Ligand exchange during cytochrome c folding.
Submillisecond folding of cytochrome c reveals that a nascent phase appears within the mixing dead time of 100 microseconds, followed by a ligand exchange reaction during which His 26/33, water and Met 80 are inter-exchanged as haem ligands through a thermodynamically controlled equilibrium. In the ligand exchange phase, the rate of formation of a misfolded histidine-histidine coordinated state (HH) decreases by two orders of magnitude as the pH is reduced from 5.9 to 4.5 due to the protonation of the misligated His 26/33. The activation energy barriers for the transitions from the histidine-water coordinated form (HW) to the histidine-methionine coordinated form and the HH form are 18 and 4 kcal mol-1 respectively, at pH 4.8. The activation energy barrier for protein to escape from the misligated HH to the HW form was measured to be 12 kcal mol-1, demonstrating the kinetic trapping effect of the misligated bis-histidine form. The development of the polypeptide tertiary structure near the haem is concomitant with the coordination of the native haem axial ligand.
Yeh
S R
SR
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Takahashi
S
S
Fan
B
B
Rousseau
D L
DL
D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009206:Myocardium / Q000201:enzymology; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013059:Spectrum Analysis, Raman; D013816:Thermodynamics
D003574:Cytochrome c Group; D008024:Ligands