Kinetics of Histidine Deligation from the Heme in GuHCl-Unfolded Fe(III) Cytochrome c Studied by a Laser-Induced pH-Jump Technique

We have developed an instrumental setup that uses transient absorption to monitor protein folding/unfolding processes following a laser-induced, ultrafast release of protons from o-nitrobenzaldehyde. The resulting increase in [H+], which can be more than 100 μM, is complete within a few nanoseconds. The increase in [H+] lowers the pH of the solution from neutrality to approximately 4 at the highest laser pulse energy used. Protein structural rearrangements can be followed by transient absorption, with kinetic monitoring over a broad time range (∼10 ns to 500 ms). Using this pH-jump/transient absorption technique, we have examined the dissociation kinetics of non-native axial heme ligands (either histidine His26 or His33) in GuHCl-unfolded Fe(III) cytochrome c (cyt c). Deligation of the non-native ligands following the acidic pH-jump occurs as a biexponential process with different pre-exponential factors. The pre-exponential factors markedly depend on the extent of the pH-jump, as expected from differences in the pK a values of His26 and His33. The two lifetimes were found to depend on temperature but were not functions of either the magnitude of the pH-jump or the pre-pulse pH of the solution. The activation energies of the deligation processes support the suggestion that GuHCl-unfolded cyt c structures with non-native histidine axial ligands represent kinetic traps in unfolding.