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Role of Met80 and Tyr67 in the Low-pH Conformational Equilibria of Cytochrome c
Citation key Battistuzzi2012
Author Battistuzzi, Gianantonio and Bortolotti, Carlo Augusto and Bellei, Marzia and Di Rocco, Giulia and Salewski, Johannes and Hildebrandt, Peter and Sola, Marco
Pages 5967–5978
Year 2012
DOI 10.1021/bi3007302
Journal Biochemistry
Volume 51
Number 30
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/H2O high-spin (HS) form and a totally unfolded bis-aquo HS state, in a single cooperative transition with an apparent pK(a) of similar to 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 similar to 4.2, and by an equilibrium between a His/OH–ligated LS and a His/H2O-ligated HS conformer, with a pK(a) of similar to 5.9. In the M80A/Y67.A 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 Omega loops formed by residues 71-85 and 40-57.
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