Multiple kinetic intermediates accumulate during the unfolding of horse cytochrome c in the oxidized state

Abstract

The unfolding kinetics of horse cytochrome c in the oxidized state has been studied at 10, 22, and 34°C as a function of guanidine hydrochloride (GdnHCl) concentration. Rapid (millisecond) measurements of far-UV circular dichroism (CD) as well as fluorescence quenching due to tryptophan to heme excitation energy transfer have been used to monitor the unfolding process. At 10°C, the decrease in far-UV CD signal that accompanies unfolding occurs in two phases. The unobservable burst phase is complete within 4 ms, while the slower phase occurs over tens to hundreds of milliseconds. The burst phase unfolding amplitude increases cooperatively with an increase in GdnHCl concentration, exhibiting a transition midpoint of 3.2 M at 10°C. In contrast, no burst phase change in fluorescence occurs during unfolding at 10°C. At 22 and 34°C, both the fluorescence-monitored unfolding kinetics and the far-UV CD-monitored unfolding kinetics are biphasic. At both temperatures, the two probes yield burst phase unfolding transitions that are noncoincident with respect to the transition midpoints as well as the dependency of the burst phase amplitudes on GdnHCl concentration. The results suggest that at least two kinetic unfolding intermediates accumulate during unfolding. One burst phase intermediate, I_U¹, has lost virtually all the native-state secondary structure, while the other burst phase intermediate, I_U², has lost both secondary structure and native-like compactness. The presence of kinetic unfolding intermediates is also indicated by the nonlinear dependence of the logarithm of the apparent unfolding rate constant on GdnHCl concentration, which is particularly pronounced at 10 and 22°C. Analysis of the burst phase unfolding transitions obtained using the two probes shows that the stabilities of I_U¹ and I_U² decrease steadily with an increase in temperature from 10 to 34°C, suggesting that the structures present in them are stabilized principally by hydrogen bonding interactions.