Formation of a biologically active, ordered complex from two overlapping fragments of cytochrome c.
A noncovalent complex of the apoprotein (1-104) and cyanogen bromide heme fragment containing residues 1 to 65, (1-65) H, has been prepared from horse heart cytochrome c. Conditions under which the redundant portions of the ferrous complex can be removed by limited trypsin digestion have been devised. The complementing fragments have been isolated from the derived complexes and four apofragments and one heme fragment have been identified in the amino acid sequence of cytochrome c. They are (39-104), (40-104), (54-104), (56-104), and (1-53)H. The formation of an ordered ferric complex composed of one heme fragment and one apofragment for the cases (1-53)H (39-104), (1-53)H-(40-104), (1-53)H-(54-104), and (1-53)H-(56-104) has been demonstrated by the quenching of the tryptophan 59 fluorescence and the regain of biological activity in a cytochrome b2 assay. The apparent dissociation constant has been estimated as less than 3 X 10(-7) M in all the aforementioned cases. Thus, the region (between residues 38 and 57) of the amino acid sequence permissible for cleavage without disruption of the ordered structure indicated by the present in vitro experiments corresponds to that (between residues 38 and 57) evolutionally deleted in the three-dimensional structure of Pseudomonas aeruginosa cytochrome c551 discovered by Dickerson et al. (Dickerson, R.E., Timkovich, R., and Almassy, R.J. (1976) J. Mol. Biol. 100, 473-491).
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001051:Apoenzymes; D001665:Binding Sites; D003574:Cytochrome c Group / Q000378:metabolism; D005033:Ethylmaleimide; D006736:Horses; D007700:Kinetics; D008961:Models, Structural; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014357:Trypsin
D000596:Amino Acids; D001051:Apoenzymes; D003574:Cytochrome c Group; D010446:Peptide Fragments; D014357:Trypsin; D005033:Ethylmaleimide