The effect of pH on the denatured state (3 M guanidine hydrochloride) was evaluated with fluorescence 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 AcH54152 (stabilizing 152 mutation added to AcH54). Changes in the compactness and the heme ligation of the denatured state, as a function of pH, were monitored through changes in Trp 59-heme fluorescence quenching. With the AcTM and AcH26 variants, no change in the fluorescence intensity occurs from pH 4 to 10. However, for the AcH54 and AcH54152 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 intensity increases again, indicating that the His 54 is displaced from the heme. The data are consistent with lysines 4 and 5 being the primary heme ligands at alkaline pH, under denaturing conditions. This conclusion was confirmed by site-directed mutagenesis. Thermodynamic analysis indicates that heme-ligand affinity in the denatured state is controlled primarily by sequence position (loop size) and that when histidines are present they inhibit lysine ligation until approximately pH 8.5 - 9.0 as compared to pH 7.5 with the AcTM variant. Thus, at physiological pH, histidine ligands provide the primary constraint on the denatured state of cytochrome c. The heme-Trp 59 distance in the denatured state of iso-1-cytochrome c, derived from analysis by Förster energy transfer theory, is ∼26/k Å at pH 4 and 10, much shorter than the random coil prediction of 56 Å. Surprisingly, the heme-Trp 59 distance in the His 54 bound conformation only drops to ∼21 Å, consistent with an extended conformation for the short polypeptide segment separating heme and Trp 59.