Titration Behavior and Tautomeric States of Individual Histidine Residues of Myoglobins

The titration behavior of individual histidine residues of myoglobins has been studied by observing the pH dependence of the chemical shifts of the nonprotonated aromatic carbon resonances in natural abundance 13C Fourier transform NMR spectra (at 15.18 MHz and 37”),of horse ferrimyoglobin, horse cyanoferrimyoglobin, and red kangaroo cyanoferrimyoglobin. In the case of the cyanoferrimyoglobins, all nonprotonated side chain carbons of aromatic amino acid residues yield detectable resonances, but only 24 of the 28 carbons of this type yield detectable resonances in the case of horse ferrimyoglobin. Eight of the 11 histidine residues of horse cyanoferrimyoglobin (and 7 of the 10 histidines of the kangaroo protein) exhibit titration behavior (pK values in the range 4.4 to 6.6). The imidazole form of each titrating histidine is predominantly (or entirely) in the NfZ-H tautomeric state. Two of the titrating resonances of the cyanoferrimyoglobins (with pK values of 5.3 and about 4.5) do not yield detectable signals in spectra of horse ferrimyoglobin. These two resonances are assigned to 0’ of His-64 and His-97 (not on a one-to-one basis). Five of the six titrating resonances of horse ferrimyoglobin have pK values (5.5, 5.7, 6.5, 6.6, and 6.6) which are consistent with those of five of the six reported pK values that were obtained from proton NMR spectra. The sixth pK (<5), observed in our 13C NMR spectra of horse ferrimyoglobin and the cyanoferrimyoglobins, does not have a detected counterpart in the reported proton NMR data. Also, the “high” pK (about 7.4 to 8.0) reported in the proton NMR studies of ferrimyoglobins has no counterpart in our 13C NMR spectra of horse ferrimyoglobin and the cyanoferrimyoglobins from horse, kangaroo, and sperm whale. One of the three nontitrating histidine CY resonances of the cyanoferrimyoglobins from horse and kangaroo (not observed in spectra of horse ferrimyoglobin) is assigned to the coordinated His-93. Our results indicate that the two uncoordinated nontitrating histidine residues are either in the imidazolium or in the NSl-H imidazole state (or a mixture of the two states). The crystal structure of myoglobin suggests that these are His-24 and His-36. We also identify the resonances of CY, C62, and @ of the two tryptophan