Kinetic evidence for an on-pathway intermediate in the folding of cytochrome c

An early folding event of cytochrome c populates a helix-containing intermediate (INC) because of a pH-dependent misligation between the heme iron and nonnative ligands in the unfolded state (U). For folding to proceed, the nonnative ligation error must first be corrected. It is not known whether I is on-pathway, with folding to the native state (N) as in U <-->INC <--> N, or whether the I must first move back through the U and then fold to the N through some alternative path (INC <--> U <--> N). By means of a kinetic test, it is shown here that the cytochrome c I does not first unfold to U. The method used provides an experimental criterion for rejecting the off-pathway I <--> U <--> N option.