Probing Protein Folding with Substitution-Inert Metal Ions. Folding Kinetics of Cobalt(III)-Cytochrome c

Ligand-substitution processes at the heme strongly influence peptide backbone dynamics during the folding of cytochrome c (cyt c). When cyt c is unfolded with guanidine hydrochloride (GuHCl) at pH 7, one of the axial ligands (Met 80) is replaced by a nitrogenous base from an amino acid residue; this misligation introduces an energy barrier with an associated folding time of several hundred milliseconds. A great deal of evidence points to His 26 or His 33 as the ligand in unfolded horse heart cyt c. Nevertheless, recent studies indicate that other bases (Lys or N-terminus in yeast cyt c) can act as ligands as well. We have found that the substitution-inert heme in the Co(III) derivative of cyt c (Co-cyt c) allows a closer look at the folding kinetics and the ligands in the unfolded form of this protein.