IDENTIFICATION OF THE PREDOMINANT NONNATIVE HISTIDINE LIGAND IN UNFOLDED CYTOCHROME-C

The heme and its two axial ligands, His18 and Met80, play a central ro le in the folding/ unfolding mechanism of cytochrome c. Because of the covalent heme attachment, His18 remains bound under typical denaturin g conditions, while the more labile Met80 ligand is replaced by an alt ernate histidine ligand. To distinguish between the two possible non-n ative histidine ligands in horse cytochrome c, variants with a His26 t o Gin or His33 to Asn substitution were prepared using a yeast express ion system. Protonation of the non-native histidine ligand in the GuHC l-denatured state results in a pronounced blue shift of the Soret heme absorbance band (low-spin to high-spin transition). While substitutio n of His26 has no effect on the apparent pK(a) of this transition (5.7 +/- 0.05), the H33N variant exhibits a substantially higher pK(a) (6. 1 +/- 0.05), indicating that His33 is the dominant sixth heme ligand i n denatured cytochrome c and that His26 (or another nitrogenous group) acts as a ligand in the absence of a histidine at position 33. The ki netics of the pH induced ligand dissociation shows two phases which we re assigned to each of the two histidine ligands on the basis of their distinct temperature dependence. Despite their nearly identical equil ibrium unfolding transitions, the two histidine mutants show differenc es in their folding kinetics. While the kinetic behavior of H26Q cyt c is very similar to that of the wild-type, the H33N mutation leads to loss of a kinetic phase with a rate in the 2-10 s(-1) range that has p reviously been attributed to the rate-limiting dissociation of a trapp ed non-native histidine, which is thus identified as His33.