HEME-BINDING TO A CONSERVED CYS-PRO-VAL MOTIF IS CRUCIAL FOR THE CATALYTIC FUNCTION OF MITOCHONDRIAL HEME LYASES

Covalent attachment of heme to the apoforms of mitochondrial cytochrom es c and c(1) requires the activity of cytochrome c heme lyase (CCHL) and cytochrome c(1) heme lyase (CC(1)HL), respectively, The two enzyme s differ in their cytochrome specificity, but they are related in sequ ence, and both contain conserved Cys-Pro-Val (CPV) motifs, By using va rious in vitro assays we investigated whether heme can bind directly t o heme lyases and whether the CPV motif may be involved in heme bindin g, Heme stabilized CC(1)HL, as a model protein, in a folded, protease- resistant conformation, stimulated the refolding of CC(1)HL after urea denaturation, and inhibited the import of the CC(1)HL precursor into mitochondria, These effects were not observed with a point mutant, CC( 1)HL(SPV), in which cysteine was replaced by serine, and with CC(1)HL( Delta CPV), in which the motif was deleted, These results show that he me lyases can bind heme directly, and they identify the CPV sequence a s a structural element important for this interaction. The phenotype o f a yeast mutant expressing CC(1)HL(SPV) is in good agreement with suc h a role of the CPV motif, The mutant cells accumulate the heme-free i ntermediate form of cytochrome c(1) and display a severe deficiency in the hole form. We suggest that the CPV motif forms a crucial part of the substrate binding site for heme.