Ferredoxin and Rubredoxin from Butyribacterium Methylotrophicum: Complete Primary Structures and Construction of Phylogenetic Trees1

J. Biochem. 106, 656-662 (1989)

Ferredoxin and Rubredoxin from Butyribacterium methylotrophicum: Complete Primary Structures and Construction of Phylogenetic Trees1

Kazuhiko Saeki,* Yoshio Yao,** Sadao Wakabayashi,* Gwo-Jenn Shen,*** J. Gregory Zeikus,*** and Hiroshi Matsubara*

* Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560; **Bio-Science Research Laboratory, Research Institute, Sankyo Co., Ltd., Shinagawa-ku, Tokyo 110; and ***Michigan Biotechnology Institute, Lansing, Michigan 48909 and Departments of Biochemistry and Microbiology, Michigan State University, East Lansing, Michigan 48824, U.S.A.

Received for publication, May 22, 1989

Complete amino acid sequences of ferredoxin and rubredoxin from Butyribacterium methylotrophicum, a methylotrophic hetero-acetogen, were determined by combination of protease digestion, Edman degradation, carboxypeptidase digestion, and/or partial acid hydrolysis. The ferredoxin was composed of 55 amino acids with a molecular weight of 5,732 excluding iron and sulfur atoms and showed a typical 2 [4Fe-4S] -type ferredoxin sequence with an internal repeat at the 14-23 and 42-51 positions. The rubredoxin was composed of 53 amino acids with a molecular weight of 5,672 excluding iron atom and showed a sequence similar to those of other anaerobic rubredoxins. The sequences were compared to those of corresponding proteins from six different bacteria to construct phylogenetic trees, which showed essentially the same topology. The relationships between the ferredoxin sequences from this bacterium and those of Clostridium thermoaceticum and Methanosarcina barked, both of which possess a carbonyl-dependent acetyl-CoA metabolic system, are also discussed.

Ferredoxin is a non-heme iron-sulfur electron carrier purification and some properties of a 2 [4Fe-4S]-type protein widely distributed among prokaryotes (1). The ferredoxin and a rubredoxin from B. methylotrophicum amino acid sequences of more than 30 bacterial ferredoxins (Saeki et al. J. Bacteriol. in press). have been determined and used in phylogenetic studies (2- In this study, we determined the amino acid sequences of 5). Rubredoxin is also a non-heme iron-sulfur protein and both ferredoxin and rubredoxin from B. methylotrophicum, substitutes for ferredoxin in some enzymatic reactions, but and compared them with those of the six other bacteria to it is not as common as ferredoxin (6). The primary struc- analyze the phylogenetic relationships. tures of both ferredoxin and rubredoxin have been determined for six bacteria; Clostridium pasteurianum (7, 8), MATERIALS AND METHODS Peptostreptococcus aerogenes (9, 10), Megasphaera elsdenii (11, 12), Chlorobium thiosulfatophilum (13, 14), Materials—Ferredoxin and rubredoxin were purified Desulfovibrio desulfuricans (15, 16), and Desulfovibrio from B. methylotrophicum as described elsewhere (Saeki et gigas (17, 18). al. J. Bacteriol. in press). All the enzymes and reagents Anaerobic bacteria which produce acetic acid, and/or used in this study were as listed previously (27). butyric acid and other fatty acid from C1-compound(s) are Protein Derivatization-After denaturation of the puri- called homo-acetogens or hetero-acetogens (19, 20), and fied proteins, the apoproteins were reduced with 2-mer- the pathway employed for utilization of C1-compound(s) captoethanol and carboxymethylated with iodoacetic acid involves a common carbonyl-dependent route for acetyl- (28). Ferredoxin was denatured at room temperature, CoA synthesis. Purification of either ferredoxin or ru- and rubredoxin at 45•Ž. bredoxin has been reported from a few acetogens (21-24). Amino Acid Analysis-The amino acid compositions of The primary structure of the [4Fe-4S]-type ferredoxin carboxymethyl (Cm-) proteins and peptides were deter- from Clostridium thermoaceticum, which contains fer- mined with an amino acid analyzer (Inca model A-5500) redoxins of both 2 [4Fe-4S]- and [4Fe-4S]-type, has been after 6N HCl hydrolysis for 24h essentially according to determined (25). Spackman et al. (29). Butyribacterium methylotrophicum is a sporeforming Sequence Determination-The amino (N-)terminal se- hetero-acetogen that grows on CO, methanol, formate, or quences of Cm-proteins and peptides were determined by sugars and is very similar to the non-sporeforming species, either manual Edman degradation or a gas-phase protein Eubacterium limosum (19, 26). We have recently reported sequencer (Applied Biosystems, model 470A) equipped 1This research was supported in part by United States Department of with an on-line connected HPLC (model 120A), which was operated according to the manufacturer's instruction. The Energy grant DE-FG02-88ER13719. Abbreviations: C-, carboxyl; N-, amino; Cm-, carboxymethyl; ODS, phenylthiohydantoin (PTH) derivatives of the amino acids octadecylsilane; PTH, phenylthiohydantoin derivative; TFA, tri- in the manual degradation were determined by HPLC (30) fluoroacetic acid. using an octadecylsilane (ODS) column (Shodex ODSpak

656 J. Biochem. B. methylotrophicum Ferredoxin and Rubredoxin 657

F511A, Showa Denko). buffer (pH 5.5) at 37•Ž. S-, C-, and A- refer to staphylococcal protease, chymotrypsin, and partial acid hydrolysis peptides, respectively. RESULTS Cm-ferredoxin (20nmol each) was digested with staphylococcal protease (2.4ƒÊg) and with chymotrypsin (2.4 Sequence Studies of Cm-Ferredoxin-The amino acid μg)in 150μl of 0.1M Tris-HCl buf6er(pH 8.0)at 37℃ for composition of Cm-ferredoxin is shown in Table IS. The 2h. Cm-rubredoxin (50nmol) was partially hydrolyzed N-terminal sequence of Cm-ferredoxin was determined by with 0.1% trifluoroacetic acid (TFA) at 24•Ž for 16h. It (20 a gas-phase sequencer up to the 35th step (Fig.1). The nmol) was also digested with chymotrypsin (2.4ƒÊg) under C-terminal sequence was determined to be -Glu-Asp by the same conditions as those for ferredoxin. The digests and carboxypeptidase Y digestion (Table ITS). partial acid hydrolysis products were, respectively, sepa- The staphylococcal protease digest of Cm-ferredoxin was rated by HPLC in 0.1% TFA with a linear gradient of 0 to separated by HPLC into five peptides, S-1 to S-5 (Fig.1S). 54% acetonitrile on an ODS column (TSK-ODS-120T, Their amino acid compositions are listed in Table IS. The Tosoh, or Shodex ODSpak F511A) at 50•Ž. In the case of amino acid compositions of peptides S-1, S-2, S-4, and S-5 insufficient separation of peptides by this method, the corresponded to residues 1-25, 26-30, 1-7, and 8-25, partially purified chymotryptic peptides were re-chromato- respectively, of the already determined N-terminal se- graphed on the same column with 10mM HCOONH4 (pH quence of the whole protein. Peptide S-2, which contained 6A) instead of TFA. the 27th residue ambiguously determined in the N- Carboxyl (C-)terminal sequences were determined by terminal sequence determination, was completely se- measuring amino acids released after incubation of Cm- quenced by manual Edman degradation. Peptide S-3 was proteins (0.4nmol per determination) with carboxypep- completely sequenced by a gas-phase sequencer and its tidase Y (60ng each) in 20ƒÊl of 0.1M pyridine-acetate C-terminal region corresponded to that of Cm-ferredoxin.

Fig.1. Summary of sequence studies of B. methylotrophicum ferredoxin. S- and C- refer, respectively, to peptides derived by staphylococcal protease and chymotryptic digestion of Cm-ferredoxin. Arrows (→), (⇒), and (⇒)show determination by a gas-phase sequencer, manual Edman degradation, and carboxypeptidase Y digestion, respectively. Dotted arrow represents ambiguous determination.

Fig.2. Summary of sequence studies of B. methylotrophicum rubredoxin. A- and C- refer, respectively, to peptides derived by partial acid hydrolysis and chymotryptic digestion of Cm-rubredoxin. Arrows (•¨), (⇒), and (⇒) show detemina- tion by a gas-phase sequencer, manual Edman degradation, and carboxypeptidase Y digestion, respectively.

Vol.106, No.4, 1989 658 K. Saeki et al.

The N-terminal sequence of Cm-ferredoxin and the com- PTH-amino acids after the 22nd residue became consider- plete sequence of peptides S-3 showed an overlap of ably low. Thus, the 22nd and 23rd residues were assumed Ala-Leu at the residues 34-35, but we could not recover any to be Asn-Gly. peptides corresponding to residues 31-33. Since the sum of The chymotryptic digest of Cm-rubredoxin was separat- amino acid compositions of either set of peptides S-1, S-2, ed into five peptides, C-1' and C-1 to C-4 by HPLC (Fig. and S-3, or peptides S-4, S-5, S-2, and S-3, did not match 5S). The amino acid compositions of peptide C-1, C-2, and that of Cm-ferredoxin (see Table IS), we digested Cm- C-3 (Table HIS) corresponded to residues 1-4, 5-11, and ferredoxin with chymotrypsin. The digest was separated by 12-30, respectively. The N-terminal sequences of peptide HPLC into three peptides, C-1 to C-3 (Figs. 2S and 3S). C-1', which had a low Met content compared to peptide C-1, The amino acid compositions of peptides C-1, C-2, and C-3 and peptide C-1 were determined up to the third step by (Table IS) corresponded to residues 1-2, 3-29, and 30-55, manual Edman degradation (Fig.2). Although the N- respectively. The N-terminal sequence of peptide C-3 was terminus of peptide C-1' could not be identified, the second determined up to the 7th step by manual Edman degrada- and third residues were identical with those of peptide C-1 tion and thus gave an overlap between peptides S-2 and S-3 and Cm-rubredoxin, implying that the Met residue in C-1' (Fig.1). The sum of amino acid compositions of peptides might be oxidized during the purification procedures. The C-1 to C-3 matched the composition of Cm-ferredoxin. peak of peptide C-3 was followed by a minor peak with an The results of the sequence studies of Cm-ferredoxin are identical amino acid composition, and thus it seems likely summarized in Fig.1. The total number of residues was 55 that the peak might contain a fl-conversion product of C-3 and the molecular weight was calculated to be 5,732 at the Asn-Gly sequence mentioned above. Peptide C-4 was excluding iron and sulfur atoms. completely sequenced by a gas-phase sequencer and its Sequence Studies of Cm-Rubredoxin-The amino acid C-terminal region corresponded to that of Cm-rubredoxin. composition of Cm-rubredoxin is shown in Table HIS. The The result gave overlaps with already determined N- N-terminal sequence of Cm-rubredoxin was determined by terminal sequences of Cm-rubredoxin and peptide A-2 at a gas-phase sequencer up to the 33rd step (Fig.2). The the residues 31-33 and 31 respectively (Fig.2). The C-terminal sequence was determined to be -Pro-Glu-Ala by sum of amino acid compositions of peptides C-1, C-2, C-3, carboxypeptidase Y digestion (Table HS). and C-4, matched the composition of Cm-rubredoxin. The partial acid hydrolysis product of Cm-rubredoxin The results of the sequence studies of Cm-rubredoxin are was separated by HPLC into three peptides, A-1 to A-3 summarized in Fig. 2. The total number of residues was 53 (Fig.4S). Their amino acid compositions are listed in Table and the molecular weight was calculated to be 5,672 HIS. The amino acid composition of peptide A-1 corre- excluding an iron atom. sponded to residues 1-19 of the already determined N-terminal sequence of Cm-rubredoxin, and that of A-3 to DISCUSSION the whole protein. Peptide A-2 was sequenced up to the 17th step by a gas-phase sequencer. In both N-terminal We report here the complete amino acid sequences of B. analysis of Cm-rubredoxin and peptide A-2, the yield of methylotrophicum ferredoxin and rubredoxin. This ru-

(A) Ferredoxins B. methylotrophicum C. pasteurianum P. aerogenes M. elsdenii C. thiosulfatophilum D. desulfuricans D. gigas C. thermoaceticum M. barkeri

(B) Rubredoxins B. methylotrophicum C. pasteurianum P. aerogenes M elsdenii C. thiosulfatophilum D. desulfuricans D. gigas Fig.3. Sequence comparison of ferredoxin and rubredoxin from B. methylotrophicum and other bacteria. Ferredoxins and rubredoxins of the following six bacteria are compared: Clostridium pasteurianum (7, 8), Peptostreptococcus aerogenes (9, 10), Megasphaera elsdenii (11, 12), Chlorobium thiosulfatophilum (13, 14), Desulfovibrio desulfuricans (ferredoxin II, 15) (16), and Desulfovibrio gigas (17, 18). In addition, Clostridium thermoaceticum ferredoxin I (25) and Methanosarcina barkeri ferredoxin (31) are also compared. Cluster or iron-binding Cys residues and residues conserved among all of the bacteriacompared are boxed by solid and dotted lines, respectively. Internal repeat sequences in B. methylotrophicum ferredoxin are underlined by thick lines.

J. Biochem. B. methylotrophicum Ferredoxin and Rubredoxin 659

TABLE I. Amino acid difference matrices to interrelate pairs from nine ferredoxins and seven rubredoxins. Values in the lower left half of the matrices are numbers of different amino acids. Values in the upper right half of the matrices are the sum of the branch lengths of the trees. (A) Ferredoxin

(B) Rubredoxin

tion. We determined previously by ESR study (Saeki, et al. J. Bacteriol. in press) that B. methylotrophicum ferredoxin had two [4Fe-4S]-clusters of essentially the same mid- point redox potential as the clusters of C. pasteurianum ferredoxin (32), which was supported by the fact that its sequence showed an alignment of eight Cys typical of Clostridium-2[4Fe-4S]-type ferredoxins. B. methylotrophicum ferredoxin has the longest internal repeat among the known ferredoxin sequences, 10 residues at positions 14-23 and 42-51, implying that the trace of an ancient gene duplication event is well conserved. Rubredoxin sequences seem to be less divergent than ferredoxin sequences; 15 amino acids are conserved in all of the seven rubredoxins, with the exception of D. desulfuricans rubredoxin which lacks seven residues in the middle of the sequence corresponding to the loop out region in the tertiary structure (33). To interrelate the proteins, numbers of amino acid (A) Ferredoxin (B) Rubredoxin replacements (D) were calculated assuming that an inser- Fig.4. Phylogenetic trees based on sequence comparison of tion, shown as a hyphen in Fig.3, represents a substitution. ferredoxin and rubredoxin. Trees are constructed,respectively, The results of calculation are listed as difference matrices essentiallyaccording to the methodof Fitch and Margoliash (34) using in Table I. From the difference matrices, phylogenetic trees aminoacid difference matrices as shownin TableI. of the two proteins were constructed, respectively, essentially according to the method of Fitch and Margoliash (34) except that amino acid difference was used (Fig.4). The bredoxin sequence represents the first report on this ferredoxin tree has very similar topology to that of the one protein from a C1-metabolizing acetogenic bacterium. The previously reported (4,5). The addition of B. methylotrophi- sequences of both proteins are compared with those from cum data suggests that the construction is successful. the six bacteria in Fig. 3. Here, five ferredoxins are 2 To examine the validity of the constructed tree, the [4Fe-4S]-type and one is a [3Fe-4S]-type from D. gigas. reconstructed distance (R) by summing the branch lengths In addition, B. methylotrophicum ferredoxin sequence is of the tree was calculated and is listed in the upper right of compared with those of C. thermoaceticum, a thermophilic the Table I. Furthermore, an index of validity (I), which is acetogen, ferredoxin I (25), and M. barkeri, a methanogen, the root square mean of the difference between D and R ferredoxin (31), because all three organisms are C1- values, was calculated by the equation given below. catabolizing anaerobes that possess a CO-dependent acetyl- CoA metabolic system. The sequences are aligned funda- mentally to match the iron-sulfur cluster binding or iron binding Cys residues, and in the case of ferredoxin, hydro- phobicity or polarity of amino acids is taken into considera- where n represents total sequences compared; Dij, number

Vol.106, No.4, 1989 660 K. Saeki et al.

of amino acid replacement between i-th and j-th sequences; distance between B. methylotrophicum and M. barkeri, and Rij, reconstructed distance between i-th and j-th which is in a separate kingdom of life and whose ferredoxin proteins. The I values for ferredoxin and rubredoxin trees is reported to have a unique [3Fe-4S]-type cluster (38), is are 1.17 and 2.11, respectively. The former value seems great. Determination of more sequences of 2 [4Fe-4S]-type reasonable for a tree of nine sequence comparison, but the ferredoxins and rubredoxins is needed to properly inter- latter value is relatively high as a comparison of seven relate acetogens and methanogens by this method. sequences. However, we conclude it to be still acceptable, because the introduction of the two Desulfovibrio proteins REFERENCES considerably raises the I value and one of them, D. desulfuricans rubredoxin, lacks seven residues in the 1. Mortenson, L. E. & Nakos, G. (1973) in Iron-Sulfur Proteins middle of themolecule as described above. (Lovengerg, W., ed.) Vol.I, pp.37-64, Academic Press, New The two phylogenetic trees independently constructed York have essentially the same topologies but with somewhat 2. Matsubara, H., Hase, T., Wakabayashi, S., & Wada, K. (1980) in different lengths of corresponding branches. This might The Evolution of Protein Structure and Function (Sigman, D. S. & Brazier, M.A. B., eds.) pp.245-266, Academic Press, New York indicate that the probability of inter species gene transfer 3. George, D. G., Hunt, L.T., Yeh, L. -S. L., & Barker, W. C. (1985) events of either ferredoxin or rubredoxin is very low, and J. Mol. Evol. 22, 20-31 that the topology of the two trees reflects the evolutionary 4. Fitch, W. M. & Bruschi, M. (1987) Mol. Biol. Evol. 4, 381-394 history of the seven bacteria compared and the difference of 5. Bruschi, M. & Guerlesquin, F. (1988) FEMS Microbiol. Rev. 54, branch lengths represents the difference of evolution rate 155-176 between the two proteins. These results are contrary to 6. Lovenberg, W. & Walker, W. M. (1978) Methods Enzymol. 53, those of Vogel et al. (35) who reported a comparison of two 340-346 7. Tanaka, M., Nakashima, T., Benson, A., Mower, H., & Yasunobu, phylogenetic trees constructed with three ferredoxins and K. T. (1966) Biochemistry 5, 1666-1681 rubredoxins from C. pasteurianum, P. aerogenes, and D. 8. Herriott, J. R., Watenpaugh, K. D., Sieker, L. C., & Jensen, L. H. gigas, and stated that the two were significantly different. (1973) J. Mol. Biol. 80, 423-432 However, this discrepancy seems superficial because the 9. Tsunoda, J. N., Yasunobu, K. T., & Whiteley, H. R. (1966) J. Biol. relative branch length ratios in the two cases are very Chem. 223, 6262-6272 similar to one another, if we focus on only the three bacteria 10. Bachmayer, H., Yasunobu, K. T., & Whiteley, H. R. (1967) Biochem. Biophys. Res. Commun. 26, 435-440 used by them. Their conclusion is probably accounted for 11. Yasunobu, K. T. & Tanaka, M. (1973) Syst. Zool. 22, 570-589 the small number of sequences compared and by the 12. Bachmayer, H., Yasunobu, K. T., Peel, J. L., & Mayhew, S. difference of evolution rate of the two proteins especially in (1968) J. Biol. Chem. 243, 1O22-1030 Desulfovibrio, even though the two have approximately the 13. Hase, T., Wakabayashi, S., Matsubara, H., Evans, M. C. W., & same number of amino acids. This might be due to stronger Jennings, J. V. (1978) J. Biochem. 83, 1321-1325 conformational constraint being needed to chelate an iron 14. Woolley, K. & Meyer, T. E. (1987) Eur. J. Biochem. 163, 161- 166 atom in rubredoxin than in ferredoxin to survive during 15. Guerlesquin, F., Bruschi, M., Bovier-Lapiere, G., Bonicel, J., & evolution. Couchoud, P. (1983) Biochimie 65, 43-47 Notably, the most recent branching points of B. meth- 16. Hormel, S., Walsh, K. A., Prickril, B. C., Titani, K., LeGall, J., & ylotrophicum in both trees are shared with the green sulfur Sieker, L. C. (1986) FEBS Lett. 201, 147-150 photosynthetic bacterium C. thiosulfatophilum. At present, 17. Bruschi, M. (1979) Biochem. Biophys. Res. Commun. 91, 623- we have no good explanation for this close relationship 628 between the two bacteria. However, it might be noteworthy 18. Bruschi, M. (1976) Biochem. Biophys. Res. Commun. 70, 615- 621 that members of Chlorobiaceae are also strictly anaerobic, 19. Zeikus, J. G., Kerby, R., & Krzycki, J. A. (1985) Science 227, can grow on CO2 and H2 under light and do not need the 1167-1173 Calvin cycle for C1 synthesis, and some of them are known 20. Ljungdahl, L. G. (1986) Ann. Rev. Microbiol. 40, 415-450 to excrete organic substances into growth culture (36). 21. Yang, S. -S., Ljungdahl, L. G., & LeGall, J. (1977) J. Bacteriol. In the ferredoxin tree the distance between B meth- 130, 1084-1090 22. Yang, S. -S., Ljungdahl, L. G., Dervartanian, D. V., & Watt, G. D. ylotrophicum and C. thermoaceticum is great, when (1980) Biochim. Biophys. Acta 590, 24-33 compared to other fermentative bacteria. This may indi- 23. Elliott, J. I. & Ljungdahl, L. G. (1982) J. Bacteriol. 151, 328-333 cate that homo- and hetero-acetogens, which have car- 24. Ragsdale, S. W. & Ljungdahl, L. G. (1984) J. Bacteriol. 157, 1-6 bonyl-dependent pathways for acetyl-CoA synthesis, do 25. Elliott, J. I., Yang, S. -S., Ljungdahl, L. G., Travis, J., & Reilly, not represent a single entity. However, we cannot exclude C.F. (1982) Biochemistry 21, 3294-3298 the possibility that the other 2[4F-4S]-type ferredoxin 26. Zeikus, J. G., Lynd, L. H., Thompson, T. E., Krzycki, J. A., (ferredoxin II) in C. thermoaceticum (23) might have a Weimer, P. J., & Hegge, P. W. (1980) Curr. Microbiol. 3, 381-386 sequence closely similar to that of B. methylotrophicum 27. Wakabayashi, S., Matsubara, H., Kim, C. H., & King, T. E. (1982) J. Biol. Chem. 257, 9335-9344 protein. Recently, Fukuyama et al. elucidated by X-ray 28. Crestfield, A. M., Moore, S., & Stein, W. H. (1963) J. Biol. Chem. crystallographic determination that the region where 238, 622-627 Clostridium 2[4Fe-4S]-type ferredoxin had a second iron- 29. Spackman, D. H., Stein, W. H., & Moore, S. (1958) Anal. Chem. sulfur cluster was occupied by an a-helix in Bacillus 30, 1190-1206 thermoproteolyticus [4Fe-4S]-type ferredoxin, and stated 30. Zimmerman, C.L., Appella, E., & Pisano, J. J. (1977) Anal. that this replacement from iron-sulfur cluster to a-helix Biochem. 77, 569-573 might be common through [4Fe-4S]-type ferredoxins 31. Hausinger, R. P., Moura, I., Moura, J. J. G., Xavier, A. V., Santos, M. H., LeGall, J., & Howard, J. B. (1982) J. Biol. Chem. 257, (37). It might be speculated that once one of the two 14192-14197 [4Fe-4S] clusters is deleted, the evolution rate becomes 32. Prince, R. C. & Adams, M. W. W. (1987) J. Biol. Chem. 262, faster in the region which does not bind the cluster. The 5125-5128

J. Biochem. B. methylotrophicum Ferredoxin and Rubredoxin 661

33. Watenpaugh, K. D., Sieker, L. C., Herriott, J. R., & Jensen, L. H. & Sistrom, W. R., eds.) pp.3-18, Plenum Press, New York (1973) Acta Cryst. B29, 943-956 37. Fukuyama, K., Nagahara, Y., Tsukihara, T., Katsube, Y., Hase, 34. Fitch, W. M. & Margoliash, E. (1967) Science 155, 279-284 T., & Matsubara, H. (1988) J. Mol. Biol. 199, 183-193 35. Vogel, H., Bruschi, M., & LeGall, J. (1977) J. Mol. Evol. 9, 111- 38. Mourn, I., Mourn, J. J. G., Huynh, B. -H., Santos, M. H., LeGall, 119 J., & Xavier, A. V. (1982) Eur. J. Biochem. 126, 95-98 36. Pfennig, N. (1978) in The Photosynthetic Bacteria (Clayton, R. K.

Supplemental Materials

Table IS. Amino acid compositions of Cm-ferredoxin, staphylococcal protease peptides and chymotrypsin peptides. Table IIS. Carboxypeptidase Y digestion of Cm-ferredoxin and Cm-rubredoxin.

Values in parentheses aremol yield of residue permol Cm-protein.

Values in parentheses are deduced from sequences. Cys was determined as Cm-cysteine. Some amino acids (eg, Ile, Val) were recovered in poor yield due to incomplete hydrolysis. n.d., not determined.

Table IIIS. Amino acid compositions of Cm-rubredoxin, mild acid cleaved peptides and staphylococcal protease peptides.

Fig. IS. Separation of staphylococcal protease digest of Cm-ferredoxin on a

TSK-ODS-120T column. Flow rate was 0.8 mVmin.

Values in parentheses are deduced from sequences. Cys was determined as Cm-cysteine. Some amino acids (eg, Ile, Val) were recovered in poor yield due to incomplete hydrolysis. n.d., not determined.

Fig. 3S. Separation of Peptides C-2 and C-3 on a Shodex

ODSpak F511A column. Flow rate was 0.8 mVmin.

Fig. 2S. Separation of chymotryptic digest of Cm-ferredoxin on a Shodex ODSpak F511A

column. Flow rate was 0.8 mVmin. Vol.106, No.4, 1989 662 K. Saeki et al.

Fig. 4S. Separation of partial acid hydrolysis product of Cm-rubredoxin on a

TSK-ODS-120T column. Flow rate was 0.8 mVmin.

Fig. 55. Separation of chymotryptic digest of Cm-rubredoxin on a Shodex ODSpak F511A

column. Flow rate was 0.8 mVmin.

J. Biochem.