LIGAND-EXCHANGE DURING CYTOCHROME-C FOLDING

Submillisecond folding of cytochrome c reveals that a nascent phase ap pears within the mixing dead time of 100 mu s, followed by a ligand ex change reaction during which His 26/33, water and Met 80 are inter-exc hanged as haem ligands through a thermodynamically controlled equilibr ium. In the ligand exchange phase, the rate of formation of a misfolde d histidine-histidine coordinated state (HH) decreases by two orders o f magnitude as the pH is reduced from 5.9 to 4.5 due to the protonatio n of the misligated His 26/33. The activation energy barriers for the transitions from the histidine-water coordinated form (HW) to the hist idine-methionine coordinated form and the HH form are 18 and 4 kcal mo l(-1) respectively, at pH 4.8. The activation energy barrier for prote in 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 misl igated bis-histidine form. The development of the polypeptide tertiary structure near the haem is concomitant with the coordination of the n ative haem axial ligand.