A two-process model describes the hydrogen exchange behavior of cytochrome c in the molten globule state with various extents of acetylation.

Acetylation of Lys residues of horse cytochrome c steadily stabilizes the molten globule state in 18 mM HCl as more Lys residues are acetylated [Goto and Nishikiori (1991) J. Mol. Biol. 222, 679-686]. The dynamic features of the molten globule state were characterized by hydrogen/deuterium exchange of amide protons, monitored by mass spectrometry as each deuteration increased the protein mass by 1 Da. Electrospray mass spectrometry enabled us to monitor simultaneously the exchange kinetics of more than seven species with a different number of acetyl groups. One to four Lys residue-acetylated cytochrome c showed almost no protection of the amide protons from rapid exchange. The transition from the unprotected to the protected state occurred between five and eight Lys residue-acetylated species. For species with more than nine acetylated Lys residues, the exchange kinetics were independent of the extent of acetylation, and 26 amide protons were protected at 60 min of exchange, indicating the formation of a rigid hydrophobic core with hydrogen-bonded secondary structures. The apparent transition to the protected state required a higher degree of acetylation than the conformational transition measured by circular dichroism, which had a midpoint at about four acetylated residues. This difference in the transitions suggested a two-process model in which the exchange occurs either from the protected folded state or from the unprotected unfolded state through global unfolding. On the basis of a two-process model and with the reported values of the exchange and stability parameters, we simulated the exchange kinetics of a series of acetylated cytochrome c species. The simulated kinetics reproduced the observed kinetics well, indicating validity of this model for hydrogen exchange of the molten globule state.