Molecular Basis Behind Inability of Mitochondrial Holocytochrome c Synthase to Mature Bacterial Cytochromes: DEFINING A CRITICAL ROLE FOR CYTOCHROME c αHELIX-1

Mitochondrial holocytochrome c synthase (HCCS) is required for cytochrome c (cyt c) maturation and therefore respiration. HCCS efficiently attaches heme via two thioethers to CXXCH of mitochondrial but not bacterial cyt c even though they are functionally conserved. This inability is due to residues in the bacterial cyt c N terminus, but the molecular basis is unknown. Human cyts c with deletions of single residues in helix-1, which mimic bacterial cyt c, are poorly matured by human HCCS. Focusing on M13 cyt c, we co-purified this variant with HCCS, demonstrating that HCCS recognizes the bacterial-like cytochrome. Although an HCCS-WT cyt c complex contains two covalent links, HCCS-M13 cyt c contains only one thioether attachment. Using multiple approaches, we show that the single attachment is to the second thiol of C(15)SQC(18)H, indicating that helix-1 is required for positioning the first cysteine for covalent attachment, whereas the histidine of CXXCH positions the second cysteine. Modeling of the N-terminal structure suggested that the serine residue (of CSQCH) would be anchored where the first cysteine should be in M13 cyt c. An engineered cyt c with a CQCH motif in the M13 background is matured at higher levels (2-3-fold), providing further evidence for helix-1 positioning the first cysteine. Bacterial cyt c biogenesis pathways (Systems I and II) appear to recognize simply the CXXCH motif, not requiring helix-1. Results here explain mechanistically how HCCS (System III) requires an extended region adjacent to CXXCH for maturation.