Comparative Study of the Primary Structures of Cytochrome B5 from Four Species* Akira Tsugitat, Midori Kobayashi, Seiji Tani, Sukei Kyo, M

Proceedings ofthe National Academy ofSciences Vol. 67, No. 1, pp. 442-447, September 1970

Comparative Study of the Primary Structures of Cytochrome b5 from Four Species* Akira Tsugitat, Midori Kobayashi, Seiji Tani, Sukei Kyo, M. A. Rashidt, Yukuo Yoshida, Toshimasa Kajihara, and Bunji Hagihara LABORATORY OF MOLECULAR GENETICS AND DEPARTMENT OF BIOCHEMISTRY, OSAKA UNIVERSITY MEDICAL SCHOOL, OSAKA, JAPAN Communicated by Britton Chance, April 1, 1970 Abstract. The primary structures of human, bovine, and chicken cytochrome bN have been determined and compared with that of the previously studied rabbit protein. One peptide containing 31 amino acid residues and another containing 10 were found common to all four species. The substitutions of amino acids between species could be accounted for mainly by single base exchange, with a few exceptional double base exchanges for the chicken. Results for bovine cytochrome b5 differ significantly from those previously reported for calf cytochrome N5. Cytochrome b5 is a hemoprotein with a role in the microsomal electron- transport system. The prosthetic group of the protein is a protoheme identical with that of hemoglobin and myoglobin.' The main interest of the work to be described resides in the relationship between the function and structure of cytochrome b5, hemoglobin, and myoglobin. Primary and tertiary structures of hemoglobin and myoglobin, together with details of their functional roles, have been reported.2-5 In contrast, little is known about cytochrome b5 except for the primary structures of two cytochromes b5.6-8 This communication describes and compares the primary structures of human, bovine, rabbit, and chicken hepatic cytochrome b5. Cytochrome b6 of rabbit liver has been crystallized9 10 and its amino acid sequence preliminarily reported.8 Crystals of rabbit cytochrome b5 suitable for X-ray analysis have been grown by dialysis against 80% saturated ammonium sulfate buffered with phosphate from pH 5.5 to 7.0. Unit cell dimensions are 111.1, 36.3, and 51.4 A. The space group is P212,21 with two molecules per asymmetric unit. Interestingly, Mathews and Strittmatter found" that bovine cytochrome b5 also crystallizes in space group P212121 with only one molecule per asymmetric unit. The bovine enzyme has been isolated and sequenced by Ozols and Strittmatter.67 Results and Discussion. The amino acid sequence of rabbit cytochrome br has been further studied and the structure was determined in more detail. The other three cytochromes were purified from human, bovine, and chicken liver after digestion of the microsomal fraction with trypsin, Nagarse, and Nagarse§ respectively.13 The apoproteins obtained by the removal of heme18 were sub- jected to the structural studies. Bovine cytochrome b5 was almost completely, 442 Downloaded by guest on October 5, 2021 VOL. 67, 1970 CYTOCHROME B5 FROM FOUR SPECIES 443

and chicken cytochrome b5 partially, sequenced using trypsin and chymotrypsin digests. Only tryptic digests of human cytochrome b5 were studied. Details are to be published elsewhere. The sequence of trypsin peptides was placed in homology with that of the other cytochromes b5 (Fig. 1). The deletion of histidine residue at the 14th position of the human enzyme24 is only tentative, because the possibility of there being an additional lysine residue after the 10th or 16th position has not been excluded experimentally.812'24 The amino acid residues are numbered from the NH2-terminus of the shortest protein (from chicken). From that point toward the left, the sequences are numbered as minus. In fact, the tryptic peptide of rabbit cytochrome b5 has 90 amino acids,8 but under severe digesting conditions with large amounts of trypsin, a dipeptide (Ser-Lys) from the COOH-terminus could be detached,8 as well as a hexapeptide (Gln,Ala,Ala,Ser,Asp,Lys) at the NH2-terminus, with no significant changes in absorption spectrum or biological activity. 12 Thus the amino-terminal hexapeptide and amino acids 82 and 83 do not seem essential for enzymatic activity. 62 out of 83 amino acids are common to the four proteins. The homogeneity percentage, 75%, is somewhat lower than that for cytochrome c (85.6%) in the same four species.'4-'7 Most strikingly, a 31-unit peptide (amino acids 35-65) and a 10-unit peptide (amino acids 70-79) were common to the four species. The first of these is more than one-third the length of the protein. In a similar study of hemoglobin and myoglobin, Ozols and Strittmatter suggested"9 20 the imidazole groups of His-60 and His-77 (Fig. 2) as possible heme-binding sites. However, there are 5-7 histidine residues in the four cytochrome b5 molecules studied here, 5 of them common to all four species, all of which can be considered possible heme-binding sites. In view of the invariant nature of the range of residues 35-65, His-36 and His-60 are perhaps better candidates than His-60 and His-77. The carboxyl groups of the glutamic acid residues might also constitute binding sites, from what we know about abnormal hemoglobin.2' The other possibilities, such as the hydroxyl group of tyrosine and the NH2 group of lysine, were excluded experimentally by Strittmatter.20'22 The differences in amino acids between rabbit cytochrome b5 and the other three animal proteins8" 2'24 could be accounted for by single base exchanges as shown in Table 1. Most of the base exchanges are found in the 5'-p ends in the codon assignments of amino acids.23 Exceptional double base exchanges are observed at least between bovine and chicken: AsnBovine (AAY)*GlXChicken (CAZ/GAZ) at position 14. Such double base changes have been more fre- quently observed in cytochrome c among these four species.14-'7 Table 2 shows the number of amino acid alterations and the possible numbers of base alterations (shown in parentheses) in cytochrome b5 and cytochrome c in the four different species, where the numerical agreement between amino acid and base alterations indicates single base exchange. The table suggests that in evolutionary terms, chicken is the most distant from the other three species. Fig. 2 shows a comparison of two sequences reported for calf liver protein by Ozols and Strittmatter8 7 and that for bovine liver protein determined by Downloaded by guest on October 5, 2021 .04

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TABLE 1. Alteration of amino acids and accompanying possible base exchanges in the code for cytochrome b5 from various animal livers. 1 VaIR,R,B (GUX) Glyc (GGX) 2 LYSR,H,B (AAZ) Argc (AGZ)* 5 ThrR,H,B (ACX) Argc (AGZ)* 9 IleR,H,B (AUY, AUA) VaIc (GUX) 10 LYSR (AAZ) GluH.B (GAZ) GInc (CAZ) 14 HiSR (CAY) AsnB (AAY) GlxC (AZ) Deletion11 16 LYSR,H,B (AAZ) Asxc AAY 20 LeUR,H,B (UUZ, CUX) Ilec (AUY, AUA) 22 LeUR,H,B (UUZ, CUX) VaIc (GUX) 24 HiSR,H,B (CAY) TyrB (UAY) 25 LYSR,H,B (AAZ) Argc (AGZ) 26 VaJR,H,B (GUX) Dec (AUY, AUA) 29 LeUR,H,B (UUZ, CMX) Dec (AUY, AUA) 34 G1URHB (GAZ) AspC (GAY) 66 G1URHB (GAZ) Alan (GCX) 67 LeUR,H,B (UUZ, CUX) MetH (AUG) 69 LYSR,H,B (AAZ) Gluc (GAZ) R, H, B, and C represent rabbit, human, bovine, and chicken cytochrome b5 respectively. X stands for U, C, A, or G; Y for V or C; Z for A or G. * CGX is also possible for the Arg codon; this seems unlikely here from the exchange moiety.

TABLE 2. Alterations of amino acids (and bases) in cytochromes b5 and c. Cytochrome Human Rabbit Bovine Chickent Human* b0° 0 Rabbit b5 3 (3) 0 c 9 (11) 0 Bovine b5 3 (3) 3 (3) 0 c 10 (11) 4 (5) 0 Chicken t b3 16 (16-17) 15 (15-17) 16 (17-18) 0 c 13 (16) 10 (11) 9 (10) 0 * Deletion of His (at 14th) was not counted. t Glx (14th) and Asx (16th) have not yet been determined; the possible number of alterations has been increased accordingly.

us.12 These two proteins are similar, but with a few significant differences. The difference in the COOH-terminal sequences may be accounted for by differences in the extraction method. However, the most striking difference is the absence of a tryptic octapeptide (18-25) in Ozols and Strittmatter's sequence. The other differences are the sequence 9-10 and the insertion of Trp at position 19 in their structure (shown as position 16 in Fig. 2). The sequence of Ile-Glu (9-10 in our work, 10-9 in Ozols and Strittmatter's) may be an experimental error on our part or on theirs. However, since we isolated by thermolysin digestion a tripeptide Ile-Glu-Lys from a tryptic peptide sequenced as Tyr-Tyr-Thr-Leu-Glu-Glu-Ile-Glu-Lys, our sequence seems the more likely. On the assumption that single base exchange is more probable than double, the sequence Ile-Glu-Lys may be justified by analogy with the rabbit, human, and chicken proteins shown in Fig. 1. Downloaded by guest on October 5, 2021 VOL. 67, 1970 CYTOCHROME BE FROM FOUR SPECIES 447

No tryptophan residue was found at position 16 in our bovine protein or in the protein from the three other species. The octapeptide 18-25 missing from Ozols and Strittmatter's structure was found in all four species. Its presence was further supported by thermolytic digestion of rabbit protein and by chy- motryptic digestion of bovine and chicken proteins. Note. After the completion of this paper, Ozols and Strittmatter published25 a correction of their sequence for calf liver cytochrome b5 and reported a peptide 18-25 similar to ours, which they had missed in their previous study. The ambiguity of the sequence (9-10) of Ile-Glu still remains, however, and needs further elucidation. * These studies were supported by research grants from the Japanese Ministry of Education, and grants awarded to A. T. from the Jane Coffin Childs Memorial Fund for Medical Research and from the National Institutes of Health, Bethesda, Md., research grant GM10982. t Requests for reprints may be addressed to Dr. A. Tsugita, Laboratory of Molecular Ge- netics, Osaka University Medical School, Osaka, Japan. $ Supported by a scholarship offered by the Japanese government (Monbusho). § Nagarse: A crystalline B. subtilis proteinase supplied by Nagarse Co., Ltd., Osaka, Japan. 1Strittmatter, P., and S. F. Velick, J. Biol. Chem., 221, 253 (1956). 2 Perutz, M. F., J. Mol. Biol., 13, 646 (1965). 3Perutz, M. F., J. C. Kendrew, and H. C. Watson, J. Mol. Biol., 13, 669 (1965). 4Perutz, M. F., Science, 140, 863 (1965). 5 Kendrew, J. C., Science, 139, 1259 (1963). 6 Ozols, J., and P. Strittmatter, J. Biol. Chem., 243, 3367 (1968). Ozols, J., and P. Strittmatter, J. Biol. Chem., 243, 3376 (1968). 8 Tsugita, A., M. Kobayashi, T. Kajihara, and B. Hagihara, J. Biochem. (Tokyo), 64, 727 (1968). 9 Kajihara, T., and B. Hagihara, J. Biochem. (Tokyo), 63, 453 (1968). 1 Kritsinger, R. H., B. Hagihara, and A. Tsugita, Biochim. Biophys. Acta, 200, 421 (1970). I' Mathews, F. S., and P. Strittmatter, J. Mol. Biol., 41, 295 (1969). 12 Tsugita, A., M. Kobayashi, S. Tani, Y. Yoshida, and B Hagihara, in preparation. 13 Hagihara, B., M. Rashid, and Y. Yoshida, in preparation. 14 Matsubara, H., and E. L. Smith, J. Biol. Chem., 238, 2732 (1962). 15 Nakashima, T., H. Higa, H. Matsubara, A. M. Benson, and K. T. Yasunobu, J. Biol. Chem., 241, 1166 (1966). 16 Chan, S. K., and E. Margoliash, J. Biol. Chem., 241, 507 (1966). 17Nedleman, S. B., and F. Margoliash, J. Biol. Chem., 241, 853 (1966). 18Ross-Fanelli, A., F. Antonini, and A. Kaputo, Biochini. Biophys. Acta, 28, 221 (1958). 19 Ozols, J., and P. Strittmatter, Proc. Nat. Acad. Sci. USA, 58, 264 (1967). 20 P. Strittmatter, J. Biol. Chem., 235, 2492 (1960). 21 Gerald, P. S., and M. L. Efron, Proc. Nat. Acad. Sci. USA, 47, 1758 (1961). 22 P. Strittmatter, J. Biol. Chem., 241, 4793 (1966). 23 Crick, F. H. C., Cold Spring Harbor Symp. Quant. Biol., 31, 1 (1966). 24 Rashid, M. A., S. Tani, M. Kobayashi, B. Hagihara, and A. Tsugita, in preparation. 2n Ozols, J., and P. Strittmatter, J. Biol. Chem., 244, 6617 (1969). Downloaded by guest on October 5, 2021