Mechanisms of mitochondrial holocytochrome c synthase and the key roles played by cysteines and histidine of the heme attachment site, Cys-XX-Cys-His

J Biol Chem. 2014 Oct 17;289(42):28795-807. doi: 10.1074/jbc.M114.593509. Epub 2014 Aug 28.


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.

Keywords: Cytochrome c; Heme; Histidine; Mitochondria; Raman Spectroscopy.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Binding Sites
  • Catalytic Domain
  • Conserved Sequence
  • Cysteine / chemistry*
  • Cytochromes c / chemistry
  • Escherichia coli
  • Heme / chemistry*
  • Histidine / chemistry*
  • Humans
  • Ligands
  • Lyases / chemistry*
  • Mitochondria / enzymology*
  • Oligonucleotides / chemistry
  • Plasmids / metabolism
  • Protein Folding
  • Pyridines / chemistry
  • Spectrophotometry, Ultraviolet
  • Spectrum Analysis, Raman
  • Sulfhydryl Compounds / chemistry


  • Ligands
  • Oligonucleotides
  • Pyridines
  • Sulfhydryl Compounds
  • Heme
  • Histidine
  • Cytochromes c
  • Lyases
  • cytochrome C synthetase
  • Cysteine