KINETIC MECHANISM OF FOLDING AND UNFOLDING OF RHODOBACTER-CAPSULATUS CYTOCHROME C(2)

In spite of marginal sequence homology, cytochrome cr from photosynthe tic bacteria and the mitochondrial cytochromes c exhibit some striking structural similarities, including the tertiary arrangement of the th ree main helices, To compare the folding mechanisms for these two dist antly related groups of proteins, equilibrium and kinetic measurements of the folding/unfolding reaction of cytochrome ct from Rhodobacter c apsulatus were performed as a function of guanidine hydrochloride (GuH Cl) concentration in the absence and presence of a stabilizing salt, s odium sulfate. Quenching of the fluorescence of Trp67 by the heme was used as a conformational probe. Kinetic complexities due to non-native histidine ligation are avoided, since cytochrome c(2) contains only o ne histidine, His17, which forms the axial heme ligand under native an d denaturing conditions. Quantitative kinetic modeling showed that bot h equilibrium and kinetic results are consistent with a minimal four-s tate mechanism with two sequential intermediates. The observation of a large decrease in fluorescence during the 2-ms dead-time of the stopp ed-flow measurement (burst phase) at low GuHCl concentration, followed by a sigmoidal recovery of the initial amplitude toward the unfolding transition region, is attributed to a well-populated compact folding intermediate in rapid exchange with unfolded molecules. A nearly denat urant-independent process at low GuHCl concentrations reflects the rat e-limiting conversion of a compact intermediate to the native state. A t high GuHCl concentrations, a process with little denaturant dependen ce is attributed to the rate-limiting Met96-iron deligation process du ring unfolding, which is supported by the kinetics of imidazole bindin g. The strong GuHCl-dependence of folding and unfolding rates near the midpoint of the equilibrium transition is attributed to destabilizati on of each intermediate and their transition states in folding and unf olding. Addition of sodium sulfate shifts the rate profile to higher d enaturant concentration, which can be understood in terms of the relat ive stabilizing effect of the salt on partially and fully folded state s.