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

Acetylation of Lys residues of horse cytochrome c steadily stabilizes the m olten 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 increa sed the protein mass by I Da. Electrospray mass spectrometry enabled us to monitor simultaneously the exchange kinetics of more than seven species wit h a different number of acetyl groups. One to four Lys residue-acetylated c ytochrome c showed almost no protection of the amide protons from rapid exc hange. The transition from the unprotected to the protected state occurred between five and eight Lys residue-acetylated species. For species with mor e than nine acetylated Lys residues, the exchange kinetics were independent of the extent of acetylation, and 26 amide protons were protected at 60 mi n of exchange, indicating the formation of a rigid hydrophobic core with hy drogen-bonded secondary structures. The apparent transition to the protecte d state required a higher degree of acetylation than the conformational tra nsition measured by circular dichroism, which had a midpoint at about four acetylated residues. This difference in the transitions suggested a two-pro cess model in which the exchange occurs either from the protected folded st ate 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 obs erved kinetics well, indicating validity of this model for hydrogen exchang e of the molten globule state.