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 cytochromes 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 enzymes differ in their cytochrome specificity, but they are related in sequence, and both contain conserved Cys-Pro-Val (CPV) motifs, By using various in vitro assays we investigated whether heme can bind directly to heme lyases and whether the CPV motif may be involved in heme binding, 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 heme lyases can bind heme directly, and they identify the CPV sequence as a structural element important for this interaction. The phenotype of a yeast mutant expressing CC(1)HL(SPV) is in good agreement with such a role of the CPV motif, The mutant cells accumulate the heme-free intermediate 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.