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 α helix-1, which mimic bacterial cyt c, are poorly matured by human HCCS. Focusing on ΔM13 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-ΔM13 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 α 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 ΔM13 cyt c An engineered cyt c with a CQCH motif in the ΔM13 background is matured at higher levels (2-3-fold), providing further evidence for α helix-1 positioning the first cysteine. Bacterial cyt c biogenesis pathways (Systems I and II) appear to recognize simply the CXXCH motif, not requiring α helix-1. Results here explain mechanistically how HCCS (System III) requires an extended region adjacent to CXXCH for maturation.
Highlights
Cytochrome c2 biogenesis involves the covalent attachment of a heme cofactor to two cysteine residues found in the conserved CXXCH motif where its His residue acts as an axial ligand to the heme iron [1,2,3]
Residue Spacing in ␣ Helix-1 of Cyt c Is Important for HCCSmediated Maturation—To determine how sequence variations in the N-terminal region of bacterial c-type cytochromes affect the holocytochrome c synthase (HCCS)-mediated biogenesis of eukaryotic cyt c, we co-expressed human HCCS with human cyt c variants that are analogous to bacterial cytochromes (Fig. 2, A–C)
Thioether Linkage in the HCCS Complex—We focused on ⌬M13 cyt c to test the hypothesis that HCCS complexes with cyt c deletion variants in ␣ helix-1 form only a single covalent attachment to heme at Cys18
Summary
Residue Spacing in ␣ Helix-1 of Cyt c Is Important for HCCSmediated Maturation—To determine how sequence variations in the N-terminal region of bacterial c-type cytochromes affect the HCCS-mediated biogenesis of eukaryotic cyt c, we co-expressed human HCCS with human cyt c variants that are analogous to bacterial cytochromes (Fig. 2, A–C). The above results suggest that ⌬M13 cyt c may only have Cys covalently attached when in complex with HCCS These results were further supported by dithionite-reduced pyridine hemochrome spectra for each of the complexes, which showed ␣ peak maxima at 552.8, 552.4, and 552.5 nm for ⌬M13, ⌬K14, and the C15S cyt c complexes, respectively (Fig. 3E), values indicative of a single thioether [31, 34, 35]. The complex with WT cyt c exhibited an ␣ peak maximum at 550.8 nm in the pyridine hemochrome spectrum (Fig. 3E), a value that is consistent with heme containing two covalent attachments [31, 32, 36] These data suggest that, once bound to HCCS, cyt c variants containing deletions in ␣ helix-1 readily form only a single thioether with heme, both reactive cysteines in the C15XXC18H19 heme attachment motif are intact. A PEP-FOLD model in which Ser is substituted with cysteine (in a ⌬M13/C15ЈA background) (Fig. 5C) shows that the S16ЈC residue is positioned where a covalent attachment might occur more frequently, that is if the ␣ helix-1 (including Phe11) anchors the first cysteine residue next to 2-vinyl of heme at the HCCS active site
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