Cytochrome c heme lyase activity of yeast mitochondria

J Biol Chem. 1998 Oct 2;273(40):25695-702. doi: 10.1074/jbc.273.40.25695.


A highly efficient in vitro system was established for measuring by high performance liquid chromatography the formation of holocytochrome c by yeast mitochondria. Holocytochrome c formation required reducing agents, of which dithiothreitol was the most effective. With biosynthetically made, pure Drosophila melanogaster apocytochrome c and Saccharomyces cerevisiae mitochondria, the activity of cytochrome c heme lyase amounted to about 800 fmol min-1 mg-1 mitochondrial protein. The kinetics were typical Michaelis-Menten (Km approximately 1 nM), as were those of mitoplasts with broken outer membranes (Km approximately 3 nM). As tested with mitoplasts, holocytochromes c from a range of species were found to be competitive inhibitors of heme lyase at physiological concentrations, providing a mechanism for controlling this concentration in vivo. Apocytochrome c associated with yeast mitochondria in two phases of Kd approximately 2 x 10(-10) and 10(-8) M, respectively, whereas mitoplasts had lost the high affinity binding. A site-directed mutant of apocytochrome c (lysines 5, 7, and 8 replaced by glutamine, glutamic acid, and asparagine) was found to be converted to holocytochrome c (Km approximately 3.3 nM; maximal activity unchanged), even though the mutations completely eliminated the high affinity binding. Thus, the high affinity binding of apocytochrome c to mitochondria is not directly related to holocytochrome c formation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoproteins / genetics
  • Apoproteins / metabolism
  • Cytochrome c Group / biosynthesis*
  • Cytochrome c Group / genetics
  • Cytochrome c Group / metabolism
  • Cytochromes c
  • Drosophila melanogaster / chemistry*
  • Enzyme Inhibitors / pharmacology
  • Hemin / metabolism
  • Kinetics
  • Lyases / antagonists & inhibitors
  • Lyases / metabolism*
  • Mitochondria / enzymology*
  • Mutagenesis, Site-Directed
  • Oxidation-Reduction
  • Protein Binding / genetics
  • Reducing Agents / pharmacology
  • Saccharomyces cerevisiae / enzymology*


  • Apoproteins
  • Cytochrome c Group
  • Enzyme Inhibitors
  • Reducing Agents
  • Hemin
  • Cytochromes c
  • Lyases
  • cytochrome C synthetase