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.

Structural Re-arrangement and Peroxidase Activation of Cytochrome c by Anionic Analogues of Vitamin E, Tocopherol Succinate and Tocopherol Phosphate

“…This is diagnostic of a molten globule organization of the hemo-protein that is characteristic of many non-native cytochrome c states induced by guanidine hydrochloride, low pH, and elevated temperature, or formed when cytochrome c binds to anionic phospholipid vesicles, micelles, polyanions, and electrodes. This high spin heme iron was also found in microperoxidases produced by tryptic digestion of cytochrome c resulting in the loss of the sixth coordination bond and markedly elevated peroxidase activity (34,35). Therefore we used electronic absorption spectroscopy to spectrally distinguish between the high spin and low spin ferric hemes (36 -39).…”

The Met80Ala and Tyr67His/Met80Ala mutants of human cytochrome c shed light on the reciprocal role of Met80 and Tyr67 in regulating ligand access into the heme pocket

“…Since coordination of His26 or His33 would require a change in the tertiary structure, it is very likely that His67, which replaces Tyr67 in the distal cavity, binds the iron atom. It is noted that the spectra of the M80A, M80A/Y67A, and M80A/Y67H mutants of yeast iso-1 cyt c at pH 7.2 revealed the presence of only a -OH-Fe-His18 species [26]. However, the low frequency spectrum of the human YHMA mutant (Fig.…”

Cytochrome c: A Multifunctional Protein Combining Conformational Rigidity with Flexibility

“…At high ionic strength the statistical coil state I converts to a molten globule, which was later termed A-state [109,110,125]. More recently, Battistuzzi et al used visible absorption and magnetic circular dichroism spectra to probe conformational transition of yeast iso-1 ferricytochrome c and three mutants (M80A, M80A/Y67H, and M80A/Y67A) in the acidic region and a low ion concentration [126]. They found that state III of the wild type first converts into a hexacoordinated high spin state, most likely with water as sixth ligand.…”