A biologically active, three-fragment complex of horse heart cytochrome c.

Abstract

A noncovalent complex of heme fragment containing residues 1 through 25, (1-25)H, and apofragment (1-65) plus apofragment (39-104) has been prepared from horse heart cytochrome c.This complex is biologically active as it can be reduced by lactate dehydrogenase and its light absorption and CD spectral and tryptophan 59 fluorescence-quenching properties are close to those of native cytochrome c.Digestion with trypsin of this complex in the ferrous form has resulted in specific removal of the redundant amino acid sequence.Two derived complexes, one active (55%) and the other inactive (45%), have been isolated.The component fragments of the active complex have been identified as (1-25)H, (28-38), and (56-104).This active ordered complex resembling cytochrome c can be reconstituted within 1 s from these three disordered fragments with an overall apparent dissociation constant <lo-'' M' and M* in the ferric and the ferrous form, respectively, at pH 7.0 and 25°C.Measurements of spectral properties and equilibrium constants and stopped flow kinetic studies suggest a pathway of high probability in assembly of the ferric complex in this system: (1-25)H (28-38):(56-104).Ligation of methionine 80 to the heme iron seems to occur in a late phase compared to assembly and folding of this complex.