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Transport of apocytochrome c across the outer mitochondrial membrane and conversion to holocytochrome c were studied in vitro. Apocytochrome c was synthesized in a cell-free homogenate from Neurospora crassa. Transfer in vitro was accomplished in a reconstituted system consisting of the postribosomal supernatant of the cell-free homogenate and of isolated and purified mitochondria from Neurospora. The reconstituted system has the following characteristics:

  • 1
    Apocytochrome c is rapidly cleared from the supernatant and holocytochrome c appears in the mitochondria with the same kinetics. More than 80% of the apocytochrome c employed is converted to holocytochrome c. No transient accumulation of apocytochrome c is found in mitochondria.
  • 2
    The heme group becomes covalently linked to apocytochrome c in the reconstituted system as demonstrated by analysis of tryptic peptide maps of the apoprotein and holoprotein.
  • 3
    Deuterohemin added to the reconstituted system but not deuteroporphyrin inhibits the formation of holocytochrome c. This inhibition is reversed by protohemin.
  • 4
    In the presence of deuterohemin about half of the apocytochrome c remains in the supernatant; the other half becomes associated with the mitochondria. The latter portion is tightly bound and is specifically released upon incubation of the mitochondria with excess apocytochrome c. It is converted to holocytochrome c after addition of protohemin.

We conclude from these observations that apocytochrome c is transported across the outer mitochondrial membrane via receptor sites. In the presence of the heme analogue deuterohemin, binding to the receptor sites on the cytoplasmic surface of the outer mitochondrial membrane still takes place but translocation does not. The latter step is apparently coupled to the covalent linkage of the heme group. We suggest that the formation of the thioether bonds between apoprotein and heme is catalysed by an enzyme in the intermembrane space and that deuterohemin can compete with protohemin for binding to the enzyme. Finally, the data indicate that it is the heme group and not the porphyrin group which is coupled to the apoprotein.