title: Characterization of equilibrium intermediates in denaturant-induced unfolding of ferrous and ferric cytochromes c using magnetic circular dichroism, circular dichroism, and optical absorption spectroscopies.
abstract: Protein unfolding during guanidine HCl denaturant titration of the reduced and oxidized forms of cytochrome c is monitored with magnetic circular dichroism (MCD), natural CD, and absorption of the heme bands and far-UV CD of the amide bands. Direct MCD spectral evidence is presented for bis-histidinyl heme ligation in the unfolded states of both the reduced and oxidized protein. For both redox states, the unfolding midpoints measured with MCD, which is an indicator of tertiary structure, are significantly lower than those measured with far-UV CD, an indicator of secondary structure. The disparate titration curves are interpreted in terms of a compound mechanism for denaturant-induced folding and unfolding involving a molten globulelike intermediate state (MG) with near-native secondary structure and nonnative tertiary structure and heme ligation. A comparison of the dependence of the free energy of formation of the MG intermediate on the redox state with the known contributions from heme ligation and solvation suggests that the heme is significantly more accessible to solvent in the MG intermediate than it is in the native state.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
authors.forename: Y G
authors.initials: YG
authors.lastname: Thomas
authors.forename: R A
authors.initials: RA
authors.lastname: Goldbeck
authors.forename: D S
authors.initials: DS
authors.lastname: Kliger
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006736:Horses; D007700:Kinetics; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry
chemical_list: D003574:Cytochrome c Group; D006418:Heme