pH-induced conformation changes and equilibrium unfolding in yeast iso-2 cytochrome c

The relationship between pH-induced conformational changes in iso-2 cytochrome c from Saccharomyces cerevisiae and the guanidine hydrochloride induced unfolding transition has been investigated. Comparison of equilibrium unfolding transitions at acid, neutral, and alkaline pH shows that stability toward guanidine hydrochloride denaturation is decreased at low pH but increased at high pH. In the acid range the decrease in stability of the folded protein is correlated with changes in the visible spectrum, which indicate conversion to a high-spin heme state--probably involving the loss of heme ligands. The increase in stability at high pH is correlated with a pH-induced conformational change with an apparent pK near 8. As in the case of homologous cytochromes c, this transition involves the loss of the 695-nm absorbance band with only minor changes in other optical parameters. For the unfolded protein, optical spectroscopy and 1H NMR spectroscopy are consistent with a random coil unfolded state in which amino acid side chains serve as (low-spin) heme ligands at both neutral and alkaline pH. However, the paramagnetic region of the proton NMR spectrum of unfolded iso-2 cytochrome c indicates a change in the (low-spin) heme-ligand complex at high pH. Apparently, the folded and unfolded states of the (inactive) alkaline form differ from the corresponding states of the less stable native protein.