Ligand exchange during unfolding of cytochrome c

The productive folding pathway of cytochrome c passes through an obligatory HW intermediate in which the heme is coordinated by a solvent water molecu le and a native ligand, His-18, prior to the formation of the folded HM sta te with both the native His-18 and Met-80 heme coordination. Two off pathwa y intermediates, a five-coordinated state (5C) and a bis-histidine state (H H), were also identified during the folding reaction, In the present work, the thermodynamics and the kinetics of the unfolding reaction of cytochrome c were investigated with resonance Raman scattering, tryptophan fluorescen ce spectroscopy, and circular dichroism, The objective of these experiments was to determine if the protein opens up and diverges into the differing h eme ligation states through a many pathway mechanism or if it passes throug h intermediate states analogous to those observed during the folding reacti on. Equilibrium unfolding results indicate that, in contrast to 5C, the sta bility of HH with respect to HW decreases as the concentration of GdnHCl in creases. The difference in their response to the denaturant indicates that the polypeptide structure of 5C is relatively loose as compared with HH in which the polypeptide is misfolded. Time-resolved resonance Raman measureme nts show that strikingly similar ligand exchange reactions occur during unf olding as were observed during folding Combined with fluorescence data, a k inetic model is proposed in which local structural rearrangements controlle d by heme ligand exchange reactions appear prior to the global relaxation o f the polypeptide chain.