Role of Met80 and Tyr67
in the Low-pH Conformational
Equilibria of Cytochrome <i>c</i>
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Abstract
The low-pH conformational equilibria of ferric yeast
iso-1 cytochrome <i>c</i> (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<sub>2</sub>O high-spin
(HS) form and a totally unfolded bis-aquo HS state, in a single cooperative
transition with an apparent p<i>K</i><sub>a</sub> 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 p<i>K</i><sub>a</sub> of ∼4.2, and by an equilibrium
between a His/OH<sup>–</sup>-ligated LS and a His/H<sub>2</sub>O-ligated HS conformer, with a p<i>K</i><sub>a</sub> 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 <i>c</i> more
susceptible to acid unfolding. This underlines the key role played
by Tyr67 in stabilizing the three-dimensional structure of cytochrome <i>c</i> by means of the hydrogen bonding network connecting the
Ω loops formed by residues 71–85 and 40–57