title: Molecular Basis Behind Inability of Mitochondrial Holocytochrome c Synthase to Mature Bacterial Cytochromes: DEFINING A CRITICAL ROLE FOR CYTOCHROME c α HELIX-1.
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.
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130.
authors.forename: Shalon E
authors.initials: SE
authors.lastname: Babbitt
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hsu
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130 kranz@biology.wustl.edu.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Kranz
mesh_terms: D020816:Amino Acid Motifs; D045304:Cytochromes c / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008190:Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D045304:Cytochromes c; D008190:Lyases; C031581:cytochrome C synthetase
keywords: cytochrome c; heme; post-translational modification (PTM); protein structure; substrate specificity; system III; thioether