title: Mechanisms of mitochondrial holocytochrome c synthase and the key roles played by cysteines and histidine of the heme attachment site, Cys-XX-Cys-His.
abstract: Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19.
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130 and.
authors.forename: Shalon E
authors.initials: SE
authors.lastname: Babbitt
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130 and.
authors.forename: Brian
authors.initials: B
authors.lastname: San Francisco
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130 and.
authors.forename: Deanna L
authors.initials: DL
authors.lastname: Mendez
authors.affiliation: the Department of Chemistry and Biochemistry, North Dakota State University, Fargo, North Dakota 58102.
authors.forename: Gudrun S
authors.initials: GS
authors.lastname: Lukat-Rodgers
authors.affiliation: the Department of Chemistry and Biochemistry, North Dakota State University, Fargo, North Dakota 58102.
authors.forename: Kenton R
authors.initials: KR
authors.lastname: Rodgers
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130 and.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Bretsnyder
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130 and kranz@biology.wustl.edu.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Kranz
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry; D004926:Escherichia coli; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D008190:Lyases / Q000737:chemistry*; D008928:Mitochondria / Q000201:enzymology*; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017510:Protein Folding; D011725:Pyridines / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D008024:Ligands; D009841:Oligonucleotides; D011725:Pyridines; D013438:Sulfhydryl Compounds; D006418:Heme; D006639:Histidine; D045304:Cytochromes c; D008190:Lyases; C031581:cytochrome C synthetase; D003545:Cysteine
keywords: Cytochrome c; Heme; Histidine; Mitochondria; Raman Spectroscopy