COMMUNICA TION
J. Biochem. 108, 7 8 (1990)
Site-Directedly Mutated Human Cytochrome c Which Retains Heme c via Only One Thioether Bond Yoshikazu Tanaka,* Ichiro Kubota," Teruo Amachi,' Hajime Yoshizumi,* and Hiroshi Matsubara*** 'Institute for Fundamental Research, Research Center, Suntory Ltd., Shimamoto-cho, Mishima-gun, Osaka 618; "Bio-Pharma Tech. Center, Suntory Ltd., Chiyoda-cho, Oura-gun, Gunma 370-05; and ***Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560 Received for publication, April 10, 1990
Although Cys-14 (human numbering) of cytochrome c was conserved during its molecular evolution and it is supposed to be essential for most cytochromes c to retain heme c via two thioether bonds, a site-directedly mutated human cytochrome c which has an alanine residue at this position and only one thioether bond through Cys-17 turns out to be functional. This shows that Cys-14 is not essential. The absorption spectrum of the atypical cytochrome c is red shifted, and similar to those of Euglena and Crithidia cytochromes c, which also have only one thioether bond [Pettigrew, G.W., Leaver, J.L., Meyer, T.E., & Ryle, A.P. (1975) Biochem. J. 147, 291-302].
Cytochrome c is a heme c-containing protein found in the mitochondria of all eukaryotic cells and which functions as an essential component of the energy-yielding mitochondrial electron transfer system. It is one of the best understood proteins with regard to structure-function and evolutionary relationships (1-3). Some residues were invariant or conserved during its molecular evolution. Various in vivo or in vitro mutated cytochromes c have been obtained and some evolutionarily invariant residues have been found to be unessential for cytochrome c to function (2-10). We previously reported that the human cytochrome c gene could complement the CYC1 (iso-1 cytochrome c gene) deficiency in yeast (11), and have implied that Cys-14, Arg-38, and Gly-84 are unessential, as judged through the use of the yeast complementation system (12). Functional expression of rat cytochrome c in yeast was also reported (13, 14). Cys-14 is one of the conserved residues and covalently binds heme c via one of two thioether linkages. Among eukaryotic cytochromes c, Euglena and Crithidia cytochromes c are exceptional having an alanine residue at this position and red shifted a peak maxima (15). While Cys -19 of yeast iso-1 cytochrome c, which correspond to Cys-14 of human cytochrome, is believed to be essential for heme c attachment (3), we tentatively implied that a mutated human cytochrome c containing Ala-14 instead of Cys-14 (C14A cytochrome c) was partly functional in yeast (12) and that Cys-14 might be important but unessential. We could not, however, exclude the possibility in the previous study (12) that some kinds of suppressional mutations occurred in the coding region of the mutated cytochrome c gene. We have now confirmed the mutation and characterized the mutated human cytochrome c, as reported in this paper. A recombinant yeast [XS-30-2BACYC1 (Mat a, Ieu2, his3, ura3, trpl, cycl::LEU2)] containing a mutated human cytochrome c gene which encodes Ala-14 instead of Cys-14 (12) was cultivated in the lactate medium [0.67% Vol. 108, No. 1, 1990
yeast nitrogen base without amino acids (Difco), 2% DL-sodium lactate, 0.05% yeast extract (Difco)] with 20 /^g/ml of histidine and uracil. A plasmid was recovered from each of eight clones after cultivation by the conventional method (16). All of the human cytochrome c genes on the plasmids were sequenced and confirmed to contain an alanine codon (GCT) instead of a cysteine codon (TGT) at the position. No other unexpected mutations were observed in the gene. The results indicate that C14A cytochrome c really functions in yeast and complements the CYC1 deficiency. The transformant was cultivated and C14A cytochrome c was purified from the cultivated yeast as described in the previous paper (11). The cytochrome c was separated into two fractions by linear NaCl gradient chromatography on a CM52 column. The ratio of the lower affinity fraction (fraction 1) and the higher affinity fraction (fraction 2) was about 1 to 4. This was also true for the recombinant native human cytochrome c (Y. Tanaka, unpublished data). The cytochrome c in fractions 1 and 2 had the same amino acid composition and expectedly contained one more alanine residue than the native human cytochrome c (Table I). One lysine residue seemed to be trimethylated, as observed in the recombinant native human and rat cytochromes c (11, 14). The amino terminal sequences were determined with a gas phase sequencer (Applied Biosystems, 470A). The amino-terminus of the cytochrome c in fraction 1 was blocked and supposed to be acetylated, like a part of rat cytochrome c produced by the recombinant yeast (14). The amino terminal sequence of fraction 2 was Gly-Asp-ValGlu-Lys-Gly-Lys-Lys-Ile-Phe-De-Met-Lys-Ala-Ser-Gln-XHis (X: no residue was detected). This clearly indicated that C14A cytochrome c was expressed in yeast and that C14A cytochrome c partly complemented the CYCl deficiency. Cys-14 thus turned out to be unessential for human cytochrome c to function in yeast. It is also interesting to note that only about 20% of the recombinant human cytochrome c was acetylated at the amino-terminus, while
Y. Tanaka et al. TABLE I.
Amino acid compositions of the cytochromes
c
Fig. 1. Visible spectra of the reduced and oxidized C14A cytochrome c. Cytochrome c oxidized with ferricyanide was resolved in 10 mM sodium phosphate buffer, pH 7.0. The cytochrome c was reduced with dithionite. The reduced and oxidized spectra are shown by solid and dotted lines, respectively. Peak maxima are also shown.
420
TMLys, trimethyllysine; n.d., not determined; the compositions of the C14A and native human cytochromes c were calculated from their sequences. Fraction 2 C14A Native human Fraction 1 Asp Thr Ser Glu Pro Gly Ala Val Met He Leu Tyr Phe
TMLys Lys His Arg Cys Trp
8.1 6.4 2.2
8.1 6.6 2.2
10.4
10.3
3.8
3.7
12.6
12.5
11
1A
3.2 2.7 6.8 6.2 4.9 2.9 0.5
3.2 2.7 6.3 6.0 4.6 2.9 0.5
16.4
16.3
2.5 2.4
2.8 2.2
n.d. n.d.
n.d. n.d.
8 7 2 10 4 13 7 3 3 8 6 5 3 0 18 3 2 1 1
8 7 2 10 4 13 6 3 3 8 6 5 3 0 18 3 2 2 1
0. 5 413
z
2 o C/3
556
400 500 WAVELENGTH
600 (nm)
REFERENCES
about 70% of the recombinant rat cytochrome c was acetylated (14), in spite of the similarity in their primary structures. The reason for this difference is uncertain at present. The absorption spectrum of the fraction 2 cytochrome c is shown in Fig. 1. The fraction 1 cytochrome c had the same absorption spectrum. The a peak maximum was red shifted to 556 ran. The atypical cytochromes c of Euglena and Crithidia also had red shifted a peak maxima. The a peak maximum of reduced pyridine ferrohemochrome was 553 nm, which was consistent with those of Euglena and Crithidia cytochromes c (15). This was also true for Euglena cytochrome c, (17, 18). The ratio of the absorbance at the reduced y peak to that at the reduced a peak was 5.7. This value is higher than that of human cytochrome c, 4.8 (11), and is similar to those of Euglena and Crithidia cytochromes c (6.0 and 5.65, respectively, 15). A red shifted absorption spectrum and a higher ratio seem to be common features of cytochromes c which retain heme c via only one thioether bond. Although the a peaks of the protozoan cytochromes c were asymmetrical (15), that of the C14A cytochrome c was symmetrical and seemed to be broader. We have clearly shown that C14A human cytochrome c is functionally expressed in yeast and that Cys-14 is unessential for human cytochrome c to function, at least partly. Cys-19 of yeast iso-1 cytochrome c is believed to be essential for covalent heme attachment and import into mitochondria (3). It is interesting that Cys-14 of heterologous human cytochrome c can be replaced by an alanine residue in yeast while Cys-19 of yeast iso-1 cytochrome c cannot be replaced by other residues (3). The ability of human cytochrome c to stabilize heme c via only one thioether bond should depend on its tertiary structure, which might differ to a certain extent from that of yeast iso-1 cytochrome c. Further characterization of C14A cytochrome c is in progress and will be reported in the future.
1. Dayhoff, M.O. (1972) Atlas of Protein Sequence and Structure Vol. 5, National Biomedical Research Foundation, Silver Spring 2. Hampsey, D.M., Das, G., & Sherman, F. (1986) J. Biol. Chem. 261, 3259-3271 3. Hampsey, D.M., Das, G., & Sherman, F. (1988) FEBS Lett. 231, 275-283 4. Sherman, F., Stewart, J.W., Parker, J.H., Inhaber, E., Shipman, N.A., Putterman, G.I., Gardisky, R.L., & Margoliash, E. (1968) J. Biol. Chem, 243, 5446-5456 5. Schweingrunber, M.E., Sherman, F., & Stewart, J.W. (1979) J. Biol. Chem. 254, 4132-4143 6. Holzshu, D., Principio, L., Conklin, K.T., Hickey, D.R., Short, J., Rao, R., McLendon, G., & Sherman, F. (1987) J. Biol. Chem. 262, 7125-7131 7. Ernst, J.F., Hampsey, D.M., Stewart, J.W., Rackovsky, S., Goldstein, D., & Sherman, F. (1985) J. Biol. Chem. 260, 1322513236 8. Das, G., Hickey, D.R., Principio, L., Taylor-Conklin, K., Short, J., Miller, J.R., McLendon, G., & Sherman, F. (1988) J. Biol. Chem. 263, 18290-18297 9. Luntz, T.L., Schejter, A., Garber, E.A.E., & Margoliash, E. (1989) Proc. Natl. Acad. Sci. U.S. 86, 3524-3528 10. Gooley, P.R. & MacKenzie, N.E. (1989) FEBS Lett. 260, 225228 11. Tanaka, Y., Ashikari, T., Shibano, Y., Amachi, T., Yoshizumi, H., & Matsubara, H. (1988) J. Biochem. 103, 954-961 12. Tanaka, Y., Ashikari, T., Shibano, Y., Amachi, T., Yoshizumi, H., & Matsubara, H. (1988) J. Biochem. 104, 477-480 13. Scarpulla, R.C. & Nye, S.H. (1986) Proc. Nad. Acad. Sci U.S. 83, 6352-6356 14. Clements, J.M., O'Connell, L.I., Tsunasawa, S., & Sherman, F. (1989) Gene 83, 1-14 15. Pettigrew, G.W., Leaver, J.L., Meyer, T.E., & Ryle, A.P. (1975) Biochem. J. 147, 291-302 16. Sherman, F., Fink, G.R., & Hicks, J.B. (1987) Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor 17. Mukai, K., Yoshida, M., Toyosaki, H., Yao, S., Wakabayashi, S., & Matsubara, H. (1989) Eur. J. Biochem. 178, 649-656 18. Mukai, K., Wakabayashi, S., & Matsubara, H. (1989) J. Biochem. 106, 479-482
J. Biochem.
J. Biochem. 108, 7 8 (1990)
Site-Directedly Mutated Human Cytochrome c Which Retains Heme c via Only One Thioether Bond Yoshikazu Tanaka,* Ichiro Kubota," Teruo Amachi,' Hajime Yoshizumi,* and Hiroshi Matsubara*** 'Institute for Fundamental Research, Research Center, Suntory Ltd., Shimamoto-cho, Mishima-gun, Osaka 618; "Bio-Pharma Tech. Center, Suntory Ltd., Chiyoda-cho, Oura-gun, Gunma 370-05; and ***Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560 Received for publication, April 10, 1990
Although Cys-14 (human numbering) of cytochrome c was conserved during its molecular evolution and it is supposed to be essential for most cytochromes c to retain heme c via two thioether bonds, a site-directedly mutated human cytochrome c which has an alanine residue at this position and only one thioether bond through Cys-17 turns out to be functional. This shows that Cys-14 is not essential. The absorption spectrum of the atypical cytochrome c is red shifted, and similar to those of Euglena and Crithidia cytochromes c, which also have only one thioether bond [Pettigrew, G.W., Leaver, J.L., Meyer, T.E., & Ryle, A.P. (1975) Biochem. J. 147, 291-302].
Cytochrome c is a heme c-containing protein found in the mitochondria of all eukaryotic cells and which functions as an essential component of the energy-yielding mitochondrial electron transfer system. It is one of the best understood proteins with regard to structure-function and evolutionary relationships (1-3). Some residues were invariant or conserved during its molecular evolution. Various in vivo or in vitro mutated cytochromes c have been obtained and some evolutionarily invariant residues have been found to be unessential for cytochrome c to function (2-10). We previously reported that the human cytochrome c gene could complement the CYC1 (iso-1 cytochrome c gene) deficiency in yeast (11), and have implied that Cys-14, Arg-38, and Gly-84 are unessential, as judged through the use of the yeast complementation system (12). Functional expression of rat cytochrome c in yeast was also reported (13, 14). Cys-14 is one of the conserved residues and covalently binds heme c via one of two thioether linkages. Among eukaryotic cytochromes c, Euglena and Crithidia cytochromes c are exceptional having an alanine residue at this position and red shifted a peak maxima (15). While Cys -19 of yeast iso-1 cytochrome c, which correspond to Cys-14 of human cytochrome, is believed to be essential for heme c attachment (3), we tentatively implied that a mutated human cytochrome c containing Ala-14 instead of Cys-14 (C14A cytochrome c) was partly functional in yeast (12) and that Cys-14 might be important but unessential. We could not, however, exclude the possibility in the previous study (12) that some kinds of suppressional mutations occurred in the coding region of the mutated cytochrome c gene. We have now confirmed the mutation and characterized the mutated human cytochrome c, as reported in this paper. A recombinant yeast [XS-30-2BACYC1 (Mat a, Ieu2, his3, ura3, trpl, cycl::LEU2)] containing a mutated human cytochrome c gene which encodes Ala-14 instead of Cys-14 (12) was cultivated in the lactate medium [0.67% Vol. 108, No. 1, 1990
yeast nitrogen base without amino acids (Difco), 2% DL-sodium lactate, 0.05% yeast extract (Difco)] with 20 /^g/ml of histidine and uracil. A plasmid was recovered from each of eight clones after cultivation by the conventional method (16). All of the human cytochrome c genes on the plasmids were sequenced and confirmed to contain an alanine codon (GCT) instead of a cysteine codon (TGT) at the position. No other unexpected mutations were observed in the gene. The results indicate that C14A cytochrome c really functions in yeast and complements the CYC1 deficiency. The transformant was cultivated and C14A cytochrome c was purified from the cultivated yeast as described in the previous paper (11). The cytochrome c was separated into two fractions by linear NaCl gradient chromatography on a CM52 column. The ratio of the lower affinity fraction (fraction 1) and the higher affinity fraction (fraction 2) was about 1 to 4. This was also true for the recombinant native human cytochrome c (Y. Tanaka, unpublished data). The cytochrome c in fractions 1 and 2 had the same amino acid composition and expectedly contained one more alanine residue than the native human cytochrome c (Table I). One lysine residue seemed to be trimethylated, as observed in the recombinant native human and rat cytochromes c (11, 14). The amino terminal sequences were determined with a gas phase sequencer (Applied Biosystems, 470A). The amino-terminus of the cytochrome c in fraction 1 was blocked and supposed to be acetylated, like a part of rat cytochrome c produced by the recombinant yeast (14). The amino terminal sequence of fraction 2 was Gly-Asp-ValGlu-Lys-Gly-Lys-Lys-Ile-Phe-De-Met-Lys-Ala-Ser-Gln-XHis (X: no residue was detected). This clearly indicated that C14A cytochrome c was expressed in yeast and that C14A cytochrome c partly complemented the CYCl deficiency. Cys-14 thus turned out to be unessential for human cytochrome c to function in yeast. It is also interesting to note that only about 20% of the recombinant human cytochrome c was acetylated at the amino-terminus, while
Y. Tanaka et al. TABLE I.
Amino acid compositions of the cytochromes
c
Fig. 1. Visible spectra of the reduced and oxidized C14A cytochrome c. Cytochrome c oxidized with ferricyanide was resolved in 10 mM sodium phosphate buffer, pH 7.0. The cytochrome c was reduced with dithionite. The reduced and oxidized spectra are shown by solid and dotted lines, respectively. Peak maxima are also shown.
420
TMLys, trimethyllysine; n.d., not determined; the compositions of the C14A and native human cytochromes c were calculated from their sequences. Fraction 2 C14A Native human Fraction 1 Asp Thr Ser Glu Pro Gly Ala Val Met He Leu Tyr Phe
TMLys Lys His Arg Cys Trp
8.1 6.4 2.2
8.1 6.6 2.2
10.4
10.3
3.8
3.7
12.6
12.5
11
1A
3.2 2.7 6.8 6.2 4.9 2.9 0.5
3.2 2.7 6.3 6.0 4.6 2.9 0.5
16.4
16.3
2.5 2.4
2.8 2.2
n.d. n.d.
n.d. n.d.
8 7 2 10 4 13 7 3 3 8 6 5 3 0 18 3 2 1 1
8 7 2 10 4 13 6 3 3 8 6 5 3 0 18 3 2 2 1
0. 5 413
z
2 o C/3
556
400 500 WAVELENGTH
600 (nm)
REFERENCES
about 70% of the recombinant rat cytochrome c was acetylated (14), in spite of the similarity in their primary structures. The reason for this difference is uncertain at present. The absorption spectrum of the fraction 2 cytochrome c is shown in Fig. 1. The fraction 1 cytochrome c had the same absorption spectrum. The a peak maximum was red shifted to 556 ran. The atypical cytochromes c of Euglena and Crithidia also had red shifted a peak maxima. The a peak maximum of reduced pyridine ferrohemochrome was 553 nm, which was consistent with those of Euglena and Crithidia cytochromes c (15). This was also true for Euglena cytochrome c, (17, 18). The ratio of the absorbance at the reduced y peak to that at the reduced a peak was 5.7. This value is higher than that of human cytochrome c, 4.8 (11), and is similar to those of Euglena and Crithidia cytochromes c (6.0 and 5.65, respectively, 15). A red shifted absorption spectrum and a higher ratio seem to be common features of cytochromes c which retain heme c via only one thioether bond. Although the a peaks of the protozoan cytochromes c were asymmetrical (15), that of the C14A cytochrome c was symmetrical and seemed to be broader. We have clearly shown that C14A human cytochrome c is functionally expressed in yeast and that Cys-14 is unessential for human cytochrome c to function, at least partly. Cys-19 of yeast iso-1 cytochrome c is believed to be essential for covalent heme attachment and import into mitochondria (3). It is interesting that Cys-14 of heterologous human cytochrome c can be replaced by an alanine residue in yeast while Cys-19 of yeast iso-1 cytochrome c cannot be replaced by other residues (3). The ability of human cytochrome c to stabilize heme c via only one thioether bond should depend on its tertiary structure, which might differ to a certain extent from that of yeast iso-1 cytochrome c. Further characterization of C14A cytochrome c is in progress and will be reported in the future.
1. Dayhoff, M.O. (1972) Atlas of Protein Sequence and Structure Vol. 5, National Biomedical Research Foundation, Silver Spring 2. Hampsey, D.M., Das, G., & Sherman, F. (1986) J. Biol. Chem. 261, 3259-3271 3. Hampsey, D.M., Das, G., & Sherman, F. (1988) FEBS Lett. 231, 275-283 4. Sherman, F., Stewart, J.W., Parker, J.H., Inhaber, E., Shipman, N.A., Putterman, G.I., Gardisky, R.L., & Margoliash, E. (1968) J. Biol. Chem, 243, 5446-5456 5. Schweingrunber, M.E., Sherman, F., & Stewart, J.W. (1979) J. Biol. Chem. 254, 4132-4143 6. Holzshu, D., Principio, L., Conklin, K.T., Hickey, D.R., Short, J., Rao, R., McLendon, G., & Sherman, F. (1987) J. Biol. Chem. 262, 7125-7131 7. Ernst, J.F., Hampsey, D.M., Stewart, J.W., Rackovsky, S., Goldstein, D., & Sherman, F. (1985) J. Biol. Chem. 260, 1322513236 8. Das, G., Hickey, D.R., Principio, L., Taylor-Conklin, K., Short, J., Miller, J.R., McLendon, G., & Sherman, F. (1988) J. Biol. Chem. 263, 18290-18297 9. Luntz, T.L., Schejter, A., Garber, E.A.E., & Margoliash, E. (1989) Proc. Natl. Acad. Sci. U.S. 86, 3524-3528 10. Gooley, P.R. & MacKenzie, N.E. (1989) FEBS Lett. 260, 225228 11. Tanaka, Y., Ashikari, T., Shibano, Y., Amachi, T., Yoshizumi, H., & Matsubara, H. (1988) J. Biochem. 103, 954-961 12. Tanaka, Y., Ashikari, T., Shibano, Y., Amachi, T., Yoshizumi, H., & Matsubara, H. (1988) J. Biochem. 104, 477-480 13. Scarpulla, R.C. & Nye, S.H. (1986) Proc. Nad. Acad. Sci U.S. 83, 6352-6356 14. Clements, J.M., O'Connell, L.I., Tsunasawa, S., & Sherman, F. (1989) Gene 83, 1-14 15. Pettigrew, G.W., Leaver, J.L., Meyer, T.E., & Ryle, A.P. (1975) Biochem. J. 147, 291-302 16. Sherman, F., Fink, G.R., & Hicks, J.B. (1987) Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor 17. Mukai, K., Yoshida, M., Toyosaki, H., Yao, S., Wakabayashi, S., & Matsubara, H. (1989) Eur. J. Biochem. 178, 649-656 18. Mukai, K., Wakabayashi, S., & Matsubara, H. (1989) J. Biochem. 106, 479-482
J. Biochem.
