CYTOCHROME-C HEME LYASE ACTIVITY OF YEAST MITOCHONDRIA

A highly efficient in vitro system was established for measuring by hi gh performance liquid chromatography the formation of holocytochrome c by yeast mitochondria. Holocytochrome c formation required reducing a gents, of which dithiothreitol was the most effective. With biosynthet ically made, pure Drosophila melanogaster apocytochrome c and Saccharo myces 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 (K-m similar to 1 nM), as were those of mitoplasts with broken outer membranes (K-m similar to 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 concentrat ion in vivo. Apocytochrome c associated with yeast mitochondria in two phases of K-d, similar to 2 x 10(-10) and 10(-8) M, respectively, whe reas mitoplasts had lost the high affinity binding. A site-directed mu tant of apocytochrome c (lysines 5, 7, and 8 replaced by glutamine, gl utamic acid, and asparagine) was found to be converted to holocytochro me c (K-m similar to 3.3 nM; maximal activity unchanged), even though the mutations completely eliminated the high affinity binding. Thus, t he high affinity binding of apocytochrome c to mitochondria is not dir ectly related to holocytochrome c formation.