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 molecule and a native ligand, His-18, prior to the formation of the folded HM state with both the native His-18 and Met-80 heme coordination. Two off pathway intermediates, a five-coordinated state (5C) and a bis-histidine state (HH), 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 fluorescence spectroscopy, and circular dichroism. The objective of these experiments was to determine if the protein opens up and diverges into the differing heme ligation states through a many pathway mechanism or if it passes through intermediate states analogous to those observed during the folding reaction. Equilibrium unfolding results indicate that, in contrast to 5C, the stability of HH with respect to HW decreases as the concentration of GdnHCl increases. 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 measurements show that strikingly similar ligand exchange reactions occur during unfolding as were observed during folding. Combined with fluorescence data, a kinetic model is proposed in which local structural rearrangements controlled by heme ligand exchange reactions appear prior to the global relaxation of the polypeptide chain.
Yeh
S R
SR
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA. SYEH@AECOM.YU.EDU
Rousseau
D L
DL
D000818:Animals; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry*; D019791:Guanidine / Q000494:pharmacology; D006418:Heme / Q000737:chemistry; D006736:Horses; D007700:Kinetics; D008024:Ligands; D009206:Myocardium / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D003574:Cytochrome c Group; D008024:Ligands; D006418:Heme; D014364:Tryptophan; D019791:Guanidine