Molecular Basis Behind Inability of Mitochondrial Holocytochrome c Synthase to Mature Bacterial Cytochromes

Abstract

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.

Engineered holocytochrome c synthases that biosynthesize new cytochromes c

“…The bacterial system I and II cytochrome c biogenesis pathways are more complex and appear to recognize only the CXXCH motif; thus, no one has engineered them to biosynthesize, for example, bis-Met or single thioether c-type cytochromes. The mitochondrial HCCS, on the other hand, recognizes an extended region besides the CXXCH motif, including α-helix 1 immediately preceding CXXCH (32,33). Recognition of this extended region may help in making new c-type cytochromes with substitutions in CXXCH.…”

A Novel 4-gene Score to Predict Survival, Distant Metastasis and Response to Neoadjuvant Therapy in Breast Cancer

“…HCCS (holocytochrome c synthase) is an enzyme that is required for mitochondrial cytochrome c maturation and therefore respiration [61]. HCCS has been implicated in the molecular mechanisms that promote embryonic heart regeneration [62,63].…”

Biosynthesis of Single Thioether c-Type Cytochromes Provides Insight into Mechanisms Intrinsic to Holocytochrome c Synthase (HCCS)

“…For Coomassie stains, proteins resolved by SDS-PAGE were stained, destained, and imaged as described previously. 13 …”

“…Although HCCS has long been recognized as the key enzyme in mitochondrial cyt c assembly 6 , investigation of its mechanism, including substrate binding, have only recently been explored (Figure 1A) 10–13 . HCCS-mediated cyt c biosynthesis is proposed to follow a four-step process: In step 1, HCCS binds noncovalently to heme (Fe 2+ ), with His154 forming an axial ligand to the heme iron (residue numbering refers to human HCCS).…”