Acid-induced unfolding and refolding transitions of cytochrome c: a three-state mechanism in H2O and D2O.
Whereas the salt-dependent conformational transition of acid-denatured horse ferricytochrome c at pH 2 is approximated by a two-state mechanism from the acid-unfolded state to the molten globule state [Kataoka, M., Hagihara, Y., Mihara, K., & Goto, Y. (1993) J. Mol. Biol. 229, 591-596], the corresponding transition in D2O has been proposed to involve a noncompact, alpha-helical intermediate state (the pre-molten globule state) [Jeng, M.-F., & Englander, S. W. (1991) J. Mol. Biol. 221, 1045-1061]. To examine the proposed difference in the conformational transitions, we carried out the HCl and DCl titrations of cytochrome c in H2O and D2O, respectively, measured by far-UV circular dichroism, tryptophan fluorescence, and Soret absorption. In both D2O and H2O, unfolding from the native state to the acid-unfolded state and subsequent refolding to the molten globule state were observed. In either solvent, the conformational transitions were well approximated by a minimal three-state mechanism consisting of the native, molten globule, and acid-unfolded states. Thus, our results did not substantiate the presence of a pre-molten globule state in D2O. Acetylation of amino groups of cytochrome c is known to stabilize the molten globule state at pH 2. On the basis of the three-state mechanism, we constructed a conformational phase diagram for the effect of pH and the degree of acetylation. This phase diagram was similar to that of the pH- and salt-dependent conformational transition of cytochrome c, suggesting that the effects of acetylation on the conformational states are similar to those of salt.
Goto
Y
Y
Department of Biology, Faculty of Science, Osaka University, Japan.
Hagihara
Y
Y
Hamada
D
D
Hoshino
M
M
Nishii
I
I
D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D017666:Deuterium Oxide; D006736:Horses; D006851:Hydrochloric Acid / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D009994:Osmolar Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D013053:Spectrophotometry; D014867:Water
D003574:Cytochrome c Group; D014867:Water; D017666:Deuterium Oxide; D006851:Hydrochloric Acid