Effect of pH on formation of a nativelike intermediate on the unfolding pathway of a Lys 73 -> His variant of yeast iso-1-cytochrome c

Previous work on a Lys 73 --> His (H73) variant of iso-l-cytochrome c at pH 7.5 [Godbole et al. (1997) Biochemistry 36, 119-126] showed that this vari ant unfolds through a nativelike intermediate that has properties consisten t with replacement of the Met 80 heme ligand by His 73. Here, the pH depend ence of the equilibrium unfolding of the wild type (WT) and H73 proteins ha ve been investigated, since a characteristic pH dependence is expected for the stability of an intermediate stabilized by histidine-heme ligation. Sta bility has been evaluated using guanidine hydrochloride and pH denaturation methods. Above pH 5, the m-values from guanidine hydrochloride denaturatio n of the WT and H73 variants remain significantly different, consistent wit h continued population of this intermediate. At pH 4.5 the m-values for the two proteins are within error the same. To assess stability at lower pH, a cid denaturation was carried out. The midpoint is about 3.3 for both protei ns but the transition is broader for the H73 protein, suggestive of interme diates again being populated during the unfolding of the H73 protein at thi s lower pH. Heme ligation by Met 80 was monitored (695 nm absorbance) durin g gdnHCl (pH 4.5 and 5.0) and acid denaturation, confirming, respectively, the absence and presence of intermediates. A thermodynamic analysis demonst rates that this complex pH dependence for the presence of histidine ligatio n induced intermediates is expected and implicates a titratable group with a pK(a) of similar to 6.6. The analysis also demonstrates when the pH depen dences of global stability and stability of an intermediate differ signific antly, population of folding intermediates as a function of pH will show no vel behavior.