Ionic strength dependence of cytochrome c structure and Trp-59 hydrogen/deuterium exchange from ultraviolet resonance Raman spectroscopy

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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 = 0.005M 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 .apprx. 229 nm for both FeII and FeIII forms of cyt c, but at .apprx. 218 nm for aqueous tryptophan. The .apprx. 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, .apprx. 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 .apprx. 30 and .apprx. 5 h, respectively, for FeII and FeIII when examined at I = 0.005. Increasing the ionic strength to 1.5 M, however, raises the half-time to .apprx. 30 h for FeIII cyt c and to a much larger value for the FeII cyt c. This variation in the protein dynamics is consistent with recent evidence that the radius of gyration of the FeIII protein increases with decreasing ionic strength (Trewhella et al., 1988).