The mitochondrial cytochrome c N-terminal region is critical for maturation by holocytochrome c synthase

► Mitochondrial holocytochrome c synthase recognises apocytochrome c N-terminus. ► N-terminus of yeast apocytochrome c/C-terminus of a bacterial cytochrome is processed. ► F15 of the N-terminus of yeast cytochrome c is essential for HCCS processing. The covalent attachment of heme to mitochondrial cytochrome c is catalysed by holocytochrome c synthase (HCCS, also called heme lyase). How HCCS functions and recognises the substrate apocytochrome is unknown. Here we have examined HCCS recognition of a chimeric substrate comprising a short mitochondrial cytochrome c N-terminal region with the C-terminal sequence, including the CXXCH heme-binding motif, of a bacterial cytochrome c that is not otherwise processed by HCCS. Heme attachment to the chimera demonstrates the importance of the N-terminal region of the cytochrome. A series of variants of a mitochondrial cytochrome c with amino acid replacements in the N-terminal region have narrowed down the specificity determinants, providing insight into HCCS substrate recognition.