ACID-INDUCED UNFOLDING AND REFOLDING TRANSITIONS OF CYTOCHROME-C - A 3-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 mechan ism from the acid-unfolded state to the molten globule state [Kataoka, M., Hagihara, Y., Mihara, K., & Goto, Y. (1993) J. Mol. Biol. 229, 59 1-596], the corresponding transition in D2O has been proposed to invol ve a noncompact, alpha-helical intermediate state (the pre-molten glob ule state)[Jeng, M.-F., & Englander, S. W. (1991) J. Mol. Biol. 221, 1 045-1061]. To examine the proposed difference in the conformational tr ansitions, we carried out the HCl and DCl titrations of cytochrome c i n H2O and D2O, respectively, measured by far-UV circular dichroism, tr yptophan fluorescence, and Soret absorption. In both D2O and H2O, unfo lding 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 minima.1 three-state mechanism consisting of the native, molten globule, and ac id-unfolded states. Thus, our results did not substantiate the presenc e 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 conformati onal 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 s alt.