Cytochrome c heme lyase activity of yeast mitochondria.
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.
Tong
J
J
Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Margoliash
E
E
D000818:Animals; D001059:Apoproteins / Q000235:genetics / Q000378:metabolism; D003574:Cytochrome c Group / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D045304:Cytochromes c; D004331:Drosophila melanogaster / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006427:Hemin / Q000378:metabolism; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000235:genetics; D019163:Reducing Agents / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D001059:Apoproteins; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D019163:Reducing Agents; D006427:Hemin; D045304:Cytochromes c; D008190:Lyases; C031581:cytochrome C synthetase