| Abstract
| - The effect of pH on the denatured state (3 M guanidine hydrochloride) was evaluated withfluorescence spectroscopy for four variants of iso-1-cytochrome c, AcTM (no surface histidines), AcH26(surface histidine at position 26), AcH54 (surface histidine at position 54), and AcH54I52 (stabilizing I52mutation added to AcH54). Changes in the compactness and the heme ligation of the denatured state, asa function of pH, were monitored through changes in Trp 59−heme fluorescence quenching. With theAcTM and AcH26 variants, no change in the fluorescence intensity occurs from pH 4 to 10. However,for the AcH54 and AcH54I52 variants the fluorescence intensity drops significantly between pH 4 and 6,consistent with His 54 binding to the heme of cytochrome c. Between pH 8 and 10 fluorescence intensityincreases again, indicating that the His 54 is displaced from the heme. The data are consistent with lysines4 and 5 being the primary heme ligands at alkaline pH, under denaturing conditions. This conclusion wasconfirmed by site-directed mutagenesis. Thermodynamic analysis indicates that heme−ligand affinity inthe denatured state is controlled primarily by sequence position (loop size) and that when histidines arepresent they inhibit lysine ligation until approximately pH 8.5−9.0 as compared to pH 7.5 with the AcTMvariant. Thus, at physiological pH, histidine ligands provide the primary constraint on the denatured stateof cytochrome c. The heme−Trp 59 distance in the denatured state of iso-1-cytochrome c, derived fromanalysis by Förster energy transfer theory, is ∼26 Å at pH 4 and 10, much shorter than the random coilprediction of 56 Å. Surprisingly, the heme−Trp 59 distance in the His 54 bound conformation only dropsto ∼21 Å, consistent with an extended conformation for the short polypeptide segment separating hemeand Trp 59.
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