Role of Met80 and Tyr67 in the Low-pH Conformational Equilibria ofCytochrome c

The low-pH conformational equilibria of ferricyeast iso-1 cytochrome c (ycc) and its M80A, M80A/Y67H, andM80A/Y67A variants were studied from pH 7 to 2 at low ionicstrength through electronic absorption, magnetic circulardichroism, and resonance Raman spectroscopies. For wild-typeycc, the protein structure, axial heme ligands, and spin state ofthe iron atom convert from the native folded His/Met low-spin(LS) form to a molten globule His/H2O high-spin (HS) formand a totally unfolded bis-aquo HS state, in a single cooperativetransition with an apparent pKa of ∼3.0. An analogouscooperative transition occurs for the M80A and M80A/Y67H variants. This is preceded by protonation of heme propionate-7, with a pKa of ∼4.2, and by an equilibrium between a His/OH−-ligated LS and a His/H2O-ligated HS conformer, with a pKa of∼5.9. In the M80A/Y67A variant, the cooperative low-pH transition is split into two distinct processes because of an increasedstability of the molten globule state that is formed at higher pH values than the other species. These data show that removal ofthe axial methionine ligand does not significantly alter the mechanism of acidic unfolding and the ranges of stability of low-pHconformers. Instead, removal of a hydrogen bonding partner at position 67 increases the stability of the molten globule andrenders cytochrome c more susceptible to acid unfolding. This underlines the key role played by Tyr67 in stabilizing the threedimensionalstructure of cytochrome c by means of the hydrogen bonding network connecting the Ω loops formed by residues71−85 and 40−57.