Proton NMR study of hemoproteins. Ionization and orientation of iron-bound imidazole in methemoglobin and metmyoglobin

Abstract Characteristics of imidazole derivatives bound to methemoglobin and metmyoglobin were investigated by proton NMR spectroscopy with concentration on the ionization of the imino proton and orientation of the imidazole ring in the heme crevice. 1. (1) The hyperfine-shifted heme methyl resonances of metmyoglobin complexes with imidazole, 4-methylimidazole and triazole were pH-dependent, while those of the N- methylimidazole complex were pH-independent. The pH-dependent shifts in the former complexes were attributed to the deprotonation of the imino proton of imidazole derivatives bound to the heme iron. In contrast, metmyoglobin complexes with 4-nitroimidazole and 4-fluoroimidazole, both of which have the ionizable NH proton, did not exhibit such pH-dependent shifts. This finding was interpreted in terms of the restricted orientation of the iron-bound 4-nitroimidazole and 4-fluoroimidazole in the heme crevice, where the imino proton is not exposed to the solvent sphere. In the methemoglobin-imidazole complex, however, the pH-dependence of the hyperfine shifts was observed, in contrast to the case of imidazole-metmyoglobin. This finding was also interpreted as arising from the specific orientation of the iron-bound exogenous imidazole in the methemoglobin heme pocket. 2. (2) Two sets of heme methyl proton resonances were detected for the 4-fluoroimidazole-metmyoglobin complex and these were attributed to the two tautomeric forms of the iron-bound 4-fluoroimidazole. The relative proportion of the two tautomers was dependent on both pH and temperature. The temperature dependence of the hyperfine shifts of the two species indicates that they are in a single spin state.