The alkaline transition of cytochrome c involves substitution of the Met80 heme ligand of the native state with a lysine ligand from a surface Ω-loop (residues 70 to 85). The standard mechanism for the alkaline transition involves a rapid deprotonation equilibrium followed by the conformational change. However, recent work implicates multiple ionization equilibria and stable intermediates. In previous work, we showed that the kinetics of formation of a His73-heme alkaline conformer of yeast iso-1-cytochrome c requires ionization of the histidine ligand (pK HL ∼ 6.5). Furthermore, the forward and backward rate constants, k f and k b, respectively, for the conformational change are modulated by two auxiliary ionizations (pK H1 ∼ 5.5, and pK H2 ∼ 9). A possible candidate for pK H1 is His26, which has a strongly shifted pK a in native cytochrome c. Here, we use the AcH73 iso-1-cytochrome c variant, which contains an H26N mutation, to test this hypothesis. pH jump experiments on the AcH73 variant show no change in k obs for the His73-heme alkaline transition from pH 5 to 8, suggesting that pK H1 has disappeared. However, direct measurement of k f and k b using conformationally gated electron transfer methods shows that the pH independence of k obs results from coincidental compensation between the decrease in k b due to pK H1 and the increase in k f due to pK HL. Thus, His26 is not the source of pK H1. The data also show that the H26N mutation enhances the dynamics of this conformational transition from pH 5 to 10, likely as a result of destabilization of the protein.