ser Leu GIY Leu GUY Asp Leu Ala LYS Ala Glu Pro Pro Arg Asp Asn Leu Leu Ala
Thr
Residue
,.
EDMAN Peptide
..--
T-l (nmol) 5 18 5 8 11 12 6 3 8 5 9 6 3 2 3 2 2 3 8 17 9
YIELD FROM AUTOMATED
TABLE
KC-2 (pm4 817 359 465 415 12 196 257 766 960 391 872 403 164 274 481
yield
-..”
91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121
Position
DEGRADATION
II
.-
Val
Ll3.l
Leu His Pro Asn Val Ser Aw Pro Asp Ile Phe Pro Glu Leu Aw Asn Leu Thr Glu Asp Asp Val 5r Asn Val Ala Ala Tyr Met
Residue
OF PEPTIDES
V8-4 b-ml) 3116 7051 2185 2266 2062 1880 1801 1801 1171 1353 1841 1633
vs-2 (nmol) 233 57 112 115 195 54 75 61 74 103 74 25 43
.._ - - -_ -
CB-2 (nmol) 36 37
t2: 22 15 18
KC-2 (pmol) 106 9 39 29 49 23 8
-.
V8-3 (nmol) 45 28 31 44 9 19
Peptide
-
yield
AN-l (pmol) 409 817 970 611 447 1034 103 449 763 179 139
T-2 (nmol) 34 34 7 26 13 20 24 22 16 18 17 29 11
F
Gln GUY LYS
LtW
Leu Phe Val Ala Asn CYS Thr LYE CYS His
10 t9
3
7 13 10 15 18
T-l (nmol) 40 30 (““,
11 8 12 11 (“,
77 78 79 80 81 82 83 84 85 86 87 88 89 90
Glu His Pro Thr Ser 5r ASP GUY Glu ASP ASP Leu Ser Glu
144 8 174 8 146 130 61 84 62 69 62 129 60 43
122 123 124 125 126 127 128 129 130 131 132 133 134 135
Ala Pro Aw Leu ASP Glu Arg Trp GUY GUY Thr Ile Tyr Phe 31 18 21 24 12 15 25 14 15 23 4 11 7 3
832 750 1139 740 1342 ( 1 AN-2 (pmol) 249 375 85 90 286 326 147 177 170 90
8
ABSORBANCE,
230nm
0
5
0
8
230nm 0 ?I
% ACETONITRILE
8
ABSORBANCE, 13 G:
8
T-2
e ul
Note. Values expressed in nanomoles were obtained with the Beckman 896 sequenator and the values expressed in picomoles with the Applied Biosystems microsequencer.
32 33 34 3.5 36 37 38 39 40 41 42 43 44 45
234
COHN
ET AL.
per codon of 1. When the two aligning regions of M. aeruginosa cytochrome c550 were compared to the aligning regions of cytochromes c5% from 11 other genera, the average minimum base difference per codon ranged from 0.76 to 1.0 for the aminoterminal region and from 0.78 to 1.07 for the carboxyl-terminal region. Since cytochrome es3 has clear antecedents in the c type cytochromes of heterotrophic bacteria it would seem to be of more ancient origin than cytochrome c550which has been found only in cyanobacteria and algae (17). The amino acid sequences of these two proteins suggest that the gene for cytochrome MINUTES es3 has been duplicated and one copy used FIG. 11. Separation of 5’. aweus V8 protease fragas the source of two major fragments for ments of peptide KC-2. the construction of the new gene for cytochrome c550. and 130 of cytochrome c550is similar to the Class 1 cytochromes have Met as a sixth carboxyl-terminal sequence, residues 55 to ligancl to the iron, resulting in a shoulder 81 of cytochrome Cam. Of the 2’7 comparable in the absorption spectrum of the oxidized positions, 9 residues are identical and 13 but not the reduced form of the cytoothers differ by a minimum base change chrome at 695 nm (18). In the Class I cytochromes cm this ligancl is attributed to the one conserved Met at residue 54. The ab0.16 sorption spectra of oxidized and reduced M. aeruginosa cytochrome c550are shown in Fig. 4 and there is a 660-nm peak in the re70 duced but not in the oxidized form. This cytochrome has only one Met at residue 119 0.12 and it is not conserved in the A. $os-aquae cytochrome c550. In the alignment of simi ilar regions in Fig. 3, Met 54 of cytochrome $4 c553is in an analogous position to His 92 of OJ cytochrome Cam. 8 Figure 5 shows the far-uv circular diz 0.08 8 chroism spectrum of cytochrome Cam. The 8 spectrum was recorded and analyzed as in Przysiecki et aL (19) to give values of 17% 9 (Y helix, 42% /3 sheet, and 41% random coil. This method is most reliable for estimating helix content, and is consistent with the sequence data which reveal regions of similarity to cytochrome c5%. Approximately one-quarter of the residues of Class I cytochromes are in the helix conformation and there is no p structure. The two 0 IO 20 30 40 similar regions in cytochrome cm and cyMINUTES tochrome cs3 correspond to the principal FIG. 12. Separation of endoproteinase Asp-N fraghelical segments in the Class I cytoments of peptide KC-2. chromes (18).
Microcystis
aeruginosa
Figures 6 through 12 show the chromatograms of the peptide separations and Table II shows the yields for sequence determinations of the peptides. ACKNOWLEDGMENTS
Financial support for this work was provided by Grant DMB-856912 from the Molecular Biology Program of the National Science Foundation and Grant GM-21677 from NIH. This is Journal Paper No. 11,666 from the Purdue University Agricultural Experiment Station.
CYTOCHROME 6. EVANS,
235
c5m P. K., AND KROGMANN,
B&hem
D. W. (1983)
Arch.
Biophys. 227,494-510.
7. YAMANAKA, T., DEKERK, H., AND KAMEN, M. D. (1967) Biochim Biophys. Acta 143,416-424. 8. KAMIMURA, Y., YAMASAKI, T., AND MATSUZAKI, E. (1977) Plant Cd Physiol 18.317-324. 9. Ho, K. K., ULRICH, E. L., KROGMANN, D. W., AND GOMEZ-LOJERO (1979) B&him Biuphys. Acta 545,236-248. 10. KINZEL, P. F., AND PESCHEK, G. A. (1983) FEBS IAt. 162,76-80. 11. BOWES, J. M., STEWART, A. C., AND BENDALL, D. S. (1983) Biochim. Biophys. Ada 725, 210219. 12. ALAM, J., SPRINKLE, J. R., HERMODSON, M. A., AND KROGMANN, D. W. (1984) Biochim. Biophys.
Acta 766,317-321. REFERENCES
1. HOLTON, R. W., AND MYERS, J. (1963) Science 142, 234-235. 2. HOLTON, R. W., AND MYERS, J. (1967) &O&&Z. Biophys. Acta 131,362-374. 3. HOLTON, R. W., AND MYERS, J. (1967) Biochim Biophys. A& 131,375-381. 4. AMBLER, R. (1982) in From Cyclotrons to Cytochromes (Kaplan, N. O., and Robinson, A. B., Eds.), pp. 263-279, Academic Press, London/ New York. 5. PETTIGREW, G. W., AND MOORE, G. R. (1987) Cytochromes c, Biological Aspects, pp. 17-27, Springer-Verlag, Berlin.
13. MAHONEY, W. C., HOGG, R. W., AND HERMODSON, M. A. (1981) J. Biol. Chem. 256,4350-4356. 14. COHN, C. L., HERMODSON, M. A., AND KROGMANN, D. W. (1989) Arch. Biochem Biqphys. 270,219226. 15. MAHONEY, W. C., AND NUTE, P. E. (1979) Arch. Biochem Biophys. 196,64-72. 16. SCHENKEIN, I., LEVY, M., FRANKLIN, E. C., AND FRANGIENE, B. (1977) Arch. B&hem, Biophys. 182,64-70. 17. DICKERSON, R. E. (1971) J. Mol. Evol. 1,26-45. 18. DICKERSON, R. E., AND TIMKOVICH, R. (1976) in The Enzymes (Boyer, P., Ed.), 3rd ed., Vol. 11, pp. 397-547, Academic Press, New York. 19. PRZYSIECKI, C. T., MEYER, T. E., AND CUSANOVICH, M. A. (1985) Biochemistry 24,2542-2549.
The Amino Acid Sequence of Low-Potential Cyanobacterium Microcystis
Cytochrome aeruginosa
c550 from the
CATHLEEN L. COHN,* JAMES R. SPRINKLE,* JAWED ALAM,* MARK HERMODSON,* TERRY MEYER,? AND DAVID W. KROGMANN*pl *Department of Biodwmistry, Purdue University, West I.&aye&e, Indiana hY9OY; and TDepartment Biochemistry, Universi@ of Arizona, Tucson, Arizona 85721 Received
August
22,1988,
and in revised
form
November
of
14,1988
The low-potential cytochrome c550has been purified from the cyanobacterium Microcystis aerugirwsa and its amino acid sequence has been determined. The protein contains 135 amino acid residues with the Cys-X-X-Cys-His heme binding site at residues 37 to 41. The sequence from residue 28 to 45 shows similarity to cytochrome cs3 residues 1 to 18 when the heme binding sites are aligned. Another region of similarity is in the carboxylterminal regions of these two proteins. The two aligning regions of cytochrome c5= correspond to helical segments in other related cytochromes. A partial sequence of cytochrome es0 from Aphanimmenmjlos-aquae was obtained and showed a 48% identity to the sequence of the M aemginosa cytochrome. The single methionine residue in cytochrome c550of M. aeruginosa occurs at position 119 but there is no methionine in this region in the A.jZos-aquae cytochrome, indicating that methionine is not the sixth ligand to the heme iron atom. Histidine 92 is a possible sixth ligand in ikl. aeruginosa cytochrome Cam. The far-uv circular dichroism spectrum indicates that this protein is approximately 17% (Yhelix, 42% p-pleated sheet, and 41% random coil. 0 1989 Academic Press, Inc.
Holton and Myers (1) first described an unusual low-potential, autooxidizable cytochrome c from Anacystis nidulans and later characterized this cytochrome in detail (2,3). Most c type cytochromes belong to Class I as defined by Ambler (4) and their redox potentials range from 0 to +500 mV (5). Cytochromes of Class III are distinguished by lower redox potential and by having several hemes per polypeptide. The cytochrome of Holton and Myers contained a single heme and had a redox potential of -260 mV. Similar low-potential cytochromes have been observed in a number of other cyanobacteria as well as in several eukaryotic algae-a red alga (6), a diatom (‘7), and a green alga (8). This cytochrome has not been found in higher plants. The presence of the cytochrome in 1 To whom
correspondence
should
cyanobacteria may depend on growth conditions (9). A functional role for cytochrome c550in the electron transport reactions supporting cyclic phosphorylation in Photosystem 1 has been postulated (10). Fractionation experiments indicate that the cytochrome may be associated with Photosystem 2 (11). We have published preliminary data on the partial amino acid sequence of this protein (12) and can now report its complete primary structure. This is the first amino acid sequence of a cytochrome of this type. EXPERIMENTAL
PROCEDURES
Materials. The collection and fractionation of Microcystis aeruginxxa and Aphanizomenon jos-aquue were described by Ho et al. (9). Cytochrome ~550 purification was as described by Alam et al. (12). A final purification of the cytochrome single symmetrical peak.
be addressed.
227
0003-9861189
by rpHPLC
yielded
$3.00
Copyright 0 1989 by Academic Presq Inc. All rights of reproduction in any form reserved.
a
COHN ET AL. TABLE I AMINO ACID COMPOSITIONS
OF CYTOCHROME
C~AND
USED IN SEQUENCE ANALYSIS
PEPTIDES
Peptides/residue
Aspartic acid Asparagine Threonine Serine Glutamic acid Glutamine Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Cysteine Methionine Tryptophan
Whole protein
34-66 T-l
106-118 T-2
62-135 KC-2
91-103 vs-2
99-109 AN-l
104-109 vs-3
110-12’7 vs-4
120-135 CB-2
21.4 (12) (9) 12.0 (12) 5.8 (6) 15.0 (12) (3) 7.0 (7) 8.0 (8) 9.1 (9) 7.1(7) 3.9 (4) 20.0 (20) 4.5 (5) 2.7 (3) 2.9 (3) 5.9 (6) 4.8 (5) 1.7 (2) 0.6 (1) (1)
5.2 (1) (4) 2.7 (3) 2.2 (1) 2.7 (1) (1) 2.0 (2) 3.2 (3) 2.6 (3) 2.0 (2) 0.6 (0) 3.7 (4) 0.4 (0) 0.3 (0) 0.9 (1) 3.6 (4) 0.7 (1) 1.0 (2) 0.1(O) (0)
4.0 (2)
13.0 (9)
3.0 (3) 2.8 (3) 7.2 (7) (0) 6.8 (7) 3.2 (3) 5.2 (5) 4.3 (4) 2.7 (3) 9.6 (10) 4.2 (5) 1.5 (2) 2.0 (2) 0.3 (0) 4.5 (5) 0.4 (0) 0.7 (1) (1)
1.9 (2) (1) 0.6 (1) 0.0 (0) 1.2 (2) (0) 0.7 (1) 0.0 (0) 0.3 (0) 0.0 (0) 0.6 (1) 2.0 (2) 0.0 (0) 0.4 (1) 0.0 (0) 0.0 (0) 0.7 (1) 0.0 (0) 0.0 (0) (0)
0.9 (0) (1)
1.0 (1) 0.2 (0) 1.0 (1) (0) 0.0 (0) 0.3 (0) 2.1(2) 1.9 (2) 0.1(O) 1.1 (1) 1.6 (2) 0.0 (0) 0.0 (0) 0.4 (0) 0.0 (0) 0.0 (0) 0.0 (0) (0)
2.1(l) (1) 0.0 (0) 0.9 (1) 1.1(l) (0) 2.9 (3) 0.5 (0) 0.0 (0) 1.0 (1) 1.0 (1) 1.1(l) 0.0 (0) 1.0 (1) 1.0 (1) 0.0 (0) 1.0 (1) 0.0 (0) 0.0 (0) (0)
1.2 (1) 0.0 (0) 0.9 (1) (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 2.0 (2) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.9 (1) 0.0 (0) 0.0 (0) (0)
4.2 (3) (1) 0.0 (0) 0.0 (0) 1.1 (1) (0) 1.0 (1) 0.1(O) 3.1(3) 3.0 (3) 0.0 (0) 2.1(2) 1.6 (2) 0.0 (0) 0.0 (0) 0.0 (0) 0.9 (1) 0.0 (0) 0.8 (1) (0)
1.3 (1) (0) 1.1(l) 0.3 (0) 1.3 (1) UN 1.1(l) 2.2 (2) 1.2 (1) 1.0 (1) 0.9 (1) 2.0 (2) 0.8 (1) 1.0 (1) 0.0 (0) 0.0 (0) 2.0 (2) 0.0 (0) 0.0 (0) (1)
134
33
13
74
13
11
6
18
15
Total
i4j
(2)
Both an Applied Biosystems microsequencer and a Beckman 890 sequenator were used. Sequencer reagents for the Applied Biosystems microsequencer
Val -
Ala
Pro A.-g W-4-(
Leu
numbers
Asp
61”
Ar‘g
Trp
130 Gly
My
T,w
Ile
Tyr
were obtained from Applied Biosystems, Inc. Reagents and procedures for the Beckman 890 sequenator are by Mahoney (13). Endoproteinase Arg-C was
Phe
t4 m-2
-I
-m-2-
FIG. 1. The amino acid sequence of cytochrome c= from M. aacginosa.
Microcgstis
aer&no.sa
CYTOCHROME
229
cm 7.0
10 1.
M.aeru@nosa
LELDEKTLTITLNDAGESVTLTSEQATEGQ
2.
A. flos-aqua8
LELPETIRTVPLhPKEGTYVLSLEPVKEE-
1.
40 KLFVANCTKCHLQGKTKTNNNVSLGLGDLAKAEPPRDN
1. 2.
70 20 LLALIDYLEHPTSYDGEDDLSELHPNVSRPDIFPELRN MKNPITXQEEEEISEI
1. 2.
110 120 LTEDDVYNVAAYMLVAP-RLDERWGGTIYF LrDEPLKAIAEHILLEELVVcTKgOEK
50
30
60
2. ELENYACAQCBAGEVIK~LQLPGLEPEALdGhLPNe 90
100 PSIKSANIE---EN 130
FIG.2. Comparison of cytochromes & from M. aeru@nosa and A. jbs-aquue. purchased from Sigma Chemical Co. and trypsin (TPCK’ treated) from U.S. Biochemicals Corp. Endoproteinase Lys-C, endoproteinase Arg-C, and endoproteinase Asp-N were from Boehringer-Mannheim Biochemicals. The other reagents and enzymes were the same as in Cohn et aL (14). Enzymatic cleavage. Trypsin digestion was performed as described by Mahoney and Nute (15). The protein was dissolved in 1 ml of 8 M urea and diluted with 4 ml of 0.1 M N)4HC03 to give a final pH of 8. A solution of TPCK-treated trypsin (1% by weight of the protein in 0.1 M NH,HCOs) was added and the reaction incubated at 3’7°C for 2 h. A second aliquot (la, w/w) of trypsin was added and the reaction incubated another 3 h. The reaction was terminated by lowering the pH to 3.3 with 1 N HCl and the digest was lyophilized. Endoproteinase Arg-C was used with a modification of the procedure of Schenkein et al. (16). Approximately 60 mg of the cytochrome was dissolved in 1 ml of 8 M ultrapure urea in a stirred reaction vessel at 37°C and 4 ml of 106 mM NHIHCOI was added. Endoproteinase Arg-C (5 U, or 0.02 mg in water) was added. A second, equal aliquot of enzyme was added
2 Abbreviations used: TPCK, L-1-ptosylamino-2phenylethyl chloromethyl ketone; CNBr, cyanogen bromide.
cytochrome
c5So
cytochrome
c55S
after 36 min and the stirring continued for 3 h. The reaction was stopped by addition of 1 N HCl. A brown, flocculent precipitate was removed and the supernatant was fractionated by rpHPLC. Endoproteinase Lys-C and endoproteinase Asp-N were used according to the instructions of the manufacturer. No detergents or urea were needed to dissolve the peptides. Other procedures for enzymatic and chemical cleavage, peptide isolation, and amino acid sequence analysis have been described (14). Peptide im.!ation and sequencing. Table I shows the composition of those peptides used in determining the sequence of the cytochrome. CNBr cleaved cytochrome cW to give two peptides which were separated by rpHPLC. Each peak was rechromatographed and aliquots were taken for sequence analyses and for determination of amino acid composition. Trypsin digestion gave peptides which were separated by rpHPLC. The two largest peaks were rechromatographed and sequenced. Digestion with endoproteinase Lys-C yielded the two peptides which were separated by rpHPLC. Each peak was chromatographed a second time and aliquots were taken for amino acid sequence and composition analysis. Peptide KC-2 of the endoproteinase Lys-C (8 mg) was hydrolyzed with Staphylococcus ou~eus V8 protease and gave 10 peptides. This digest produced 10 major peaks, three of which were used in the sequence determination.
28 45 E G Q K L P V A N C T K C H L Q G K”“““” 1 D G A S I
9s
18 E S & g C A S C fi M G G K"""""
130
PNVSRPDIFPELRNLTEDDVYNVAAYMLVAPRLDERU.'.' 55 EAFGG-------B-&SAE~IEA~&N~V~--AQAEKGbj".'
81
FIG.3. Alignment of similar regions of cytochrome cm with cytochrome cW from M. aerugima
230
COHN
200
300
FIG. 4. Absorption
200
spectra
FIG. 5. CD spectrum ruginosa
210 220 230 NANOMETERS of cytochrome
AL.
400 WAVELENGTH, of cytochrome
Another sample of peptide KC-2 (10 mg) was digested with endoproteinase Asp-N. The resolution of this digest by rpHPLC allowed the identification of peaks AN-l and AN-2 by their amino acid compositions and these were sequenced. The sequence of cytochrome cW was derived as follows. The N-terminal sequence of the intact protein and of fragment CB-1 from cyanogen bromide cleavage gave the identities of residues 1 through 38. Fragment CB-2 gave the carboxyl-terminal sequence from residue 120 to 135. Peak T-l from the trypsin digest gave residues 34 through 66. An N-terminal sequence of peptide KC-2 from the endoproteinase Lys-C digest gave residues 62 to 102. The KC-2 peptide served as the source of all but one (T-2) of the peptides used in the remaining sequence. The KC-2 peptide was digested with S. aureus V8 protease and this digest, on chromatographing, yielded peptides M-2 (residues 91 to 103), VS-3 (residues 104 to log), and V8-4 (residues 110 to 126). The overlap between V8-2 and V8-3 was provided by the peptide AN-l isolated from endoproteinase-Asp-N digest of EK-2. This peptide contained residues 99 to 109. The overlap between V8-4 and VS-
190
ET
240
cW from
500 nm cW from
600
700
M. aerugnosa
5 was provided by T-2 from the trypsin digest. The cyanogen bromide fragment CB-2 which contains residues 120 to 135 completed the sequence. All positions in the sequence were verified by sequencing peptides a second time or by sequencing other peptides from this protein. The carboxyl terminus proved resistant to carboxy-peptidase analysis. One more peptide was isolated from an endoproteinase Asp-N digest of the
I
I/
1’ I
-0 8 16 24 M I NUTES
250 M. aeFIG. 6. Final
purification
of cytochrome
cm.
MicrocyAs I
aerugkoaa
CEI-2 CB-1 1 ii
i!
60
3 i! I Ii I I !! I I !
I
I
,I II I : : I ,
, j 1 ! i 1 i i i i i i ! i ! i ! \ i 1 ! i I i, ,.2
I
0 FIG. chrome
7. Separation cbW.
231
cm
The sequence is compared to that of the cytochrome from M. aeruginosa (Fig. 2). The identity between the 118 sites of the A.jlosaquae cytochrome with those of M. aeruginosa cytochrome is 48%. The sequences of the cytochromes c5= from these two organisms are 6’7% identical (14). When the amino acid sequences of cytochrome c550and cytochrome c553of M. aeruginosa (14) are compared (Fig. 3), two regions of sequence similarity are evident. The heme binding amino-terminal region of cytochrome cg3, residues 1 through 18, is similar to the heme binding region of cytochrome Cam, residues 28 through 45. Of these 18 residues, 9 are identical and 4 others differ by a minimum base change per codon of 1. The region between residues 93
I
i! i! j!,’
CYTOCHROME
1 I I I
I .o
I
8 16 22 MI NUTES of *CNBr
fragments
of cyto-
I I 1 , 1 I I I I I I I
0.8
cytochrome which yielded residues 119 to 135 on sequence analysis. Both of the C-terminal peptides ended cleanly and the composition analyses in Table I are consistent with the phenylalanine tyrosine carboxy1 terminus.
-
c30
-I
30
70
/60
I / / 11
I
/
w rl E
I 50 g
I /
RESULTS
AND
DISCUSSION
The amino acid sequence of cytochrome c550from M aeruginosa is given in Fig. 1. The primary structure contains 135 amino acids. The amino acid composition calculated from the complete sequence is in agreement with the composition cletermined by amino acid analysis of the hydrolyzed protein which is shown in Table I. Cytochrome cm0 is a polypepticle chain of 135 residues with a calculated molecular weight of 14,917 to which a heme residue and two hydrogens should be added for a total molecular weight of 15,537. Cytochrome c550from A. JEos-aquae was available in modest amounts and a partial sequence for this molecule was obtained.
2 40 s I I
/ I
I
30
/
20
I I
0.2 /
IO
/ I / ~: I
I
I
16
32
48
0 64
MINUTES
FIG. 8. Separation chrome cm.
of trypsin
fragment
T-l
of cyto-
Residue
Leu Glu Leu Asp Glu LYS Thr Leu Thr Ile Thr Leu Asn Asp Ala GUY Glu Ser Val Thr Leu Thr Ser Glu Gln Ala Thr Glu GUY Gln LYS
Position
1 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 30 31
N-terminus CB-1 (nmol 36 32 33 25 31 44 15 28 18 32 17 28 17 13 21 24 18 7 23 10 12 5 6 12 7 13 5 11 13 8 12
Peptide
yield
-_-..-.-
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76
Position
Ile Asp 5r Leu
Lt?U
LYS Thr Asn Asn Asn Val Ser Leu GIY Leu GUY Asp Leu Ala LYS Ala Glu Pro Pro Arg Asp Asn Leu Leu Ala
Thr
Residue
,.
EDMAN Peptide
..--
T-l (nmol) 5 18 5 8 11 12 6 3 8 5 9 6 3 2 3 2 2 3 8 17 9
YIELD FROM AUTOMATED
TABLE
KC-2 (pm4 817 359 465 415 12 196 257 766 960 391 872 403 164 274 481
yield
-..”
91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121
Position
DEGRADATION
II
.-
Val
Ll3.l
Leu His Pro Asn Val Ser Aw Pro Asp Ile Phe Pro Glu Leu Aw Asn Leu Thr Glu Asp Asp Val 5r Asn Val Ala Ala Tyr Met
Residue
OF PEPTIDES
V8-4 b-ml) 3116 7051 2185 2266 2062 1880 1801 1801 1171 1353 1841 1633
vs-2 (nmol) 233 57 112 115 195 54 75 61 74 103 74 25 43
.._ - - -_ -
CB-2 (nmol) 36 37
t2: 22 15 18
KC-2 (pmol) 106 9 39 29 49 23 8
-.
V8-3 (nmol) 45 28 31 44 9 19
Peptide
-
yield
AN-l (pmol) 409 817 970 611 447 1034 103 449 763 179 139
T-2 (nmol) 34 34 7 26 13 20 24 22 16 18 17 29 11
F
Gln GUY LYS
LtW
Leu Phe Val Ala Asn CYS Thr LYE CYS His
10 t9
3
7 13 10 15 18
T-l (nmol) 40 30 (““,
11 8 12 11 (“,
77 78 79 80 81 82 83 84 85 86 87 88 89 90
Glu His Pro Thr Ser 5r ASP GUY Glu ASP ASP Leu Ser Glu
144 8 174 8 146 130 61 84 62 69 62 129 60 43
122 123 124 125 126 127 128 129 130 131 132 133 134 135
Ala Pro Aw Leu ASP Glu Arg Trp GUY GUY Thr Ile Tyr Phe 31 18 21 24 12 15 25 14 15 23 4 11 7 3
832 750 1139 740 1342 ( 1 AN-2 (pmol) 249 375 85 90 286 326 147 177 170 90
8
ABSORBANCE,
230nm
0
5
0
8
230nm 0 ?I
% ACETONITRILE
8
ABSORBANCE, 13 G:
8
T-2
e ul
Note. Values expressed in nanomoles were obtained with the Beckman 896 sequenator and the values expressed in picomoles with the Applied Biosystems microsequencer.
32 33 34 3.5 36 37 38 39 40 41 42 43 44 45
234
COHN
ET AL.
per codon of 1. When the two aligning regions of M. aeruginosa cytochrome c550 were compared to the aligning regions of cytochromes c5% from 11 other genera, the average minimum base difference per codon ranged from 0.76 to 1.0 for the aminoterminal region and from 0.78 to 1.07 for the carboxyl-terminal region. Since cytochrome es3 has clear antecedents in the c type cytochromes of heterotrophic bacteria it would seem to be of more ancient origin than cytochrome c550which has been found only in cyanobacteria and algae (17). The amino acid sequences of these two proteins suggest that the gene for cytochrome MINUTES es3 has been duplicated and one copy used FIG. 11. Separation of 5’. aweus V8 protease fragas the source of two major fragments for ments of peptide KC-2. the construction of the new gene for cytochrome c550. and 130 of cytochrome c550is similar to the Class 1 cytochromes have Met as a sixth carboxyl-terminal sequence, residues 55 to ligancl to the iron, resulting in a shoulder 81 of cytochrome Cam. Of the 2’7 comparable in the absorption spectrum of the oxidized positions, 9 residues are identical and 13 but not the reduced form of the cytoothers differ by a minimum base change chrome at 695 nm (18). In the Class I cytochromes cm this ligancl is attributed to the one conserved Met at residue 54. The ab0.16 sorption spectra of oxidized and reduced M. aeruginosa cytochrome c550are shown in Fig. 4 and there is a 660-nm peak in the re70 duced but not in the oxidized form. This cytochrome has only one Met at residue 119 0.12 and it is not conserved in the A. $os-aquae cytochrome c550. In the alignment of simi ilar regions in Fig. 3, Met 54 of cytochrome $4 c553is in an analogous position to His 92 of OJ cytochrome Cam. 8 Figure 5 shows the far-uv circular diz 0.08 8 chroism spectrum of cytochrome Cam. The 8 spectrum was recorded and analyzed as in Przysiecki et aL (19) to give values of 17% 9 (Y helix, 42% /3 sheet, and 41% random coil. This method is most reliable for estimating helix content, and is consistent with the sequence data which reveal regions of similarity to cytochrome c5%. Approximately one-quarter of the residues of Class I cytochromes are in the helix conformation and there is no p structure. The two 0 IO 20 30 40 similar regions in cytochrome cm and cyMINUTES tochrome cs3 correspond to the principal FIG. 12. Separation of endoproteinase Asp-N fraghelical segments in the Class I cytoments of peptide KC-2. chromes (18).
Microcystis
aeruginosa
Figures 6 through 12 show the chromatograms of the peptide separations and Table II shows the yields for sequence determinations of the peptides. ACKNOWLEDGMENTS
Financial support for this work was provided by Grant DMB-856912 from the Molecular Biology Program of the National Science Foundation and Grant GM-21677 from NIH. This is Journal Paper No. 11,666 from the Purdue University Agricultural Experiment Station.
CYTOCHROME 6. EVANS,
235
c5m P. K., AND KROGMANN,
B&hem
D. W. (1983)
Arch.
Biophys. 227,494-510.
7. YAMANAKA, T., DEKERK, H., AND KAMEN, M. D. (1967) Biochim Biophys. Acta 143,416-424. 8. KAMIMURA, Y., YAMASAKI, T., AND MATSUZAKI, E. (1977) Plant Cd Physiol 18.317-324. 9. Ho, K. K., ULRICH, E. L., KROGMANN, D. W., AND GOMEZ-LOJERO (1979) B&him Biuphys. Acta 545,236-248. 10. KINZEL, P. F., AND PESCHEK, G. A. (1983) FEBS IAt. 162,76-80. 11. BOWES, J. M., STEWART, A. C., AND BENDALL, D. S. (1983) Biochim. Biophys. Ada 725, 210219. 12. ALAM, J., SPRINKLE, J. R., HERMODSON, M. A., AND KROGMANN, D. W. (1984) Biochim. Biophys.
Acta 766,317-321. REFERENCES
1. HOLTON, R. W., AND MYERS, J. (1963) Science 142, 234-235. 2. HOLTON, R. W., AND MYERS, J. (1967) &O&&Z. Biophys. Acta 131,362-374. 3. HOLTON, R. W., AND MYERS, J. (1967) Biochim Biophys. A& 131,375-381. 4. AMBLER, R. (1982) in From Cyclotrons to Cytochromes (Kaplan, N. O., and Robinson, A. B., Eds.), pp. 263-279, Academic Press, London/ New York. 5. PETTIGREW, G. W., AND MOORE, G. R. (1987) Cytochromes c, Biological Aspects, pp. 17-27, Springer-Verlag, Berlin.
13. MAHONEY, W. C., HOGG, R. W., AND HERMODSON, M. A. (1981) J. Biol. Chem. 256,4350-4356. 14. COHN, C. L., HERMODSON, M. A., AND KROGMANN, D. W. (1989) Arch. Biochem Biqphys. 270,219226. 15. MAHONEY, W. C., AND NUTE, P. E. (1979) Arch. Biochem Biophys. 196,64-72. 16. SCHENKEIN, I., LEVY, M., FRANKLIN, E. C., AND FRANGIENE, B. (1977) Arch. B&hem, Biophys. 182,64-70. 17. DICKERSON, R. E. (1971) J. Mol. Evol. 1,26-45. 18. DICKERSON, R. E., AND TIMKOVICH, R. (1976) in The Enzymes (Boyer, P., Ed.), 3rd ed., Vol. 11, pp. 397-547, Academic Press, New York. 19. PRZYSIECKI, C. T., MEYER, T. E., AND CUSANOVICH, M. A. (1985) Biochemistry 24,2542-2549.