Publication Details (including relevant citation information):
Biochemistry (1996) 35: 16852-16862.
Sauder JM, MacKenzie NE, Roder H
In spite of marginal sequence homology, cytochrome c2 from photosynthetic bacteria and the mitochondrial cytochromes c exhibit some striking structural similarities, including the tertiary arrangement of the three main helices. To compare the folding mechanisms for these two distantly related groups of proteins, equilibrium and kinetic measurements of the folding/unfolding reaction of cytochrome c2 from Rhodobacter capsulatus were performed as a function of guanidine hydrochloride (GuHCl) concentration in the absence and presence of a stabilizing salt, sodium 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 c2 contains only one histidine, His17, which forms the axial heme ligand under native and denaturing conditions. Quantitative kinetic modeling showed that both equilibrium and kinetic results are consistent with a minimal four-state mechanism with two sequential intermediates. The observation of a large decrease in fluorescence during the 2-ms dead-time of the stopped-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 denaturant-independent process at low GuHCl concentrations reflects the rate-limiting conversion of a compact intermediate to the native state. At high GuHCl concentrations, a process with little denaturant dependence is attributed to the rate-limiting Met96-iron deligation process during unfolding, which is supported by the kinetics of imidazole binding. The strong GuHCl-dependence of folding and unfolding rates near the midpoint of the equilibrium transition is attributed to destabilization of each intermediate and their transition states in folding and unfolding. Addition of sodium sulfate shifts the rate profile to higher denaturant concentration, which can be understood in terms of the relative stabilizing effect of the salt on partially and fully folded states.
Address (URL): http://pubs.acs.org/doi/abs/10.1021/bi961976k