title: Ionic strength dependence of cytochrome c structure and Trp-59 H/D exchange from ultraviolet resonance Raman spectroscopy.
abstract: Ultraviolet resonance Raman spectra are reported for cytochrome c (cyt c) in FeII and FeIII oxidation states at low (0.005 M) and high (0.9-1.5 M) ionic strength. With 200-nm excitation the amide band intensities are shown to remain constant, establishing that redox state and ionic strength have no influence on the alpha-helical content. The tyrosine 830/850-cm-1 doublet, however, shows a loss in 830-cm-1 intensity at I = 0.005 M for the FeIII protein, suggesting a weakening or a loss of H-bonding from an internal tyrosine, probably Tyr-48, which is H-bonded to a heme propionate group in cyt c crystals. Excitation profiles of tryptophan peak at approximately 229 nm for both FeII and FeIII forms of cyt c, but at approximately 218 nm for aqueous tryptophan. The approximately 2200-cm-1 red shift of the resonant electronic transition is attributed to the Trp-59 residue being buried and H-bonded. Consistent with this Trp environment, the H-bond-sensitive 877-cm-1 Trp band is strong and sharp, and the 1357/1341-cm-1 doublet has a large intensity ratio, approximately 1.5, for both FeII and FeIII cyt c. The 877-cm-1-band frequency shifts to 860 cm-1 when the Trp indole proton is replaced by a deuteron. This band was used to show that Trp H/D exchange in D2O is much faster for FeIII than FeII cyt c. The half-time for exchange at room temperature is estimated to be approximately 30 and approximately 5 h, respectively, for FeII and FeIII when examined at I = 0.005.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, Princeton University, New Jersey 08544-1009.
authors.forename: G Y
authors.initials: GY
authors.lastname: Liu
authors.forename: C A
authors.initials: CA
authors.lastname: Grygon
authors.forename: T G
authors.initials: TG
authors.lastname: Spiro
mesh_terms: D000818:Animals; D003574:Cytochrome c Group / Q000032:analysis*; D004735:Energy Transfer; D004795:Enzyme Stability; D005290:Ferric Compounds / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D006736:Horses; D006860:Hydrogen Bonding; D009206:Myocardium / Q000201:enzymology; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D014364:Tryptophan / Q000032:analysis*; D014443:Tyrosine / Q000032:analysis
chemical_list: D003574:Cytochrome c Group; D005290:Ferric Compounds; D005296:Ferrous Compounds; D014443:Tyrosine; D014364:Tryptophan