Synthesis of a heme fragment of horse cytochrome c which forms a productive complex with a native apofragment.
To investigate the amino acid sequence of apocytochrome c recognized by yeast mitochondrial cytochrome c synthetase, a labeled apofragment containing residues 1 to 25 of horse cytochrome c, N alpha-[3H]acetyl-(1-25), and an analog containing glycine in place of cysteines 14 and 17, N alpha-[3H]acetyl-[14-Gly, 17-Gly] (1-25), have been synthesized using the Merrifield's improved solid phase method (Mitchell, A. R., Ericksen , B. W., Ryabtsev , M. N., Hodges , R. S., and Merrifield, R. B. (1976) J. Am. Chem. Soc. 98, 7357-7362) and then purified to homogeneity. Upon incubation with yeast mitochondria in the presence of hemin, a radioactive species, produced from N alpha-[3H]acetyl-(1-25) and not from N alpha-acetyl-[14-Gly, 17-Gly] (1-25), formed a complex with native apofragment (23-104). This semisynthetic complex was indistinguishable from the native complex in resistance to trypsin upon reduction with ascorbate and by ion-exchange chromatography. The radioactive species, dissociated from the complex was identical with native heme fragment N alpha-acetyl-(1-25)H by reverse-phase high pressure liquid chromatography. Treatment of this radioactive heme fragment with silver sulfate and then with dithiothreitol generated the original apofragment . Thus, if it is assumed that the mitochondrial enzyme catalyzing this covalent attachment of heme to synthetic apo-N alpha-[3H]acetyl-(1-25) is a cytochrome c synthetase, the results may be interpreted as indicating that the amino acid sequence of residues 1 to 25 of horse cytochrome c would contain the principal recognition site of the enzyme.
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000378:metabolism*; D003574:Cytochrome c Group / Q000138:chemical synthesis* / Q000378:metabolism*; D045304:Cytochromes c; D006418:Heme / Q000378:metabolism*; D006736:Horses; D008190:Lyases; D008928:Mitochondria / Q000201:enzymology; D008958:Models, Molecular; D010446:Peptide Fragments / Q000138:chemical synthesis; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014166:Transferases / Q000378:metabolism
D001059:Apoproteins; D003574:Cytochrome c Group; D010446:Peptide Fragments; D006418:Heme; D045304:Cytochromes c; D014166:Transferases; D008190:Lyases; C031581:cytochrome C synthetase