title: Role of Met80 and Tyr67 in the low-pH conformational equilibria of cytochrome c.
abstract: The low-pH conformational equilibria of ferric yeast iso-1 cytochrome c (ycc) and its M80A, M80A/Y67H, and M80A/Y67A variants were studied from pH 7 to 2 at low ionic strength through electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies. For wild-type ycc, the protein structure, axial heme ligands, and spin state of the iron atom convert from the native folded His/Met low-spin (LS) form to a molten globule His/H(2)O high-spin (HS) form and a totally unfolded bis-aquo HS state, in a single cooperative transition with an apparent pK(a) of ~3.0. An analogous cooperative transition occurs for the M80A and M80A/Y67H variants. This is preceded by protonation of heme propionate-7, with a pK(a) of ~4.2, and by an equilibrium between a His/OH(-)-ligated LS and a His/H(2)O-ligated HS conformer, with a pK(a) of ~5.9. In the M80A/Y67A variant, the cooperative low-pH transition is split into two distinct processes because of an increased stability of the molten globule state that is formed at higher pH values than the other species. These data show that removal of the axial methionine ligand does not significantly alter the mechanism of acidic unfolding and the ranges of stability of low-pH conformers. Instead, removal of a hydrogen bonding partner at position 67 increases the stability of the molten globule and renders cytochrome c more susceptible to acid unfolding. This underlines the key role played by Tyr67 in stabilizing the three-dimensional structure of cytochrome c by means of the hydrogen bonding network connecting the Ω loops formed by residues 71-85 and 40-57.
authors: Department of Chemistry, University of Modena and Reggio Emilia, via Campi 183, 41100 Modena, Italy. gianantonio.battistuzzi@unimore.it
authors: Gianantonio
authors: G
authors: Battistuzzi
authors: Carlo Augusto
authors: CA
authors: Bortolotti
authors: Marzia
authors: M
authors: Bellei
authors: Giulia
authors: G
authors: Di Rocco
authors: Johannes
authors: J
authors: Salewski
authors: Peter
authors: P
authors: Hildebrandt
authors: Marco
authors: M
authors: Sola
mesh_terms: D045304:Cytochromes c / Q000737:chemistry / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D008715:Methionine / Q000737:chemistry* / Q000502:physiology; D011487:Protein Conformation; D058767:Protein Unfolding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology; D014443:Tyrosine / Q000737:chemistry* / Q000502:physiology
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine; D045304:Cytochromes c; D008715:Methionine