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Nuclear magnetic resonance titration curves of histidine ring protons. V. Comparative study of cytochrome c from three species and the assignment of individual proton resonances.

Abstract A single histidine C-2 proton resonance is seen to shift with change in pH in the NMR spectra of horse ferrocytochrome c, as previously found for horse ferricytochrome c (Cohen, J. S., Fisher, W. R., and Schechter, A. N. (1973) J. Biol. Chem. 249, 1113–1118). The pKa value (6.5) of this histidine residue in the reduced form is almost the same as that (6.4) in the oxidized form, and only a small upfield shift is observed in the reduced form, indicating a slightly more electronically shielded environment in this case. No titrating resonance is observed in the NMR spectra of tuna ferriand ferrocytochrome c in the pH range 5 to 8. Since histidine residue 33 in horse cytochrome c is replaced by a tryptophan residue in the analogous position in tuna cytochrome c, it can be concluded that the titrating resonance observed for the horse protein can be assigned to histidine residue 33. A sharp resonance appears in the spectra of tuna ferricytochrome c in the region of absorption of histidine ring C-2 protons when the pH is adjusted below pH 3.5. This resonance can be assigned to histidine residue 26, the only unliganded histidine residue present (histidine 18 is liganded to the iron atom and does not absorb in this region). A similar resonance is observed under the same conditions in spectra of horse ferricytochrome c. Its attribution to histidine residue 26 enables a consistent assignment of the 2 observed histidine ring C2 proton resonances to the 2 unliganded histidine residues in the horse protein. The imidazole side chain of histidine 26 is believed to be hydrogen-bonded to the peptide bond carbonyl oxygen atom of proline 44 (Dickerson, R. E., et al. (1971) J. Biol. Chem. 246, 1511–1533). Presumably, the protonation of this imidazole side chain, leading to the breakage of this hydrogen bond, is one of the first steps in the acid-induced conformational transition of cytochrome c, which has a midpoint at pH 2.5. A comparative study of the pH-dependent chemical shifts of resonances in the spectra of yeast ferricytochrome c enables the three C-2 proton resonances observed to be assigned to the 3 unliganded histidine residues present in this case (26, 33, 39). The similarity of the pH-induced chemical shifts of the analogous histidine C-2 proton resonances in tuna, horse, and yeast cytochrome c indicates a similarity in the environments of the histidine residues in these cytochromes from widely divergent species.