Biophys J, January 2000, p. 405-415, Vol. 78, No. 1

Fast Events in Protein Folding: Structural Volume Changes Accompanying the Early Events in the NI Transition of Apomyoglobin Induced by Ultrafast pH Jump

Stefania Abbruzzetti,^* Elisa Crema,^* Laura Masino,^* Arnaldo Vecli,^* Cristiano Viappiani,^* Jeanne R. Small, Louis J. Libertini,^§ and Enoch W. Small^§

 ^*Dipartimento di Fisica, Università di Parma, and  Istituto Nazionale per la Fisica della Materia, Parco area delle Scienze n. 7A, 43100 Parma, Italia;  Department of Chemistry and Biochemistry, Eastern Washington University, MS 74, Cheney, Washington USA; and  ^§Quantum Northwest, Inc., Spokane, Washington USA

Ultrafast, laser-induced pH jump with time-resolved photoacoustic detection has been used to investigate the early protonation steps leading to the formation of the compact acid intermediate (I) of apomyoglobin (ApoMb). When ApoMb is in its native state (N) at pH 7.0, rapid acidification induced by a laser pulse leads to two parallel protonation processes. One reaction can be attributed to the binding of protons to the imidazole rings of His24 and His119. Reaction with imidazole leads to an unusually large contraction of 82 ± 3 ml/mol, an enthalpy change of 8 ± 1 kcal/mol, and an apparent bimolecular rate constant of (0.77 ± 0.03) × 10¹⁰ M¹ s¹. Our experiments evidence a rate-limiting step for this process at high ApoMb concentrations, characterized by a value of (0.60 ± 0.07) × 10⁶ s¹. The second protonation reaction at pH 7.0 can be attributed to neutralization of carboxylate groups and is accompanied by an apparent expansion of 3.4 ± 0.2 ml/mol, occurring with an apparent bimolecular rate constant of (1.25 ± 0.02) × 10¹¹ M¹ s¹, and a reaction enthalpy of about 2 kcal/mol. The activation energy for the processes associated with the protonation of His24 and His119 is 16.2 ± 0.9 kcal/mol, whereas that for the neutralization of carboxylates is 9.2 ± 0.9 kcal/mol. At pH 4.5 ApoMb is in a partially unfolded state (I) and rapid acidification experiments evidence only the process assigned to carboxylate protonation. The unusually large contraction and the high energetic barrier observed at pH 7.0 for the protonation of the His residues suggests that the formation of the compact acid intermediate involves a rate-limiting step after protonation.

Biophys J, January 2000, p. 405-415, Vol. 78, No. 1
© 2000 by the Biophysical Society   0006-3495/00/01/405/11  $2.00

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